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KONZO AND CASSAVA TOXICITY
A STUDY OF ASSOCIATED NUTRITIONAL FACTORS IN THE POPOKABAKA
DISTRICT, DEMOCRATIC REPUBLIC OF CONGO
Delphin DIASOLUA NGUDI
Academic Year 2004-2005
KONZO AND CASSAVA TOXICITY: A STUDY OF ASSOCIATED
NUTRITIONAL FACTORS IN THE POPOKABAKA DISTRICT,
DEMOCRATIC REPUBLIC OF CONGO
KONZO-ZIEKTE EN DE TOXICITEIT VAN MANIOK: EEN STUDIE VAN DE VOEDINGSFACTOREN IN POPOKABAKA DISTRICT,
DEMOCRATISCHE REPUBLIEK KONGO
Door
Delphin DIASOLUA NGUDI, M.Sc.
Thesis submitted in fulfilment of the requirements for the degree
of Doctor (Ph.D.) in Applied Biological Sciences
Proefschrift voorgedragen tot het bekomen van de graad van
Doctor in the Toegepaste Biologische Wetenschappen
Op gezag van: Rector: Prof. Dr. Apr. A. DE LEENHEER
Decaan: Prof. Dr. Ir. Herman VAN LANGENHOVE
Promotoren:Prof. Dr. Patrick KOLSTEREN
Prof. Dr. Ir. Fernand LAMBEIN
CITATION
“A person who has food has many problems.
A person who has no food has only one problem”
Chinese saying
DEDICATION
To Thérèse Luntala and our sons Berdit, Gaël and Beni
In memory of my Grand Oncle André Banketa and my father Bernard Ngudi-a-nkama
Promotoren: Prof. Dr. Patrick KOLSTEREN
Vakgroep Voedselveiligheid en Voedselkwaliteit, Universiteit Gent Voedings unit, Prins Leopold Instituut voor Tropische Geneeskunde, Antwerpen Prof. Dr. Ir. Fernand LAMBEIN Instituut Planten Biotechnologie voor Ontwikkelingenlanden, Universiteit Gent
Decaan: Prof. Dr. Ir. Herman VAN LANGENHOVE
Academiejaar 2004 - 2005
Delphin DIASOLUA NGUDI
KONZO AND CASSAVA TOXICITY
A STUDY OF ASSOCIATED NUTRITIONAL FACTORS IN THE POPOKABAKA
DISTRICT, DEMOCRATIC REPUBLIC OF CONGO
Proefschrift
voorgedragen tot het bekomen van de graad van
Doctor in de Toegepaste Biologische Wetenschappen
Op gezag van de rector,
Prof. Dr. Apr. A. DE LEENHEER
Nederlandse vertaling titel:
KONZO-ZIEKTE EN DE TOXICITEIT VAN MANIOK: EEN STUDIE VAN DE
VOEDINGSFACTOREN IN POPOKABAKA DISTRICT, DEMOCRATISCHE
REPUBLIEK KONGO
Diasolua Ngudi, D. (2005). Konzo and cassava toxicity: a study of associated nutritional factors in the Popokabaka District, Democratic Republic of Congo. Ph D. thesis Universiteit Gent, Belgium, 160 p ISBN 90-5989-073-6 The author and the promoter give the authorisation to consult and to copy parts of this work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in this work should be obtained from the author.
Acknowledgments
I would like to express my gratitude to all the many people who and institutions that have
contributed to this research. I am particularly grateful to my promoter Prof Dr Patrick
Kolsteren for his encouragement, his guidance and constructive criticisms. My special thanks
to Prof Dr Ir Fernand Lambein, the co-promoter of this thesis for accepting me to work under
his supervision. I am indebted to him for improving my skills, including my laboratory
techniques and for sharing his knowledge and experience on nutritional and neuro-
toxicological disease. I also thank Prof. Em. Dr. Ir André Huyghebaert, my promoter until his
retirement for handling the administrative issues of my enrolment to the university.
I thank Dr J. Howard Bradbury from the Australian University and chairperson of the
Cyanide Cassava Disease Network for his advices and for graciously providing me kits for
analyses of cyanide and thiocyanate. I thank Dr Yu-Haey Kuo (Dianna) for the initiation to
the use of HPLC instruments and for her meticulous attention in scrutiny of manuscripts. I
thank Prof Dr Thorkild Tylleskär for inspiring and encouraging me to initiate this research.
Dr Theophile Ntambwe, former Director of PRONANUT has to be thanked for his support
and follow up of my scholarship file. I am thankful to Prof Dr JP Banea Mayambu, Director
of PRONANUT for the encouragement, guidance and support. I am indebted to both of them
for providing me field facilities. The staff and colleagues of PRONANUT are greatly
acknowledged for their enthusiasm and encouragement.
Prof Dr Simon Malele ma Ludani from the Australia’s University of Southern Queensland/
Dubai and Dr Fabienne Ladrière from Médecins du Monde are thanked for their assistance
and for reading and improving this thesis. I am deeply grateful to the members of the
examination committee: Prof Dr Ir Georges Hofman, Prof Dr Ir Colin Janssen, Prof Dr Geert
Callewaert, Prof Dr Armand Christophe and Dr Ir Bruno De Meulenaer. Their criticism of
this work was very constructive.
The Belgian Cooperation and Development Ministry has exceptionally granted me a mixed
scholarship through the Belgian Technical Cooperation (BTC) in April 2002. I thank Mr
Marino Orban, Mr G. Kasende and Mrs Sarah Stijnen of BTC for their efficient management
of my dossier. Congolese Ministry of Health through PRONANUT, OXFAM – Destelbergen
(Katrien Goddemaer), GTZ (Nour Salua) and anonymous relatives and friends provided also
financial support to conduct part of my program.
Prof Em Marc Van Montagu, Prof Ann Depicker, Prof Dr Lieve Gheysen and staff of IPBO
are thanked for the hospitality and their interest in my research.
Konzo and cassava toxicity
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Exceptional thanks to Ir. Anne-Marie De Winter, Regine Haspeslagh, Veerle Van Ongeval, Ir.
Ann Peters, Christine Graveel, Jean-Pierre Dubois, Ing. Alfons Lenaert, Myriam De Vos and
Ing. Yves De Jonge for their assistance, continuous interest and encouragement.
I am indebted to the Visie newspaper and to the VRT-film crew Jo Frere and Jasmine De
Bruycker for the interest in the topic of my research and for their objective reporting on the
disease on the disease konzo and on our research.
Piet Meyvaert, Paul Meirsman, Lieve Van Wijmeersch, Fernand Verhoeven, Erik Verhaegen,
Willy Mpoyi, Louis Kitenge, Raf Nunga, Elie and Blaise Ndosimau, Sera and Emmanuel
Kisuesue, Ir Bernard Lelou, Prof Dr Emmanuel Biey, Dr José Biey, Dr Thomas Mpiana, Dr
Clément Mulenda Tshamala, Michel Fazili and other friends, not mentioned by name, are
acknowledeged for their help and friendship.
I would like to express my cheerfully thanks to my mother, to my sisters Lili, Micheline and
Euphrasie,to my sister in law Makiese and to my brothers in law Malueki, Nsinga, Ntondo
and Yende for taking care of our sons while we were out of the country and far from them.
Many thanks to my uncle Prof Dr Mamingi Nlandu, to my brothers Domi, Sivis and Nsimba,
to my cousin Dimbu, to my sisters in law Annie, Julie and Elisée and to all my nephews and
nieces.
To my sons Berdit, Gäel and Beni, I have to apologise for not giving them all the attention
they deserved during the many busy years that research entailed. I thank Thérèse Luntala for
often coping with my absence concerning things of life and for her advice, help and love.
Last but not the least; I thank the authorities and the population of Popokabaka for their
enthousiasm and collaboration during my field trips.
Gent, June 2005
Delphin Diasolua Ngudi
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III
CONTENTS
Acknowledgments......................................................................................................................I
CONTENTS........................................................................................................................... III
List of figures ........................................................................................................................... V
List of tables............................................................................................................................VI
Abbreviations and acronyms..............................................................................................VIII
Samenvatting ........................................................................................................................... X
Résumé .................................................................................................................................XIII
Summary............................................................................................................................ XVII
I Literature review.............................................................................................................. 2
I.1 Introduction.................................................................................................................... 2
I.1.1 Paraparesis and neurodegenerative diseases, what is the meaning? ...................... 2
I.1.2 The “hidden endemias” .......................................................................................... 3
I.2 Konzo.............................................................................................................................. 7
I.2.1 Background information on konzo......................................................................... 7
I.2.2 Clinical features and differential diagnosis.......................................................... 10
I.2.3 Epidemiology ....................................................................................................... 14
I.2.4 Infection or toxico-nutritional etiology? .............................................................. 15
I.3 Dietary exposure to cyanide from cassava .................................................................. 17
I.3.1 Cassava................................................................................................................. 17
I.3.2 Cyanide toxicity ................................................................................................... 27
I.4 Conclusion.................................................................................................................... 39
I.4.1 Rationale of the research ...................................................................................... 40
I.4.2 Objectives............................................................................................................. 40
II Occurrence of konzo and dietary pattern .................................................................... 43
II.1.1 Introduction .......................................................................................................... 43
Konzo and cassava toxicity
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II.1.2 Materials and Methods ......................................................................................... 43
II.1.3 Results .................................................................................................................. 47
II.1.4 Discussion and conclusion ................................................................................... 54
III Cassava food quality and safety.................................................................................... 59
III.1 Food Safety and Amino Acid Balance in Processed Cassava "cossettes" ............... 59
III.1.1 Introduction ...................................................................................................... 59
III.1.2 Materials and methods ..................................................................................... 62
III.1.3 Results and discussion...................................................................................... 66
III.2 Residual cyanogens, free and total amino acid profiles of cooked cassava leaves
"saka- saka”......................................................................................................................... 79
III.2.1 Introduction ...................................................................................................... 79
III.2.2 Materials and methods ..................................................................................... 80
III.2.3 Results and discussion...................................................................................... 82
III.2.4 Conclusions ...................................................................................................... 92
IV Dietary cyanogen and sulphur metabolites excretion................................................. 95
IV.1.1 Introduction ...................................................................................................... 95
IV.1.2 Material and methods ....................................................................................... 96
IV.1.3 Results .............................................................................................................. 98
IV.1.4 Discussion ...................................................................................................... 100
V General discussion and conclusions............................................................................ 105
V.1 Occurrence of konzo............................................................................................... 106
V.2 Cassava foods and sulphur metabolites ................................................................. 108
V.3 Conclusions and recommendations........................................................................ 111
References ............................................................................................................................. 114
Curriculum Vitae ..................................................................................................................... ii
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List of figures
Figure I-1: Health zones of Kwango District in Bandundu Province, DRC............................. 8
Figure I-2: From left to right: mild form, moderate form and severe form of konzo in young
subjects ............................................................................................................................. 12
Figure I-3 : Cassava roots harvested and cassava plant in the field........................................ 18
Figure I-4: Location of cassava production, 1996 (Scott et al, 2000)..................................... 23
Figure I-5: Summary of traditional cassava processing in Africa (from Banea-Mayambu,
1997c)............................................................................................................................... 27
Figure I-6: Cyanogenesis from linamarin (McMahon et al, 1995) ......................................... 29
Figure I-7: Basic processes involved in the metabolism of cyanide (ATSDR, 1997) ............ 35
Figure I-8 : Cysteine catabolism ............................................................................................. 37
Figure II-1: Distribution of onset of konzo from 1980 to 2002.............................................. 49
Figure III-1: Flow diagram of cassava cossettes processing .................................................. 60
Figure III-2: Free amino acids in cassava cossette samples ................................................... 74
Figure III-3: Protein amino acids profile of the raw and cooked cassava leaves ................... 87
Konzo and cassava toxicity
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List of tables
Table I-1: Characteristic features of four tropical myeloneuropathies (Tylleskär et al, 1994c)5
Table I-2: WHO criteria for konzo versus newly suggested criteria (with permission and from
Tshala-Katumbay, 2001a) ................................................................................................ 11
Table II-1: Socio-demographic variables and 24hr recall food consumption of participants
among the high prevalence of konzo health area (n = 224) and the low prevalence of
konzo health area (n =263)............................................................................................... 46
Table II-2: Distribution of konzo cases per health area .......................................................... 49
Table II-3 : Degree of disability on walking and age distribution of konzo patients by gender
.......................................................................................................................................... 50
Table II-4: 24-hour recall of household food intake frequencies (%) .................................... 51
Table II-5: Seasonal food consumption availability (%) listed by the respondents ............... 53
Table III-1: Cyanogens content in cassava cossettes (mg HCN equivalent kg - 1 dry weight)67
Table III-2: Estimated daily cossettes and total cyanogens intake ......................................... 69
Table III-3: Total protein amino acids content in cassava cossettes (mg g - 1dry weight)...... 72
Table III-4: Amino acid scoring pattern of different cossette samples .................................. 73
Table III-5. Free protein amino acids content in cassava cossettes (mg g - 1 dry weight)....... 75
Table III-6. Essential Amino Acid (EAA) requirements and estimated daily intake ............. 77
Table III-7: Cyanogen content in raw and cooked cassava leaves (mg HCN equivalent kg-1
dry weight) ....................................................................................................................... 84
Table III-8: Protein content and amino acid composition of raw and cooked pounded cassava
leaves (g kg-1 dry weight) ................................................................................................. 86
Table III-9: Comparison of the essential amino acid contents of different raw and cooked
pounded cassava leaves samples and their amino acid score with the recommended FAO
reference ........................................................................................................................... 89
Konzo and cassava toxicity
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Table III-10: Free amino acid and trigonelline content in raw and cooked cassava leaves (g
kg-1 dry weight) ................................................................................................................ 91
Table IV-1: Distribution of konzo- affected households in each health area with the number
of konzo patients given in brackets .................................................................................. 98
Table IV-2: Total cyanogens in cassava flour, thiocyanate and taurine in urine samples
collected in three konzo prevalence areas of Popokabaka (DRC). .................................. 99
Konzo and cassava toxicity
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Abbreviations and acronyms
α-ABA α-Amino butyric acid
AMPA α-Amino-3hydroxy-5-methyl-isoxazole-4-propionic acid
ATC 2-Aminothiazoline-4-carboxylic acid
ATSDR Agency for Toxic Substances and Disease Registry
BOAA ß-Oxalylaminoalanine
ß-ODAP ß-N-oxalyl-α,ß-diaminopropionic acid
BTC/CTB Belgian Technical Cooperation/ Coopération Technique Belge
CEPLANUT Centre National de Planification de Nutrition Humaine
CI confidence interval
CN- Cyanide
CNS Central nervous system
D. R. C. Democratic Republic of Congo
EAA Essential amino acids
FAO Food and Agriculture Organisation
GABA γ-Amino butyric acid
HCN Hydrogen cyanide
HNL α-Hydroxynitrile lyase
HPLC High Performance Liquid Chromatography
HTLV-1 Human T cell lymphocyte virus Type I
IITA International Institute for Tropical Agriculture
KCN Potassium cyanide
NaCN Sodium cyanide
OCN- Cyanate
OR Odds Ratio
PITC Phenylisothiocyanate
Ppm Part per million
Prhz Popokabaka rural health zone
PRONANUT Programme National de Nutrition
R. D. C. République Démocratique du Congo
SAA Sulphur containing amino acids
SCN- Thiocyanate
SD Standard deviation
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SPSS Statistical Package for Social Science
TAN Tropical ataxic neuropathy
TSP/HAM Tropical spastic paraparesis/ Human T cell lymphocyte virus
Type I-associated myelopathy
UDPG Uridine diphosphoglucose
UK United Kingdom
UNICEF United Nations for Infants and Children Emergency Funds
UNU United Nations University
USA United States of America
WHO World Health Organisation
Konzo and cassava toxicity
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Samenvatting
Konzo is een neurologische aandoening die gekenmerkt wordt door een plots beginnende en
blijvende verlamming van de benen. Dit komt vooral voor bij kinderen en vrouwen op
vruchtbare leeftijd. Wortels van bittere maniok (Manihot esculenta Crantz) en daarvan
afgeleide producten die een hoog gehalte aan cyanogenen bevatten, kunnen acute cyanide
vergiftiging veroorzaken met symptomen als braken, duizeligheid, maagpijn, flauwte,
hoofdpijn en diarree. Konzo werd toegeschreven aan een langdurige chronische inname van
cyanide in bittere maniokwortels die onvoldoende geroot werden. Maniok is een belangrijke
basis voor het dagelijkse voedsel van meer dan een half miljard mensen verspreid over de
ganse wereld, nochtans bevat het cyanogeen glycosiden, voornamelijk linamarine dat na
enzymatische omzetting tot cyanohydrine aanleiding kan geven tot het giftige blauwzuur
(HCN) na verdere enzymatische of spontane omzetting. Alhoewel er voldoende aanwijzingen
zijn voor een verband tussen de ziekte konzo en de consumptie van bittere cassava, blijft het
pathologisch mechanisme van de ziekte onduidelijk. Men vermoedt dat zowel HCN als zijn
metabolieten (2-aminothiazolin-4-carbonzuur, cyanaat en isothiocyanaat) een rol spelen in de
pathologie maar er is geen proefdier model om dit te bevestigen. Wel staat vast dat de
enzymatische omzetting van cyanide naar thiocyanaat (80% van het cyanide wordt langs die
weg gedetoxifieerd) gebruik maakt van zwavel afkomstig van de zwavelhoudende
aminozuren methionine en cysteine.
In dit werk wordt het voorkomen van de konzo ziekte in Popokabaka, in de Bandundu
provincie van de Democratische Republiek Kongo bestudeerd. Uit diezelfde regio kwam het
eerste gepubliceerde verslag over konzo in 1938. Mogelijke associaties tussen de ziekte met
gezinsgebonden factoren en met het eetpatroon van de bevolking die hoofdzakelijk maniok
eet, werden onderzocht. Konzo komt nog steeds voor in die regio met een incidentie van 1,3
‰ in 2002. Meer vrouwen dan mannen waren aangetast en er werd geen informatie over de
Konzo and cassava toxicity
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levensverwachting van de patiënten gevonden. Zowel geslacht als burgerlijke stand
vertoonden een statistische associatie met de kans op konzo. Maniok was het dominante
bestanddeel van de voeding in praktisch elk gezin en werd minstens eenmaal per dag gegeten
als luku, een dikke pasta van maniokmeel en water. Als bijgerechten werd voornamelijk
maniok bladeren en lokaal geteelde bonen gegeten. Alhoewel die bijgerechten rijker zijn aan
eiwit is de kwaliteit van dit eiwit laag door een gebrek aan zwavelhoudende aminozuren.
De ‘cossettes’, de gerote en gedroogde maniok wortels die het voornaamste
voedingsbestanddeel vormen en de maniokbladeren die het voornaamste bijgerecht vormen in
de regio werden onderzocht. De dagelijkse hoeveelheid werd bepaald, het gehalte aan en de
dagelijkse inname van cyanogeen en de hoeveelheid zwavelhoudende aminozuren vereist
voor de detoxificatie ervan werd berekend. De vrije en totale aminozuren in de maniok
produkten werden bepaald om eventuele inherente toxinen op te sporen, om de kwaliteit van
de proteïne te bepalen en om te kunnen vergelijken met de dagelijkse vereisten voor kinderen
en volwassenen. Er werd berekend dat kinderen van 1 tot 9 jaar dagelijks 0,4 tot 1,1 mg HCN
equivalenten innemen in 241 tot 389 g maniok produkten, hoofdzakelijk van ‘cossettes’.
Matig actieve volwassen mannen en vrouwen namen 0,6 tot 1,5 mg HCN equivalenten in per
dag van 390 tot 532 g maniok produkten bereid uit ‘cossettes ‘, in de veronderstelling dat
60% van de dagelijkse energievereisten voldaan worden door maniokwortels die 1,6 tot 2,8
mg HCN equivalenten bevatten per kg droog gewicht. We vonden geen potentieel giftige niet-
proteïne aminozuren in maniokwortels. Lysine en leucine zijn de limiterende aminozuren
terwijl methionine slechts voor 13% bijdraagt in het lage gehalte van zwavelhoudende
aminozuren van de ‘cossettes’. Er werd berekend dat, indien alleen maniok gegeten wordt, de
dagelijkse behoefte aan methionine voor kinderen van 1 tot 9 jaar slechts voor 60% voldaan
is, terwijl aan de dagelijkse behoefte van de volwassenen wel voldaan wordt indien 60% van
de dagelijkse energiebehoeften komt uit maniokprodukten (het nationale gemiddelde). De
Konzo and cassava toxicity
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maniokbladeren die wel rijker zijn aan proteïne maar eveneens arm zijn aan methionine,
kunnen dit gebrek bij kinderen in gebieden met konzo niet compenseren. De maniokbladeren
als bijgerecht kunnen ook een bijkomende bron van cyanide zijn indien dit onvoldoende lang
gekookt wordt wegens een gebrek aan brandhout, de enige beschikbare brandstof voor de
bereiding. Zwavelhoudende aminozuren zijn noodzakelijk voor de detoxifiëring van cyaniden
afkomstig van maniokwortels die onvoldoende geroot werden, maar ook van de bladeren die
onvoldoende lang gekookt werden.
In een epidemiologische studie werden urinestalen en stalen van de maniokbloem van
verschillende gezinnen onderzocht met de bedoeling de voedselveiligheid na te gaan, de
inname van cyanogenen te berekenen en een mogelijk verband na te gaan met de
zwavelmetabolieten taurine en thiocyanaat in de urine. Drie generaties na het eerste
gedocumenteerd voorkomen van konzo in deze regio blijft er nog steeds een hoog risico voor
de blootstelling aan cyanide van maniokbloem. In de helft van de gezinnen is het
cyanidegehalte in maniokbloem hoger dan de door de Wereld Gezondheid Organisatie en de
FAO aanbevolen grens van 10µg HCN equivalent/g bloem. De helft van de urinestalen
bevatten meer dan 300 µmol/l thiocyanaat. Dit wijst op een grote overmaat aan cyanide
inname Alhoewel men geen significante correlatie vond, wijst de lage concentratie taurine, de
eindmetaboliet van de zwavelhoudende aminozuren, op een hoog verbruik van zwavel voor
de detoxifiëring van cyanide met vorming van thiocyanaat.
De bevolking van Popokabaka is nog steeds blootgesteld aan een te hoog cyanogeen gehalte
uit de maniokvoeding en misschien ook aan cyanogenen uit de omgeving. Dit verhoogt het
risico voor konzo in die regio waar die verlammende ziekte nog steeds voorkomt. Een beter
gevarieerd en gebalanceerd dieet, dat rijker is aan methionine, is noodzakelijk voor een meer
efficiënte detoxifiëring van cyanide in het menselijke lichaam.
Konzo and cassava toxicity
XIII
Résumé
Konzo est un désordre neurologique caractérisé par un début soudain d’une paralysie
permanente des membres inférieurs. Les enfants d’au moins trois ans et les femmes en âge de
procréer sont les plus affectés. La consommation des tubercules de manioc et de leurs
produits dérivés contenant une grande quantité de cyanogènes peut causer une intoxication
pouvant se manifester par des vomissements, des nausées, des vertiges, des douleurs
abdominales, la faiblesse, des maux de tête et la diarrhée. Konzo a été attribué à une haute
consommation de cyanure des tubercules de manioc amer insuffisamment traité. Le manioc,
aliment de base très important pour plus d’un demi milliard d’habitants de la planète, contient
des glucosides cyanogéniques, principalement la linamarine qui après conversion
enzymatique en cyanohydrines, peut libèrer spontanément ou enzymatiquement un prodruit
toxique, l’acide cyanhydrique. Bien que le lien évident entre la maladie konzo et la
consommation du manioc amer insuffisammnet traité soit établi, les mécanismes
pathogéniques du konzo restent encore à élucider. L’acide cyanhydrique et ses métabolites
(acide 2-aminothiazoline-4-carboxylique, cyanate, thiocyanate) ont été suspectés de jouer un
rôle dans la pathogénicité du konzo chez l’être humain mais il n’y a pas encore de modèle
animal pour s’en assurer ou le confirmer. En effet, la conversion enzymatique du thiocyanate
à partir du cyanure (environ 80 % de cyanure est transformé par cette voie) nécessite le soufre
provenant des acides aminés soufrés, la méthionine et la cystéine.
Notre travail rapporte l’apparition des cas de konzo à Popokabaka, province de Bandundu, R.
D. Congo, une des régions incluse dans la première publication sur le konzo en 1938. Nous
décrivons l’association entre le konzo et les facteurs socio-économiques liés au ménage et les
habitudes alimentaires des populations consommant le manioc. Le konzo continue à sévir
dans cette région de Popokabaka avec une incidence de 1,3 ‰ en 2002. La paralysie affecte
plus de femmes que d’hommes et nous n’avons trouvé aucune publication sur l’espérance de
Konzo and cassava toxicity
XIV
vie (ou sur la mortalité) des personnes atteintes de konzo. Le genre et l’état civil des chefs de
ménage sont associés au degré de prévalence de konzo dans les différentes localités de la
région. L’alimentation dans la contrée est largement dominée par le manioc. Presque tous les
ménages consomment au moins une fois par jour du luku, pâte obtenue après cuisson et
malaxage de la farine de manioc dans de l’eau bouillante. Les principaux aliments
d’accompagnement du luku, les feuilles de manioc et le niébé, sont limités en acides aminés
soufrés.
Nous avons analysé les cossettes de manioc (produit dérivé des tubercules de manioc roui et
séché) et les feuilles de manioc, respectivement aliment de base et principal aliment
d’accompagnement dans la région, pour estimer la quantité de cyanogènes résiduels
consommée journellement et la quantité d’acides aminés soufrés disponibles pour la
détoxification de ces cyanogènes. Nous avons déterminé les acides aminés libres et totaux
dans les cossettes et les feuilles de manioc pour détecter la présence inhérente des acides
aminés non protéiniques potentiellement toxiques, pour évaluer la qualité de leur protéine
alimentaire et la comparer aux besoins recommandés en acides aminés des enfants et des
adultes. Nous avons trouvé une consommation journalière estimée de 0,4 à 1,1 mg HCN
équivalent dans 241 à 389 g de luku et de 0,6 à 1,5 mg HCN équivalent dans 390 à 532 g de
luku respectivement chez les enfants âgés de 1 à 9 ans et chez les adultes (masculin et
féminin) avec une activité modérée lorsque 60 % de l’énergie alimentaire journalière requise
provient du manioc de 1,6 et de 2,8 mg HCN équivalent par kg de produit sec. Aucun acide
aminé non protéinique potentiellement toxique n’a été détecté dans les produits dérivés du
manioc. La lysine et la leucine sont des acides aminés limitants et le contenu en méthionine
est très bas contribuant pour environ 13 % du total des acides aminés soufrés dans les
cossettes de manioc. Les enfants âgés de 1 à 9 ans ne peuvent pas s’attendre à satisfaire leurs
besoins recommandés en méthionine tandis que les adultes peuvent les satisfaire à partir de la
Konzo and cassava toxicity
XV
quantité requise calculée pour satisfaire 60 % de l’énergie alimentaire journalière mais pas
assez pour la détoxication de cyanure et le métabolisme normal. Les feuilles de manioc,
quantitativement riches en protéines mais limitées en acides aminés soufrés, ne peuvent pas
compenser la déficience alimentaire en acides aminés soufrés occasionnée par l’aliment de
base dans les régions affectées par le konzo. Nous concluons aussi que les feuilles de manioc
peuvent être une autre source non négligeable de cyanogène alimentaire dans cette région. En
effet les feuilles de manioc requièrent pour leu détoxication une longue cuisson et avec, le
manque d’électricité et de gaz, la rareté de bois de chauffage pour la préparation des aliments,
il y a risque d’écourter le temps de cuisson et par conséquent de consommer des aliments
insuffisamments cuits.
Les acides aminés soufrés sont essentiels pour la détoxication des cyanogènes résiduels
contenus dans le manioc (tubercules ou feuilles) insuffisamment traité ou cuit.
Les échantillons de manioc prélevés auprès des ménages ainsi que ceux des urines obtenus
des participants sélectionnés au hasard pour une étude épidémiologique que nous avions
effectuée ont été examinés pour évaluer l’innocuité de la farine de manioc prête à la cuisson
puis à la consommation, pour déterminer la charge en cyanogène et apprécier la relation
potentielle entre les métabolites soufrés urinaires, la taurine et le thiocyanate. Il y a un risque
élevé d’exposition alimentaire aux cyanogènes dû à la consommation du manioc
insuffisamment traité dans cette région où le konzo est rapporté depuis trois générations. La
farine de manioc prélevée dans plus de la moitié des ménages contenait un taux de cyanogène
au dessus du seuil de 10 ppm fixé par la FAO et l’OMS. Les urines de plus de la moitié des
participants contenaient plus de 300 umol de thiocyanate par litre. Ce qui suggère qu’il y a
une charge importante en cyanure. Par ailleurs, ces urines accusaient aussi une concentration
très basse en taurine (produit du métabolisme des acides aminés soufrés), suggérant que le
soufre est préférentiellement orienté dans la détoxication du cyanure par la formation du
Konzo and cassava toxicity
XVI
thiocyanate, bien que nous n‘avons presque pas trouvé de corrélation entre la taurine urinaire
et le thiocyanate.
La population de Popokabaka reste toujours grandement exposé aux cyanogènes alimentaires
du manioc et peut être aussi aux cyanogènes environnementaux. La prévention de risque élevé
d’appariton de cas de konzo dans la région requiert une alimentation suffisante, variée et
équilibrée particulièrement riche en méthionine pour permettre une détoxication effective du
cyanure par l’organisme en laissant assez d’acides aminés soufrés pour le reste des besoins
métaboliques de l’organisme.
Konzo and cassava toxicity
XVII
Summary
Konzo is a neurological disorder characterised by sudden onset of paralysis of the legs, which
occurs particularly in children and women of childbearing age. Consumption of cassava
(Manihot esculenta Crantz) and its products that contain large amounts of cyanogens may
cause acute cyanide poisoning with symptoms of vomiting, nausea, dizziness, stomach pains,
weakness, headache and diarrhoea. Konzo has been attributed to the high dietary cyanide
exposure from insufficiently processed roots of bitter cassava. Cassava which is an important
staple food for more than half a billion inhabitants worldwide contains cyanogenic glycosides,
mainly linamarin that after enzymatic conversion to cyanohydrins, may release spontaneously
or enzymatically the toxic hydrogen cyanide (HCN). Although evidence linking the disease
with consumption of bitter cassava has been established, the pathogenic mechanism of konzo
remains unclear. HCN and its metabolites (2-aminothiazoline-4-carboxylic acid, cyanate and
thiocyanate) have been suspected to play a role in the pathogenicity in humans but there is no
animal model to ascertain or to confirm this. The enzymatic conversion of cyanide into
thiocyanate (about 80 % of cyanide is transformed by this route) requires sulphur arising from
the sulfur containing amino acids (SAA) methionine and cysteine.
In this work, we reporte the occurrence of konzo disease in Popokabaka, Province of
Bandundu, D. R. Congo, one of the areas included in the first published report on konzo in
1938. We described associated household factors involved in the disease and the dietary
pattern of the cassava consuming populations. Konzo is still occurring in this area with an
incidence rate of 1.3‰ in 2002. The disease affected a larger proportion of females than
males but we found no reports on the life expectancy of konzo patients. Gender and marital
status of the heads of household were associated with the degree of prevalence of konzo. The
diet was largely dominated by cassava and almost all households consumed at least once daily
the luku, a stiff porridge from the cassava flour. Major foods such as cassava leaves and
Konzo and cassava toxicity
XVIII
cowpeas consumed as side–dishes to the staple food luku are of poor quality in protein
especially in SAA.
We analysed processed cassava roots ‘cossettes’, as the major staple food and cassava leaves,
as the major side-dish to the staple food in the region to estimate the quantity of daily intake
of cyanogen and for calculate the amount of SAA required for its detoxification. We
determined free and total amino acids in the cassava products to investigate the presence of
potentially toxic inherent nonprotein amino acids, to evaluate the dietary protein quality and
to compare with the amino acid requirements of children and adults. We estimated that
children (1-9yr) consumed daily about 0.4 to 1.1 mg of HCN equivalent in 241 to 389 g of
cassava product from the ‘cossettes’ and moderately active female or male consumed 0.6 to
1.6 mg of HCN equivalent in 390 to 532 g cassava product from the ‘cossettes’ when 60% of
the daily energy requirement is provided by cassava roots containing between 1.6 and 2.8 mg
HCN equivalent per kg dry weight. No potentially toxic nonprotein amino acids were detected
in cassava products. Lysine and Leucine were the limiting amino acids and the methionine
content was very low and contributed only about 13 % of the total SAA in the ‘cossettes’. We
found that children of 1 to 9 years old cannot expect to meet methionine requirement whereas
adults can meet SAA requirement from the calculated quantity required to satisfy 60 % of the
daily energy from the staple food. Cassava leaves that were found to be quantitatively rich in
protein but this protein is of poor quality with SAA as the most limiting amino acids, cannot
compensate for the dietary deficiency in SAA in the staple food in konzo affected areas. We
concluded also that cassava leaves could be an additional source of dietary cyanogen in the
region. The leaves require prolonged cooking and with the unavailability of electricity or gas
and scarcity of firewood they are consumed after a short cooking time.
SAA are essential for detoxification of the residual cyanogens in the insufficiently processed
cassava roots and also in the improperly cooked cassava leaves.
Konzo and cassava toxicity
XIX
In an epidemiological study, samples of cassava flour from households and samples of urine
obtained from selected participants were examined to monitor the safety of the flour intended
to be consumed, to check cyanogen overload and to assess a potential relation between
urinary sulphur metabolites taurine and thiocyanate. There is a high risk of dietary cyanogen
exposure from cassava flour in this region where konzo was first reported three generations
ago. Cassava flour from more than half of the households had total cyanogen content above
the WHO/FAO recommended safe limit (10 μg HCN equivalent/kg cassava flour). The urine
samples from half of the participants contained more than 300 μmol/l of thiocyanate. This
suggested a high cyanide overload. The low concentration in urinary taurine found suggested
that more sulphur is directed to the detoxification of cyanide by formation of thiocyanate,
although urinary taurine and thiocyanate were slightly or not correlated.
The populations of Popokabaka are still highly exposed to cyanogen dietary from cassava and
perhaps to environmental cyanogens. The increased risk of konzo in this region where the
paralytic disease is still occurring requires a more efficient post harvest processing and a
better balanced diet, particularly richer in methionine, to allow efficient detoxification of
cyanide in the body.
Konzo and cassava toxicity
1
CHAPTER I:
LITERATURE REVIEW
Konzo and cassava toxicity
2
I Literature review
I.1 Introduction
I.1.1 Paraparesis and neurodegenerative diseases, what is the
meaning?
Paraparesis is a common form of neurological disability in developing countries (Howlett,
1994). It is a slight paralysis or weakness of both legs resulting in mild to moderate loss of
bilateral lower extremity motor function, which may be a manifestation of spinal cord
diseases; peripheral nervous system diseases; muscular diseases; intracranial hypertension;
parasagittal brain lesions; and other conditions. Paraparesis often progresses to paraplegia,
paralysis of the legs and lower part of the trunk. Symptoms are mild and may include spastic
paraparesis of the lower limbs, ataxia hypertonia (excessive muscle tone), mild peripheral
neuropathy, and problems of urinary incontinence (Parker, 2004). Leprosy, poliomyelitis,
tuberculosis and trauma are the main causes, but a heterogeneous group of diseases with
paraparesis also exists whose occurrence is limited to the tropics and whose etiology is still
unknown (Howlett, 1994).
In the 19th century, neurologists recognised that the muscle weakness could be due to primary
disorders of muscle or secondary to loss of neuromuscular integrity, as it happens when
peripheral nerves are cut or when motor neurones degenerate. Furthermore, it was observed
that there are forms of motor neurone degeneration which selectively affect upper motor
neurone (e.g.: primary lateral sclerosis, hereditary spastic paraplegia, tropical spastic
paraparesis (TSP), neurolathyrism, konzo) or lower motor neurone (e.g.: spinal muscular
atrophies, hereditary bulbar palsy), or combination of upper and lower motor neurone (e.g.:
amyotrophic lateral sclerosis) (Donaghy, 1999; Swash et al, 1999; Talbot, 2002).
Konzo and cassava toxicity
3
Neurodegeneration corresponds to any pathological condition primarily affecting neurons. In
practice, neurodegenerative disorders represent a large group of neurological disorders with
heterogeneous clinical and pathological expressions affecting specific subsets of neurons in
specific functional anatomic systems; they arise for unknown reasons and progress in a
relentless manner (Przedborski et al, 2003). A number of mechanisms appear to contribute to
the neurodegenerative process, including alterations in calcium homeostasis in the
endoplasmic reticulum which contribute to neuronal excitotoxicity and apoptosis, and
unregulated calpain (cysteine endopeptidase; EC 3.4.22.17) proteolysis, initiated by the
dysregulation of calcium ion homeostasis. Mitochondrial disfunction may also be linked to
neurodegenerative disease through free radical generation, impaired calcium buffering and the
mitochondrial permeability transition. Apoptotic and necrotic cell death are both observed in
neurodegenerative diseases. Another mechanism may be the disorganization of the
cytoskeleton leading to neuronal degeneration (Sigma-RBI®, 2001).
Motor neurone disease is a term introduced by Brain in 1962, intending to unify under one
umbrella various idiopathic degenerative motor system diseases (Swash et al, 1999). A large
number of diseases of diverse aetiology may selectively affect the motor neurons in the
central nervous system (CNS) and, among the hundreds of different neurodegenerative
disorders. So far most attention has been given to only a handful of diseases including
Alzheimer disease, Parkinson disease, Huntington disease, and amyotrophic lateral sclerosis
(Tshala-Katumbay, 2001a; Talbot, 2002; Przedborski et al, 2003). Many of the less common
or less publicized neurodegenerative disorders, though not less devastating, have remained
essentially ignored or neglected (Przedborski et al, 2003).
I.1.2 The “hidden endemias”
Tropical myeloneuropathies (Table I-1) is a term proposed by Román et al (1985) for the
“hidden endemias” of the neurodegenerative diseases that predominantly affect the spinal
Konzo and cassava toxicity
4
cord and peripheral nerves with poorly known aetiology such as Tropical Spastic
Paraparesis/Human T cell Lymphocyte Virus Type I-Associated Myelopathy (TSP/HAM),
tropical ataxic neuropathy (TAN), neurolathyrism and konzo, diseases in which a virus or a
natural toxin causes selective upper motor neurone impairment (Tylleskär, 1994c; Verma and
Bradley, 2001). Upper motor neurone signs are the result of an interruption in the neural
pathway above the anterior horn cell. Characteristic of an upper motor neurone disease are:
• Weakness – the extensors are weaker than the flexors in the arms, but the reverse is
true in the legs with weakness more pronounced in flexor muscles.
• Muscle wasting is absent or slight – muscle wasting is prominent in a lower motor
neurone lesion
• Hyper-reflexia and clonus in upper motor neurone disorder – reflexes are absent or
reduced in a lower motor neurone lesion
• Spasticity
• No fasciculations in upper motor neurone disorder – fasciculations occur in a lower
motor neurone lesion.
Tropical Spastic Paraparesis/Human T cell Lymphocyte Virus Type I-Associated Myelopathy
(TSP/HAM) is a neurological disease characterised by slowly progressive spastic paraparesis
with insidious onset in adulthood. It has been found all around the world (except at the
Artics), mainly in tropical and subtropical regions. The diagnostic criterion of TSP/HAM is
seropositivity to HTLV-I (Cassab and Penalva-de-Oliveira, 2000; Maloney et al 2000;
Zaninovic’, 2001).
TAN is a form of tropical myeloneuropathy which was first described in Nigeria but also
occurs in other parts of the tropics. Its clinical presentation is characterized by gradual onset
of ataxia due to posterior column loss. The clinical diagnosis requires at least two of the
following features: bilateral optic atrophy, deafness, predominantly posterior column
Konzo and cassava toxicity
5
myelopathy and polyneuropathy. The occurrence of TAN is associated with chronic moderate
dietary cyanide exposure arising from cassava (Howlett, 1994).
Table I-1: Characteristic features of four tropical myeloneuropathies (Tylleskär et al, 1994c)
Konzo Tropical ataxic
neuropathy
Neurolathyrism HTLV-I
associated
myelopathy
Geographical area Africa Africa Asia/ Africa Worldwide
Occurrence epidemic
and endemic
endemic epidemic and
endemic
endemic
Highest prevalence 3 % 3 % 3 % 0.1 %
Familial clustering yes yes yes yes
Type of onset acute slow acute slow
Course permanent progressive permanent progressive
High incidence age
group
< 40 > 40 < 40 > 40
Main neurological
findings:
Gait abnormality
Spastic
paraparesis
Ataxia
Spastic
paraparesis
Spastic
paraparesis
Peripheral
neuropathy
no yes no common
Sphincter
involvement
no no rare yes
Optic atrophy rare yes no no
Deafness no common no no
Etiology Attributed
to weeks of
high
cyanide
exposure
from
cassava
Attributed to
prolonged,
varying cyanide
exposure from
cassava
Caused by
months of high
grass pea
(Lathyrus sativus)
consumption
Caused by
chronic HTLV-I
infection
Konzo and cassava toxicity
6
Neurolathyrism is an upper motor neurone disease caused by excessive and prolonged
consumption of grass pea (chickpea), Lathyrus sativus, which contains the glutamate
analogue neurotoxin ß–N-oxalyl-α,ß-diaminopropionic acid (ß-ODAP) also known as ß-
Oxalylaminoalanine(BOAA). ß-ODAP is an excitotoxic amino acid that presumably acts on
the neuronal glutamate receptors. Neurolathyrism is epidemic and endemic in geographic
areas subject to famine and drought such as Afghanistan, Bangladesh, China, Ethiopia, Nepal
and India. Neurolathyrism is characterized by spastic paraparesis of the legs with or without
sphincter disturbances (Spencer, 1999; Getahun et al, 1999).
Konzo is a distinct disease entity with selective upper motor neuron damage which is
characterised by a sudden onset of an irreversible, a non-progressive and symmetrical spastic
paraparesis or, in severely affected subjects, tetraparesis (Howlett et al, 1990; Tylleskär,
1994b; Banea-Mayambu, 1997c; Tshala-Katumbay, 2001a; Mwanza et al, 2005). Konzo has
been attributed to the high dietary cyanide exposure from insufficiently processed roots of
bitter cassava (Manihot esculenta Crantz) and reported from remote rural areas of
Mozambique, Tanzania, Cameroon, Angola, the Central African Republic and the Democratic
Republic of Congo (DRC) (Trolli, 1938; Cliff et al, 1985; Howlett et al, 1990; Banea et al,
1992a; Tylleskär et al, 1992; Tshala-Katumbay, 2001b; Bonmarin et al, 2002; Ernesto et al,
2002a). Konzo has only been reported from cassava growing and consuming areas but
affected populations constitute only a fraction of the total of over 500 million cassava-
consuming populations of the tropics. Konzo has some similarities to neurolathyrism but there
is no geographical overlap of the two diseases (Howlett et al, 1990; Tylleskär et al, 1994c;
Lambein et al, 2004). Prevalence rates for konzo vary between studies; rates between 1 and 30
per 1000 have been reported (Tylleskär et al, 1992). The total number of confirmed konzo
cases in reported studies exceeds 4000 (Tylleskär, 1994a; Bradbury, 2004). The D. R. C.
covers the largest reported number of konzo cases. The Health Ministry of D. R. C. estimated
Konzo and cassava toxicity
7
the number of konzo cases in D. R. C. to be around 100,000 (R. D. C., 2000). The age and the
sex distribution of konzo show a distinct pattern. No child under the age of 2.5 years, of
which most are breast-fed, has ever been found to contract konzo. Women of child bearing
age and children 3-13 years of age run the highest risk of contracting konzo. No case of konzo
has been reported from nearby urban populations (Banea-Mayambu et al, 1997a).
I.2 Konzo
I.2.1 Background information on konzo
Formerly called epidemic spastic paraparesis (Carton et al, 1986; Rosling, 1988), konzo is a
neurological disorder that gives rise to crippling spastic paralysis of both legs (paraparesis) or
of both legs and arms (tetraparesis) in severely affected subjects (Tshala-Katumbay, 2001a;
Mwanza et al, 2005). It is an upper motor neuron disease which was first described in the
former Belgian Congo (present D. R. C.) by Dr Trolli in a published report that summarised
regrouped observations in Kwango district about two affections of unknown origin; epidemic
spastic paraparesis, “konzo” of the people in Kwango and a syndrome with oedema and
dyschromic cutaneous lesions (Trolli, 1938).
In 1936, Dr Tessitore, a district medical officer in Kahemba area reported an outbreak of an
affection that he called “amyotrophic lateral sclerosis” of which he described several cases. In
1937, Dr Mercken noted around Feshi, area neighbouring Kahemba some cases of affection
which evoke symptoms of Heine-Medin disease. Some times later in 1937, Drs Doucet and
Orlovitch reported other cases in Moyen-Wamba (the present Popokabaka and Mwela
Lemba), another Feshi neighbouring area (Figure I-1). Some subjects reported having been
affected earlier during outbreaks of the year 1928 or 1929 and 1931- 1932. The affection
appeared periodically in those areas and was well known by the local population who named
it “konzo”.
Konzo and cassava toxicity
8
Figure I-1: Health zones of Kwango District in Bandundu Province, DRC
Konzo and cassava toxicity
9
The word “konzo” originally khoondzo has its origin from kiyaka, the language spoken in
Kwango district and had three meanings (Trolli, 1938; Van der Beken, 1993). It means a
fetish used with traps to catch wild animals by weakening their legs, the trap itself and, tied
legs. This latter meaning is illustrated by a famous proverb in Kiyaka:
“mene, yakele khosi mutu, khoondzo watholula bidiimbu”,
This means “I was strong, but khoondzo (fetish) has weakened my legs”. As a consequence of
this proverb, konzo has come to mean “trapped” or “weakened “or “tied” legs. It is in this
sense that konzo is used to denote the paralytic disease and thus, a local belief that the disease
is related to a bad destiny or sorcery (Van der Beken, 1993).
Lucasse (1952) who did not read the report of Trolli, described and suggested the first clinical
description of konzo observed in some affected subjects 14 years ago in Kwango district, as
follows:
Bilateral paresis of the lower limbs which is accompanied by spasms in the adductor and
flexor muscles of the lower part of the body giving rise to vicious attitudes of lower limbs and
sometimes of spine (lordosis).
After a period with no further reports on konzo, the National Planning Centre of Human
Nutrition of Zaire (present D. R. C.) CEPLANUT (currently PRONANUT) in 1982 reported
hundreds of cases of spastic paraparesis from an outbreak that started in 1978 in the Central
part of the Bandundu province, neighbouring Kwango district (CEPLANUT, 1982). Another
outbreak occurred in 1983, in the northern part of the above area and up to now, outbreaks of
konzo are still occurring in the Bandundu province and other parts of D. R. C. (Tshala-
Katumbay, 2001b; Bonmarin et al, 2002).
Other outbreaks of konzo have been described in several other parts of Africa, especially in
Mozambique, Tanzania, Central African Republic, and Cameroon (Cliff et al, 1985; Howlett
et al, 1990; Tylleskär et al, 1994c; Ernesto et al, 2002a).
Konzo and cassava toxicity
10
Konzo is now accepted as the scientific name for a distinct human disease entity which is
characterised by a sudden onset of a non-progressive and irreversible spastic paraparesis in a
person formerly without other symptoms (Howlett et al, 1990; Tylleskär, 1994b).
I.2.2 Clinical features and differential diagnosis
I.2.2.1 Clinical features and classification
Konzo is a distinct type of upper motor neuron disease with a typical clinical picture of
crippling spastic paraparesis (WHO, 1996). The clinical picture of konzo is identical in all
studies (Trolli, 1938; Lucasse, 1952; Carton et al, 1986; Howlett et al, 1990; Tylleskär et al,
1995; Cliff and Nicala, 1997; Tshala-Katumbay, 2001b; Bonmarin et al 2002; Ernesto et al,
2002a). The disease typically occurs in an apparently healthy person and there is no
prodromal phase or triggering illnesses. The onset is characterized by an abrupt paraparesis
occurring the first days of the illness. A common history is that of a healthy person who goes
to bed feeling well and wakes up during the night or early morning unable to stand or walk.
The paraparesis may also occur abruptly during or after manual work or a long walk. Initial
symptoms are often described as heaviness, trembling or weakness of the legs associated with
difficulty or inability to stand. Other complaints that may appear over time include weakness
in the arms or hands, difficulty in articulating speech, and blurring of vision. Sensory
symptoms of radicular low back pain, and paresthesia in the legs, can also be present but these
usually clear in the first weeks or months. Incontinence is typically absent. The disease affects
mainly children and women of childbearing age (Howlett, 1994; Tylleskär, 1994b; WHO,
1996).
Although the main clinical picture of konzo consists of the sudden onset of a non progressive
and symmetrical spastic paraparesis of the legs in affected subjects, the diagnosis of konzo is
based on the WHO criteria (WHO, 1996) given in the Table I-2.
Konzo and cassava toxicity
11
These criteria are easy to use in the field to screen the population. However Tshala-Katumbay
(2001a) suggested a new version with more operational criteria in comparison with the WHO
criteria (Table I-2).
The degree of physical disability caused by konzo was classified by Lucasse (1952) and later
amended by WHO (1996) as follows:
• Mild form: when the patient does not need to regularly use any walking aid
• Moderate form: when the patient regularly uses one or two stick(s) or crutches
• Severe form: when the patient is bedridden or unable to walk without living support.
Table I-2: WHO criteria for konzo versus newly suggested criteria (with permission and from
Tshala-Katumbay, 2001a)
Criteria WHO New version
1 Visible symmetric spastic
abnormality of gait while walking
or running
Sudden onset of a non –progressive
bilateral and symmetric spastic
abnormality of gait while walking or
running
2 History of onset less than one week
followed by a non-progressive
course in a formerly healthy person
Bilaterally exaggerated knee or ankle
jerks
3 Bilaterally exaggerated knee or
ankle jerks without signs of disease
of the spine
Absence of objective sensory and genito-
urinary symptoms
4 Absence of grass pea (Lathyrus
sativus) consumption
Living under conditions of sub-acute or
chronic exposure to cyanogens and
undernutrition at the onset
Konzo and cassava toxicity
12
Figure I-2: From left to right: mild form, moderate form and severe form of konzo in young
subjects
This classification (Figure I-2) is easy to use even by paramedical agents unfamiliar with the
symptoms but it is sometimes difficult to distinguish between slightly konzo affected persons
(mild form) with non-affected persons.
I.2.2.2 Differential diagnosis
Konzo with its upper motor neuron manifestations can be confused with other diseases. Using
the WHO criteria, konzo by its spastic paraparesis can easily be distinguished from causes of
flaccid paraplegia such as poliomyelitis, leprosy or trauma (Howlett, 1994). The commonest
neurological diseases to be considered in its differential diagnoses include neurolathyrism,
TAN and TSP/HAM (Table I-1).
Konzo and cassava toxicity
13
Konzo is clinically very similar to neurolathyrism but differs from TAN and TSP/HAM
(Tylleskär et al, 1994c; Cliff and Nicala, 1997; Zannovic’, 2001; Lambein et al, 2004). The
socio-economic conditions of konzo and neurolathyrism patients are very similar as well.
Both diseases can be considered a sign of poverty, monotonous diet and illiteracy (Getahun et
al, 2002b). Neurolathyrism only differs from konzo with the somewhat higher age of onset,
predominance of males among the affected, sphincter involvement in some cases and the
absence of cranial nerve involvement (Tylleskär et al, 1994c). There is no geographical
overlap between the consumption of the grass pea and cassava and therefore there is no
geographical overlap of the two diseases. It would be difficult to make a differential diagnosis
between neurolathyrism and konzo if both disorders occurred in the same population
(Tylleskär et al, 1994c; Lambein et al, 2004).
Konzo and TAN have been attributed to dietary cyanide exposure from consumption of
insufficiently processed cassava roots, but rates of exposure differ in both diseases (Howlett et
al, 1990). In contrast to konzo, TAN is a progressive disorder with slow onset that mainly
affects older adults. Furthermore, konzo involves damage to upper motor neurone, whereas
TAN is mainly caused by damage of sensory neurons in the spinal cord resulting in ataxia.
TAN rarely progresses to inability to walk, whereas a high proportion of the konzo-affected
subjects are unable to walk. About half of the TAN cases have optic atrophy which is rare
among konzo cases. About one in five TAN cases has exaggerated reflexes, a sign that occurs
in all konzo cases (Howlett, 1994; Tylleskär et al, 1994c).
TSP/HAM is clinically possible to distinguish from both konzo and neurolathyrism, although
the clinical features of TSP/HAM include typical signs that are similar to both diseases such
as muscle weakness in the legs, hyperflexia, clonus and extensor plantar responses.
TSP/HAM is characterised by a chronic progressive spastic paraparesis with sphincter
disturbances, not to mild sensory loss, absence of spinal cord compression, urinary
Konzo and cassava toxicity
14
incontinence, impotence and seroposivity for HTLV-I antibodies (Tylleskär, 1994b; Cliff and
Nicala, 1997; Cassab and Penalva-de-Oliveira, 2000; Maloney et al 2000; Zaninovic’, 2001).
I.2.3 Epidemiology
The occurrence of konzo is limited to geographical pockets in rural Africa and the majority of
cases occur in epidemic outbreaks during the dry season (Rosling, 1997; Bonmarin et al,
2002). Sporadic cases also occur, but they are also restricted to dry or war periods with
monotonous cassava diet. Not a single case has been identified in an urban population
(Tylleskär et al, 1995). More than 4000 cases have been confirmed from reported studies. Of
these, more than half of the cases are from D. R. C. but the reported number of cases is
undoubtedly underestimated, as case detection is incomplete in the remote rural areas
affected. The Health Ministry of D. R. C. has estimated the number of konzo cases in D. R. C.
to be around 100,000 (R. D. C., 2000). Two extensive epidemic outbreaks, each numbering
more than 1000 cases have been brought to the attention of the scientific community; the first
reported outbreak occurred in Kwango district in the southern part of Bandundu province of
D. R. C. in 1936-37, and the second in Nampula Province of Mozambique in 1981. Other
smaller outbreaks have been reported from very poor remote rural population of Central
African Republic, Mozambique, Tanzania, Cameroon, Angola, and Democratic Republic of
Congo (D. R. C.) (Howlett et al, 1990; Banea-Mayambu et al, 1992a; Tylleskär et al, 1994c;
Tshala-Katumbay, 2001b; Bonmarin et al, 2002; Ernesto et al, 2002a).
Konzo primarily affects children above the age of three and women in the fertile age group.
Adult males are less frequently affected. No breast-fed child (in affected populations, the
breast feeding period extends beyond two years of age) has been found to have contracted
konzo. A pronounced familial clustering of cases of konzo has been noted in all affected
populations. Prevalence varies between studies and between most affected villages.
Prevalence rates of between 1 and 30 per 1000 have been recorded (Howlett, 1994; Cliff et al,
Konzo and cassava toxicity
15
1997b). The peculiar geographical, seasonal and age variation in occurrence as well as the
abrupt onset have facilitated epidemiological studies for possible etiological factors in konzo
(Howlett, 1994; Tylleskär et al, 1995; WHO, 1996; Banea-Mayambu et al, 1997a; Rosling,
1997).
I.2.4 Infection or toxico-nutritional etiology?
“The etiology of konzo is unclear. Information given by local people is useless. Often, they
say the disease is sent either by God or by the ndoki, the bad spirit of enemies” stated
Tessitore in 1930s (Trolli, 1938). Three generations later after the first report, the etiology of
konzo has not been established with certainty. The infectious etiology was proposed (Trolli,
1938; Lucasse, 1952; Carton et al, 1986) but konzo patients do not show any signs of
infections and are sero-negative to HTLV-I and other retroviruses (Tylleskär et al, 1996;
Tshala-Katumbay et al, 2001b). The facts that outbreaks are restricted to rural areas without
any secondary cases along connecting roads or in neighbouring urban areas argues against
infectious etiology. So far, all studies on konzo have failed to demonstrate an infectious
etiology (WHO, 1996; Rosling, 1997).
A toxico-nutritional hypothesis was suggested in 1930s by Georgiades who observed
similarities between konzo and lathyrism and recommended further studies on cassava
concerning the processing and detoxification methods used before its consumption. Konzo
might be caused by cyanide exposure resulting from consumption of insufficiently processed
cassava roots and simultaneous low dietary intake of sulphur containing amino acids
providing substrate to thiocyanate conversion (Trolli, 1938; Cliff et al, 1985). This hypothesis
is supported by a consistent association between temporal and geographical occurrence of
konzo and the chain of events that leads to high cyanide and low sulphur intake. This chain is:
• Intensive cultivation of bitter cassava varieties in poor rural areas,
Konzo and cassava toxicity
16
• A cassava dominated diet, which is brought about by having a farming system
dominated by bitter cassava
• Shortcuts in processing as indicated by high residual levels of cyanogens in cassava
products consumed,
• High cyanide intake indicated by high urinary and serum thiocyanate levels,
• Low intake of foods rich in sulphur containing amino acids indicated by low urinary
inorganic sulphate levels (Tylleskär, 1994d; Tylleskär et al, 1995; Cliff et al, 1997a;
Rosling, 1997; Banea-Mayambu et al, 1997c)
High consumption of cassava is by itself not sufficient to cause konzo and within the affected
populations, cassava is consumed daily by everybody but only certain percentage of the
population acquire konzo (Tylleskär, 1994b). The underlying cause inducing the high
exposure to cyanide and unbalanced diet include drought, intensive trade of cassava by poor
farmers and collapse of the socio-economic fabric due to political conflicts and civil war
(Banea-Mayambu et al, 1997a; Banea-Mayambu et al, 1997b; Cliff et al, 1997b; Tshala-
Katumbay et al, 2001b). So far, the resulting diet and toxic exposure associated to konzo is
similar in all areas and konzo has not been reported from any area lacking this unbalanced
diet (Rosling, 1997).
Although the etiology and the exact cellular malfunctions induced by overconsumption of
cassava products remain unclear, cyanide (CN-), 2-aminothiazoline-4-carboxylic acid (ATC),
cyanate (OCN-) and thiocyanate (SCN-) have been suspected playing a role in the pathogenic
mechanism of konzo (Spencer, 1999; Tor-Agbidye et al, 1999):
• CN- has been suggested as a causal factor in some series of neurological disorders
because of its potential inhibitor effect on mitochondrial energy transformation
secondarily inducing neuronal dysfunction. CN- is unlikely to be responsible since the
Konzo and cassava toxicity
17
outcome of sub-lethal cyanide intoxication is Parkinsonism, with changes in basal
ganglia, cerebellum and cerebral cortex;
• ATC, a minor cysteine-dependent metabolite of cyanide, has not yet been investigated
for its systemic toxicity. However, its intracerebroventricular injection in rats induces
seizures and hippocampal damage, neither of which are known to occur in konzo;
• OCN-, a normal human metabolite that is produced by the spontaneous degradation of
urea (carbomoylation), is known to cause neurodegenerative disease in humans and
animal (WHO, 2004); but these neurological conditions appear to be more closely
related to TAN rather than to konzo (Tor-Agbidye et al, 1999);
• SCN- is generally considered a major, innocuous detoxification product of cyanide.
SCN- is constantly elevated in subjects with konzo (Tylleskär, 1994, Banea-Mayambu,
1997c; Tshala-Katumbay, 2001b). Since experimental evidence shows that SCN-
increases glutamate binding to the α-amino-3-hydroxy-5-methyl-isoxazole-4-
propionic acid (AMPA) receptor and potentiates AMPA-mediated responses. This
might secondarily induce excitotoxic effects and hence neuronal disfunction or cell
death. The potential role of thiocyanate in konzo merits attention (Tor-Agbidye et al,
1999).
I.3 Dietary exposure to cyanide from cassava
I.3.1 Cassava
I.3.1.1 Classification and botany
Cassava (Manihot esculenta Crantz) is the English name given to the manioc plant, a hardy
perennial shrub belonging to the family Euphorbiaceae and ranging in height between 1 – 3
m depending on growing conditions (Brough, 1991). There are many cultivars or varieties
Konzo and cassava toxicity
18
under cultivation. Cassava varieties are usually classified into sweet and bitter cultivars but no
morphological or other taxonomic characteristics seem to be associated with this
classification (Nweke and Bokanga, 1994). The genus Manihot incorporates over 200 species
of which Manihot esculenta is the most important, from the nutritional and economic points
of view (Nartey, 1978).
The shrub may have multibranched or unbranched stems, grey, green or brown in colour, with
large palmate leaves (Figure I-3). The primary leaves are unlobed, whereas the secondary
leaves are 3-lobed, and subsequent leaves develop, the lobes increase in number, reaching the
number of lobes characteristic of the cultivar. Mature stem cuttings which are universally
used as propagules and planted erect or at a particular angle for vegetative regeneration of
plants, give rise to roots at the cut end via callus tissue formation, and adventitious roots form
at nodes in the soil.
Figure I-3 : Cassava roots harvested and cassava plant in the field
Konzo and cassava toxicity
19
The roots are initially fibrous, but gradually undergo enlargement. At maturity, they become
fusiform, long and slender, occasionally globose appendices of the stem in the upper
rhizosphere, seldom penetrating deeply into the soil. The mature root tuber (Figure I-3)
possesses three distinct regions, namely, the phelloderm or peel, the cortex or flesh, and the
central vascular core. The peel is generally 1-4mm thick and composed of outer epidermis, a
sub-epidermis and an inner layer readily separable from the bulk of the tuber. The cortex
consists of a mass of parenchyma cells and constitutes the region of carbohydrate storage.
Generally, the cortex lacks xylem vessels, and is therefore without fibre, but older tubers
develop hardened xylem vessels, giving rise to stringy tubers, undesirable for food. Root
pigmentation may vary with respect to variety, from light yellow, brown to pink and
intermediate shades, whereas cortex pigmentation varies from white, yellow to pink. The
tubers are the most valuable part of the plant, although in some countries the leaves are used
as green vegetable (Nartey, 1978; Brough, 1991).
I.3.1.2 Introduction and distribution of cassava in Africa
Cassava is believed to have been introduced originally in the Gulf of Benin in 1562 and along
the Congo River in 1611 from where it spread to the west coast of Africa (Nartey, 1978;
Carter et al, 1997). Later introductions in the islands of Reunion, Madagascar and Zanzibar
led its spread in East Africa. Finally, it spread inland in all directions to encompass the region
of Lake Tanganyika (Nartey, 1978; Brough, 1991).
The Portuguese first brought cassava to Africa in the form of flour or “farinha”. The
Tupinamba Indians of eastern Brazil had taught them techniques of cassava preparation and
production and, they had developed a liking for the various processed forms. Cassava flour
was used as a provision for ships plying between Africa, Europe and Brazil. The first mention
of cassava cultivation in Africa dates back to 1568. At first, it was cultivated with the sole
purpose of supplying slave ships, until 1600. In the late 19th century cassava had been
Konzo and cassava toxicity
20
successfully incorporated into many farming systems of Central Africa instead of millet, yam
and plantain, the former staple food in most areas along the Congo River (Carter et al, 1997).
The ultimate wide distribution of cassava in the whole of tropical Africa was motivated by the
ability of the crop to withstand locust attacks, and to tolerate drought, poor soils and weeds.
These characteristics, together with the fact that the crop can be left without harvesting over
several years, made it a useful security against periods of famine (Nartey, 1978).
In the late 19th and 20th centuries, colonial administrators encouraged diffusion and increased
cultivation of cassava. The encouragement by the colonial governments may often have taken
place in a manner insensitive to the applicability of cassava to local farming systems and food
habits. Moreover colonial governments displayed an ambivalent attitude towards cassava.
Whilst it was introduced as an anti-famine and anti-locust crop, cassava was also thought to
promote laziness, soil depletion and malnutrition. Post-independence diffusion of the crop in
Africa has primarily been the result of local processes of migration and agricultural change.
There is ample evidence of the willingness of African farmers to experiment with the search
for new crops and varieties. Cassava’s special characteristics make it well adapted to farmers’
risk aversion strategies and allow it to be grown under a great diversity of circumstances and
changing economic conditions. The consumption of cassava leaves, in frequent rather than
sporadic form, was probably an African invention (Carter et al, 1997).
Currently, cassava is grown on wide scale between latitudes 30° north and south, the so called
“cassava belt”, an ecological zone which coincides with many of the less developed countries
where cassava is adapted to the prevailing conditions (Nartey, 1978).
I.3.1.3 Importance and advantages
Cassava (Manihot esculenta Crantz) is a shrub widely grown for its tuberous roots in tropical
regions of Africa, Asia and Latin America. Sweet and bitter cultivars are produced as food,
feed and for industrial uses.
Konzo and cassava toxicity
21
Cassava roots form a staple food for an estimated 500 million people in the tropics and the
leaves are commonly consumed as a vegetable in several areas. Cassava ranks fourth on the
list of major food crops in developing countries after rice, wheat and maize (FAO, 1990).
Cassava is of great importance for food security in Africa in general, and D. R. C in
particular. Cassava possesses a number of useful agricultural traits. The crop is a relatively
efficient producer even under adverse environmental conditions such as erratic low rainfall
and low soil fertility. Cassava productivity in terms of calories per unit land area is
significantly higher than that of other staple food crops. The edible portion in percent of dry
weight of the root crop is high. Cassava is resistant to locust damage and most pests. Growth
of cassava requires a low input in the timing of labour. Except being sensitive to drought
shortly after planting, cassava requires no special planting or harvesting dates. The roots can
be stored in the ground without harvesting for a lengthy period of time, up to three years or
more after the formation of the edible roots is complete. Hence, cassava cultivation serves as
something like a household food bank that can be drawn upon when adverse agro-climatic
conditions or civil unrest limit the availability of and access to other food (Koch et al, 1994;
Scott et al, 2000).
In sub-Saharan Africa, cassava provides daily food products for nearly half of the continent’s
population. DRC is the country with the highest per capita consumption in the world, about
60 % of total daily energy intake is provided by cassava (FAO, 1990). In addition, cassava
leaves contribute 20 % of the protein in Congolese diets. The Congolese staple food
production takes mainly place on traditional farms. The Congolese smallholder farming is
characterized by reliance on family labour, on a small stock of physical capital and on a large
area of land. Women play a predominant role in farming, processing, and marketing. In rural
areas, most traditional subsistence food crops have become important as cash crops and urban
food demand is the driving force behind this evolution. Farms without an adequate access to
Konzo and cassava toxicity
22
markets are generally characterized by low levels of cash income and surplus production
(Goossens, 1996). Cassava production is a commercial activity, and not merely a subsistence
agricultural activity. Cassava is an important source of cash income for poor farmers as well
as for prosperous ones. Both rich and poor farmers often sell a higher proportion of cassava
than from any other crop or income earning activity. The proportion of cassava production
marketed is a good indicator of the level of diversification in the crop production activity of
an area (Nweke and Bokanga, 1994).
I.3.1.4 Production and consumption levels
“Cassava is apparently emerging from its obscurity in the tropics and is marching northward
and southward to fill new roles in temperate climates”, is the assessment of Franklin D.
Martin quoted by Nartey (1978).
In the last two decades, cassava production grew at a more modest pace, 1.8 percent annually.
Production of cassava grew by 27 percent between 1983 and 1996 to 164 million metrics ton.
On a per capita basis, production of cassava in developing countries remained virtually
constant at 37 kg per capita, supported mainly by the per hectare production growth in Sub-
Saharan Africa. Production tends to be highly skewed toward particular regions. Slightly
more than half the global production of cassava takes place in sub- Saharan Africa, followed
by Southeast Asia with 23 percent and Latin America with 20 percent (Figure I-4).
Most statistics do not usually distinguish between sweet and bitter varieties; in some, sweet
varieties are not included as they are commonly grown as a secondary crop for home
consumption. Brazil is the largest producer of cassava in the world, but most of the crop is
consumed locally and exports are only a small portion of the total output. The same pattern
applies to other important producers, such as Nigeria, Indonesia, D. R. C., India and
Colombia. Cassava does not form an important part of the staple diet in Thailand, and that
Konzo and cassava toxicity
23
country is the world’s largest exporter of cassava products. In contrast, Africa does not export
much cassava because production is almost entirely consumed as food.
Figure I-4: Location of cassava production, 1996 (Scott et al, 2000)
Sub-Saharan Africa, 51.6%
Latin America, 19.5%
Other South Asia, 0.2%
Southeast Asia, 23.0%
China, 2.2%
India, 3.5%
In the last few years most of the important producers have greatly increased their production.
Surplus production of cassava products enters international trade in different forms, such as
chips, broken dried roots, meal, flour and tapioca starch. Dried cassava roots and meal are
used as raw material for compound animal feed, while cassava starch is used for industrial
purposes, particularly the paper and textile industry; grocery tapioca is used solely for human
consumption. The principal markets for cassava products are the European Community, USA,
UK and Japan (FAO, 2004).
Konzo and cassava toxicity
24
Between 1983 and 1996, increase of consumption of cassava as food has been particularly
rapid in Sub- Saharan Africa at 3.1 percent per year. The region has experienced low and
negative economic growth and booming populations (Scott et al, 2000).
Cassava is the staple food in most of D. R. C., and especially in Bandundu province where a
great number of konzo cases have been reported (Trolli, 1938; Banea-Mayambu et al, 1992a;
Tylleskär et al, 1994c; Tshala-Katumbay, 2001b; Bonmarin et al, 2002).
Between 1987 and 1990, the annual production of fresh cassava roots in Bandundu was 4
million metric tons while the national production was estimated at 17 million tons. The annual
production per household was 7.9 tons and the quantities of cassava produced are ten to
twenty times larger than for other crops in Bandundu. The cassava production per farm is
relatively stable during the year: between 560 kg and 680 kg per month in Bandundu. Farm
purchases of cassava are highest in October and November, when fields are prepared and
available labour to harvest roots is limited. Sales are highest from September to February,
when receipts from other crops are low. Nearly all households buy cassava from time to time
but the quantities are generally small and are used for immediate consumption or to be resold.
Only seven percent of the rural households are not self supporting for cassava (Goossens,
1996)
I.3.1.5 Problems associated with cassava
I.3.1.5.1 Nutritional value
Cassava roots contain around 30 to 40 % of dry matter of which starch and sugars account for
approximately 90 %. This renders cassava root, an excellent source of carbohydrate but has
extremely low levels of protein and fat (Bradbury and Holloway, 1988)
Unlike the roots, which are essentially a source of carbohydrate, fresh cassava leaves are a
good source of proteins and vitamins which can provide a valuable supplement to
Konzo and cassava toxicity
25
predominantly starchy diets (Hahn, 1989). The nutrient content of cassava leaves is
comparable with other green leaves and other vegetables generally regarded as good protein
sources (Mbemba and Remacle, 1992). Vitamin A, thiamine, riboflavin, niacin and vitamin C
are of high concentration in the fresh leaves (Ravindran and Ravindran, 1988; Almazan and
Theberge, 1989)
I.3.1.5.2 Constraints in production
Besides the advantages on the production of cassava, there are many production constraints
which can include diseases, pest, weeds, soil and agronomic factors, and socio-economic
factors. The major diseases of cassava are leaf diseases, stem diseases and tuber rot.
Vertebrate pest, nematodes, mites and insects may attack or infect the roots and render them
susceptible to rot-causing organisms. Cassava can be seriously affected by early weed
infestation. Weed competition in cassava crops reduces canopy development, tuberization and
tuber yield. The important soil and agronomic factors that affect cassava production are soil
temperature and moisture (if above 30° C and if drought is frequent), soil erosion and low soil
fertility (continuous cultivation of cassava without adequate erosion control measures, can
result in severe and irreversible soil degradation), and poor cultural practices. The main socio-
economic factors affecting cassava production relate to inadequate resource allocation and
infrastructure (IITA and UNICEF, 1990).
I.3.1.5.3 Post-harvest deterioration
Cassava roots are extremely perishable. They can be kept in the ground prior to harvesting for
up to about 2 years, but once they have been harvested (removed from the stem) they begin to
deteriorate within 40 – 48 hours (IITA and UNICEF, 1990). The fresh tubers in general have
high moisture content, usually between 50 and 70 %, and hence have a relatively low
mechanical strength. They also have a very high respiratory rate, and the resultant heat
Konzo and cassava toxicity
26
production softens the texture, which leads to damage. Unlike the other tuber crops, cassava
roots do not exhibit exogenous dormancy, have no function in propagation, and possess no
bud primordial from which regrowth can occur. For these reasons, cassava roots are more
perishable than other tuber crops. Mechanical damage during the harvesting and handling
stages also renders cassava root unsuitable to long-term storage. Deterioration of cassava has
an adverse effect on the processed product, and thus the crop must be stored properly (IITA
and UNICEF, 1990; Ravi et al, 1996, Ravi and Aked, 1996).
I.3.1.5.4 Cassava in human nutrition
Cassava root is an important starchy crop grown in the tropics, which constitutes the staple
food of about half of billion people and the leaves are consumed as vegetable. Roots and
leaves of cassava contain high level of cyanogenic glycosides mainly linamarin and to a lesser
extent lotaustralin which can be hazardous to the consumer and of which the potential toxicity
is a public health concern (Essers, 1995; Padmaja, 1995). Roots from the sweet varieties may
be eaten raw or cooked by boiling, steaming or roasting. Roots from the bitter varieties
required processing to remove the cyanogenic compound before consumption. The processing
methods generally adopted comprise combinations of activities such as peeling, boiling,
steaming, slicing, grating, soaking or steeping, fermenting, pounding, roasting, drying and
milling (Hahn, 1989; Padmaja, 1995). They can be transformed in the form of flour or gari
(granules) and then consumed as fufu (stiff porridge), chikwangue (wrapped steamed boiled
paste much stiffer than fufu), tapioca, dried gari or stiff paste of gari (Figure I-5).
Consumption of cassava leaves is of great significance in the nutrition of a population
subsisting primarily on cassava. The most widely practiced processing for use of leaves as
food involves crushing, parboiling in water, washing and cooking. Sometimes additional
ingredients such as pepper, palm oil, fish, peanut or other aromatic ingredients are added. The
leaves, in some parts, are sun dried then cut or pounded and finally cooked. Cooked cassava
Konzo and cassava toxicity
27
leaves are served and consumed as the main side-dishes to the processed cassava roots like
fufu, chikwangue or boiled cassava root (Hahn, 1989).
Figure I-5: Summary of traditional cassava processing in Africa (from Banea-Mayambu,
1997c)
I.3.2 Cyanide toxicity
I.3.2.1 Introduction
Cyanide (CN-) most commonly occurs as hydrogen cyanide (HCN) and its salts sodium
cyanide (NaCN) and potassium cyanide (KCN). Cyanides comprise a wide range of
compounds of varying degrees of chemical complexity, all of which contain a CN moiety, to
which humans are exposed in gas, liquid, and solid form from a broad range of natural and
anthropogenic sources. Cyanogenic glycosides, producing hydrogen cyanide upon hydrolysis,
are found in a number of plant species. Cyanides are also produced by certain bacteria, fungi,
Fresh cassava roots
Soaking Heaping Cutting
Solid state fermentation
Grating
Fermentation in water
Moist fermentation
Pounding Drying Drying
-
Pressing
Boiling Pounding Pounding
Drying
Roasting
Paste Flour Flour
Pounding
Granules Flour
Konzo and cassava toxicity
28
and algae. Minute amounts of cyanide in the form of vitamin B12 (cyanocobalamine) are a
necessary requirement in the human diet (ATSDR, 1989).
HCN, a colourless or pale liquid or gas with a faint bitter almond-like odour, has a molecular
weight of 27.03 and a boiling point of 25.7° C. It is miscible with water and alcohol and
slightly soluble in ether.
Cyanide is released to the environment from numerous sources. Metal finishing and organic
chemical as well as iron and steel production, and automobile exhaust are major sources of
cyanide releases in the atmosphere. Workers in a wide variety of occupations may be exposed
to cyanides. The general population may be exposed to cyanides by inhalation of
contaminated air, ingestion of contaminated drinking water, and/or consumption of a variety
of foods (ATSDR, 1989).
Among the general population, subgroups with the highest potential for exposure to cyanide
include active and passive smokers, individuals involved in large scale processing of food
high in cyanogenic glycosides and individuals consuming foods high in cyanogenic
glycosides (WHO, 2004).
I.3.2.2 Occurrence of cyanogenic glycosides
The cyanogenic glycosides are a group of nitrile-containing secondary plant compounds that
yield cyanide (cyanogenesis) following enzymatic breakdown. They are amino acid-derived
plant constituents and their functions remain to be determined in many plants; however, in
some plants they have been implicated as herbivore deterrents and as transportable forms of
reduced nitrogen (Kakes, 1994). Whereas most plants produce a small amount of cyanide
associated with ethylene production, between 3000 – 12000 plant species produce sufficient
quantities of cyanogenic compounds to be considered toxic (Poulton, 1990). The
concentrations of cyanogenic glycosides can vary widely as a result of genetic and
environmental factors, location, season, and soil types (Ermans et al, 1980). Several
Konzo and cassava toxicity
29
economically important plants are highly cyanogenic, including white clover, flax (containing
linamarin), almonds, sorghum, wild lima bean, rubber tree, and cassava. The agronomically
most important cyanogenic food crop however, is cassava (McMahon et al, 1995; White et al,
1998; Vetter, 2000).
I.3.2.3 Cyanogenesis in cassava
All cassava tissues, with the exception of seeds, contain cyanogenic glycosides mainly
linamarin and lesser amounts of lotaustralin in about 10 to 1 ratio. An acyanogenic cassava
cultivar has never been found (Bokanga, 1994). Cyanogenic glycosides are
compartmentalised within the cell vacuole while the complementary hydrolytic enzymes are
located within the cytosol of the same cells (White et al, 1994). The amino acids valine and
isoleucine are the precursors used in the synthesis of linamarin and lotaustralin, respectively.
The initial step in the biosynthesis of linamarin is the N-hydroxylation of valine followed by
the formation of 2-methyl-propanal oxime and its dehydration to yield 2-methylpropionitrile.
Figure I-6: Cyanogenesis from linamarin (McMahon et al, 1995)
Konzo and cassava toxicity
30
The addition of oxygen forms acetone cyanhydrin which is then glycosylated (by a soluble
UDPG-glucosyltransferase) to form linamarin (Conn, 1994). No HCN is released from intact
cyanogenic plants, the substrates (cyanogenic glycosides) and the enzymes must be located in
different compartments of the cell. Cyanogenesis is initiated in cassava when the plant tissue
is damaged. The generation of cyanide from linamarin is a two-step process involving the
initial deglycosylation of linamarin and the cleavage of acetone cyanhydrin to form acetone
and cyanide (Figure I-6). These reactions are catalysed by linamarase (a ß-glucosidase) and
by α-hydroxynitrile lyase (HNL). Since acetone cyanhydrin may enzymatically be broken
down by HNL as well as a spontaneously decompose at pH slightly >4.0 or temperature >
30°C, it has been generally assumed that the linamarase is the rate-limiting step (McMahon et
al, 1995; White et al, 1998; Vetter, 2000). In spite of the relative instability of acetone
cyanohydrin, it can coexist with intact glycosides and HCN in differently processed cassava
products. Therefore, cyanogens in cassava products can exist in three forms:
• Cyanogenic glycosides (linamarin and lotaustralin),
• Acetone cyanohydrin,
• Free HCN (Tewe, 1994).
Cassava tubers vary widely in their cyanogenic glycosides content, although most varieties
contain 15 to 400 mg HCN equivalent/ kg fresh weight. Occasionally, varieties with high
cyanide content (1300 to 2000 mg/ kg) are also encountered (Hahn, 1989).
Cassava leaves also contain high concentrations of cyanogenic glucosides and the values fall
mostly in the range of 1000 to 2000 mg HCN equivalent/ kg on a dry matter basis (Bokanga,
1994). Very high values up to 4500 mg/ kg have been occasionally reported. The high content
of cyanogenic glycosides in cassava is however a factor restricting its utilization as a food
(Padmaja, 1995).
Konzo and cassava toxicity
31
I.3.2.4 Effect of processing on cyanogenic glycosides in cassava
Cassava roots are processed by a number of methods that vary widely from region to region.
Generally, all those techniques are intended to reduce toxicity and improve palatability and
storability (Tewe, 1994). Adequate processing of cassava is of prime importance in
eliminating the toxic glycosides and converting cassava into a safe food. Cyanide–yielding
substances of cassava are normally reduced to negligible levels by effective processing
(Rosling, 1988). Cassava roots provide an important source of dietary energy but they have
some limitations. Firstly roots are readily perishable if they are not processed and they cannot
be stored like cereals or other tubers (potatoes, yam). Secondly roots from the bitter varieties
cannot be consumed raw and they are unsuitable for roasting and boiling as fresh roots
because of the high levels of potentially toxic cyanogenic glycosides (linamarin and
lotaustralin). Therefore, cassava roots from such varieties must be processed before
consumption to reduce the content of toxic cyanogenic glycosides and their degradation
products (acetone cyanhydrin and free cyanide) in the final food product. Because of their
high water content, harvested roots rot if they are not processed shortly after harvesting. The
processing considerably reduces the water content (about 50 - 70 % in the freshly harvested
tuber) and thus facilitates transportation. The processing serves to make the starch of the
cassava root suitable for consumption as a major food component in the form of boiled paste,
flour or granules in the many different dishes prepared according to cultural preferences.
Cultural preferences vary a lot and the choice of processing method is thus aimed at obtaining
a cassava food product which is safe to eat and has a desired taste, flavour and texture.
To be considered as safe for consumption, cyanogens should be removed by processing to a
level below 10 mg equivalent HCN per kilogram (ppm) dry weight of cassava product, the
recommended safe limit set by FAO/WHO (1991)
Konzo and cassava toxicity
32
Although the processing steps are different for each product, they permit the glucosidase to
interact with the cyanogens and the release of cyanide. The processing leads to two end
products mostly depending on the locally available processing resources (Hahn, 1989;
O’Brien et al 1995; Ravi and Abed, 1996). Dry chips or cossettes and flour are the main
products where sunlight is abundant and wet paste is the main product where water supply is
abundant. These products need additional home preparation. Cooked paste, steamed and
toasted granules are relatively more advanced that enter the marketing system in ready-to-
serve forms, although toasted granules may need minimal preparation by soaking in hot or
cold water. These products are usually more convenient and attractive to urban consumers,
and competitive with food grains in the market place. Cassava is more often processed into
first group products for home use and into second group products for sale. However cassava is
widely marketed in form of cossettes in D. R. C. and most of the cassava products pass
through fermentation or soaking stage. The period of the fermentation usually lasts a number
of days, and varies depending on the product, processing technique and on the market for
which the product is intended for sale (Nweke and Bokanga, 1994). But where market access
has been improved, the fermentation period tends to decline; e.g. in D. R. C., the completion
of a new tarmac road to the capital city of Kinshasa resulted in an increased demand due to
improved market access. This caused the farmers producing cassava cossettes with a reduced
soaking period from three or four days to one or two days (Tylleskär et al, 1991).
Retting of cassava roots by steeping them in water causes higher losses in total cyanogens
and, makes them soft and causes the cells to rupture, releasing linamarase. Cyanogenic
glycoside removal can be enhanced by direct leaching into the soaking water (Muzanila et al,
2000). Wet fermentation has been reported to facilitate the breakdown of cyanogenic
glycosides to low total cyanogen levels, up to 13,5 % reduction during the first of day
soaking, and 65% reduction the second day of soaking (O’Brien et al, 1992) or even in one
Konzo and cassava toxicity
33
experiment up to 90 % reduction after 4 days of fermentation (Padmaja, 1995). The efficiency
of fermentation or soaking of cassava root has been well documented as one of the best
methods for cyanogen elimination (O’Brien et al, 1992; Padmaja, 1995; Ravi and Padmaja,
1997). Sun-drying of cassava is generally considered to be the least efficient of the various
categories commonly practiced in Africa (Mlingi et al, 1995; Essers et al, 1996). The
cyanogenic potential of heap fermented cassava roots was significantly lower than those from
sun dried (Zvauya et al, 2002). The short-cut method of alternate pounding and drying of
cassava roots resulted in a sharp decline in glycoside levels but high cyanohydrin levels may
remain if the products are not sufficiently dried (Mlingi et al, 1995). Processing steps such as
crushing and pounding may be incorporated prior to sun-drying to increase the efficiency of
cyanogen removal. However, sun-drying alone as a processing method of highly cyanogenic
cassava varieties remains inadequate if levels are to be reduced to the recommended FAO/
WHO safe limit set at 10 mg HCN equivalent /kg dry (Essers et al, 1996; Bainbridge et al,
1998).
Fresh cassava leaves contain very high levels of cyanogenic glycosides, usually 5 to 20 times
more than the amount present in the edible parts of the roots. Effective detoxification
processing is required prior to consumption. Bokanga (1994) found after pounding cassava
leaves a reduction of the cyanogenic potential by 63 to 73 %. The rapid removal of cyanogens
from cassava leaves can be attributed to the presence in the leaves of a high level of
linamarase activity, to the extensive mechanical damage imparted to the leaves during
pounding thereby facilitating the contact between linamarase and linamarin and promoting
cyanogenesis. The removal of cyanide by altering the cooking time and initial water
temperature in the preparation of pounded cassava leaves showed that starting with water at a
temperature of 27°C, brought down the total cyanide level 3.8 times more effectively than
starting with already boiling water and, increasing cooking time from half hour to one and
Konzo and cassava toxicity
34
half hour resulted in a 2.5-fold more effective reduction (Essers, 1989). Gradual heat applied
during cooking of pounded cassava leaves accelerates the evaporation of hydrogen cyanide
(boiling point at 25.7°C) and starting cooking at 27°C will increase progressively the
temperature to reach the maximum linamarase activity reported to be 55°C. Putting the leaves
into boiling water will immediately reduce drastically the activity of linamarase and may
therefore prevent liberation of cyanide from its glycosydic bond (Essers, 1989; Bokanga,
1994).
I.3.2.5 Metabolism of cyanogens in Humans
During cassava processing, cyanogenic glycosides (linamarin and lotaustralin) break down
into glucose and acetone cyanohydrin through the activity of the endogenous enzyme
linamarase. Acetone cyanohydrin gradually breaks down into HCN spontaneously
(temperature and pH dependant) or enzymatically (Figure I-6). These cyanogens (linamarin,
acetone cyanohydrin and HCN) can be reduced to negligible levels by effective processing,
but insufficiently processed cassava products contain varying amounts of cyanogens.
Following consumption, any of the three types of cyanogens may result in cyanide exposure
(Essers et al, 1992; Rosling, 1994). However the fate of the cyanogens will differ during
digestion in the gut and metabolism in the body (Carlsson et al, 1995).
The ingested linamarin is thought to be hydrolysed to glucose and acetone cyanhydrin in the
intestinal tract; hydrogen cyanide is then produced by a catalytic reaction in the intestine and
rapidly absorbed from the intestine to the blood (Sreeja et al, 2003). A part of ingested
linamarin has been found to pass through the human body unchanged, absorbed directly in the
intestine and excreted intact in urine (Carlsson et al, 1995; Carlsson et al, 1999; Sreeja et al,
2003). Ingested residual cyanhydrins are assumed to break down to cyanide in the alkaline
environment of the gut (Tylleskär et al, 1992). Cyanide is rapidly absorbed by the
gastrointestinal tract and distributed throughout the body by the blood. The major portion of
Konzo and cassava toxicity
35
cyanide in blood is sequestered in the erythrocytes, and a relatively small proportion is
transported via plasma to the target organs (liver, lungs, kidney, brain, central nervous
system). Although cyanide can interact with substances such as methemoglobin in the
bloodstream, the majority of cyanide metabolism occurs within the tissues. Cyanide is
metabolized in mammalian systems by one major route and several minor routes (Figure I-7).
Figure I-7: Basic processes involved in the metabolism of cyanide (ATSDR, 1997)
Konzo and cassava toxicity
36
The major route of metabolism for cyanide is detoxification in the liver by the mitochondrial
enzyme rhodanese (thiosulphate-sulphurtransferase, EC 2.8.1.1), which catalyses the transfer
of the sulfane sulphur of thiosulphate to the cyanide ion to form the less toxic thiocyanate
(Figure I-7). About 80 % of cyanide is detoxified by this route. The rate-limiting step is the
amount of thiosulphate which is produced by ß-mercaptopyruvate resulting from
transamination of cysteine. While rhodanese is present in the mitochondria of all tissues, the
species and tissue distributions of rhodanese are highly variable. In general the highest
concentrations of rhodanese are found in the liver, kidney, brain and muscle, but the supply of
thiosulphate is limited.
Cyanide is principally excreted as thiocyanate in the urine but the limiting factor in cyanide
metabolism is the concentration of the sulfur containing substrates primarily thiosulphate, but
also cystine and cysteine as product of methionine (essential amino acid) and cysteine
catabolism (Figure I-8). There are several pathways for cysteine catabolism. The more
important catabolic pathway is that via a cytochrome –P450-coupled enzyme, cysteine
dioxygenase that oxidises the cysteine sulphydyl to sulfinate, producing the intermediate
cysteinesulfinate. Cysteinesulfinate can serve as a biosynthetic intermediate undergoing
decarboxylation and oxidation to produce taurine, the bile salt precursor. The enzyme
cystathionase can also transfer the sulphur from one cysteine to another generating
thiocysteine and pyruvate. Transamination of cysteine yields ß-mercaptopyruvate which then
reacts with sulphite (SO32-) to produce thiosulphate and puryvate. Both thiocysteine and
thiosulphate can be used by the enzyme rhodanese to incorporate sulphur into cyanide,
thereby detoxifying the cyanide to thiocyanate (Hoffer, 2002; Komarnisky et al, 2003;
Stipanuk, 2004).
Konzo and cassava toxicity
37
Figure I-8 : Cysteine catabolism
Methionine
Cysteine
Pyruvate
+
Thiocysteine
ß-Mercaptopyruvate
Thiosulphate
+
cyanide
Cysteine
+ or Thiocyanate
Thiocyanate
Cysteinesulfinate
Hypotaurine
Taurinerhodanese
Cysteine
NH4+ H20
Pyruvate
SO32-
3-mercaptopyruvate sulphurtransferase
aminotransferase
α-ketoglutarateglutamate
Konzo and cassava toxicity
38
The level of thiocyanate normally present in body fluids is low but increases with chronic
exposure to cyanide and with smoking habits (Vesey et al. 1999, Kussendrager and Van
Hooijdonk, 2000). Thiocyanate remains the most useful chemical biomarker for dietary
cyanogen intake because it is a very stable metabolite that can be determined with relatively
cheap, specific and sensitive methods (Rosling, 1994, Ressler and Tatake, 2001). Urinary
thiocyanate is commonly used to check cyanogen overload in a population using cassava
roots and cassava products as staple food (Haque & Bradbury, 1999, Ernesto et al. 2002a).
Cyanide can also be metabolized by several minor routes, including the combination of
cyanide and hydroxycobalamin (vitamin B12) to yield cyanocobalamin (vitamin B12) and the
non-enzymatic combination of cyanide with cystine, forming 2-aminothiazoline-4-carboxylic
acid (ATC) which is excreted via the urine. However in protein-deficient subjects, in whom
sulfur amino acids are low, cyanide may conceivably be converted to cyanate (Tor-Agbidye et
al, 1999).
I.3.2.6 Effects of cassava toxicity in humans
The toxicity of cassava arises from the release of cyanide during hydrolysis of cyanogenic
glycosides by the glucosidases of intestinal microflora. Intact linamarin has also been reported
to be absorbed through the intestinal mucosa. Cyanide can also be released in vivo by
glucosidases of the liver and other tissues, causing in situ cytotoxicity (Padmaja, 1995).
Cyanide is a potent toxin that acts by inhibiting cellular respiration.
Cyanide toxicity occurs when the capacity for conversion of cyanide to thiocyanate is acutely
exceeded. This leads to inhibition of cytochrome oxidase and prevents cell respiration and
oxidative respiration. Acute toxicity results from the ingestion of lethal amounts of cyanide.
Doses of 50 to 100 mg are reported to be lethal to adults. Acute cassava poisoning, sometimes
leading to the death of whole families, has been occasionally reported in humans after
consumption of bitter cassava roots or inadequately processed cassava, usually at times when
Konzo and cassava toxicity
39
the normal eating habits are affected by famine. More common are incidences of chronic
cyanide toxicity due to prolonged consumption of insufficiently processed cassava. Chronic
toxicity of cassava has been implicated in several diseases such as tropical ataxic neuropathy,
endemic goiter and konzo. Many of these conditions result from the consumption of poorly
processed cassava.
I.4 Conclusion
Konzo is a paralytic disease rarely reported and little known even in the affected area. This
symmetric paralysis of both legs affects mainly women at childbearing age and children
above three years old, among the poor rural population of remote areas of Sub-Saharan Africa
where cassava is the staple food. Affected persons live far from the big city where decisions
are made and they are of no particular interest for political authorities. Literature on konzo is
limited to some epidemiological consideration. Evidence linking the disease with high
consumption of improperly processed cassava roots has been established. In addition, a low
intake of sulphur amino acids (methionine and cysteine) needed for the metabolic
detoxification of cyanide in the human body has also been thought to be an important co-
factor in the development of konzo (Cliff et al, 1985).
Up to date, there is no medicine to cure this crippling non-progressive and irreversible
disease. Nevertheless, some investigators (Ernesto et al, 2002b) have proposed the following
strategies to prevent and to eliminate konzo:
• Introduction of other staples, vegetables, pulses and fruits to decrease the daily
cyanide intake and broaden the diet of the people,
• Improved processing of cassava roots to produce products that have less residual
cyanide,
• Introduction of low cyanide, high yielding, well-adapted, disease-resistant varieties of
cassava,
Konzo and cassava toxicity
40
• and improved early warning systems of a possible konzo epidemic.
I.4.1 Rationale of the research
Konzo is a neglected and an emerging neurological crippling disease that affects the poor
segments of remote rural communities of sub-Saharan Africa. There is convincing evidence
linking konzo with high cyanide exposure (Tylleskär, 1994a; Ludolph and Spencer, 1996):
First, heavy dietary reliance on bitter cassava is strongly associated with the development of
konzo. Secondly, there is a consistent association between shortcut soaking cassava
processing and outbreaks of konzo. Thirdly, the bitter cassava contains cyanogenic
glycosides, mainly linamarin and to a lesser extent lotaustralin which upon hydrolysis release
the mitochondrial toxin cyanide, a potent inhibitor of cytochrome C oxidase (complex IV of
the mitochondrial respiratory chain). Also, in affected populations, the excretion of urinary
thiocyanate and the ratio thiocyanate/ inorganic sulphur in the blood of affected populations
are increased. (Tylleskär et al, 1991; Banea et al, 1992b; Tylleskär et al, 1992).
Definite confirmation of an etiologic role of cyanide in konzo by identification of the
mechanism in an experimental animal model, or a quasi experimental preventive intervention
is lacking. There is therefore a need to know if a wild plant (food), a vitamin deficiency,
another toxin in cassava or some other factor may be contributing to or be an essential factor
in the aetiology of konzo (Tylleskär, 1994a, Bonmarin et al, 2002).
I.4.2 Objectives
I.4.2.1 General objective
• Identify associated nutritional factors involved in konzo
I.4.2.2 Specific objectives
• To review the literature on konzo and its relation to cassava dietary exposure.
Konzo and cassava toxicity
41
• To determine the prevalence and associated dietary factors with Konzo.
• To assess dietary intake with special emphasis on intake of sulfur amino acids.
• To quantify the daily intake of cyanogen, and to estimate the amount of sulphur amino
acids required for their detoxification in konzo affected areas.
• To determine free amino acids in order to evaluate the presence of inherent potentially
toxic nonprotein amino acids in the cassava products.
• To develop total protein amino acids profiles of cassava products in order to evaluate the
dietary protein quality and to compare them with the amino acid requirements of children
and adults.
• To monitor the level of dietary exposure to cyanogens from cassava in the selected konzo
affected community.
• To assess a potential relationship between urinary thiocyanate as biomarker of daily
cyanogen exposure and taurine as modulator of neuroexcitation.
Konzo and cassava toxicity
42
CHAPTER II:
OCCURRENCE OF KONZO AND DIETARY PATTERN*
* This chapter will be submitted for publication in Tropical Medicine and International Health as: Delphin Diasolua Ngudi, Jean-Pierre Banea-Mayambu., Fernand Lambein and Patrick Kolsteren. Konzo and dietary pattern in cassava-consuming populations of Popokabaka, Democratic Republic of Congo
Konzo and cassava toxicity
43
II Occurrence of konzo and dietary pattern
II.1.1 Introduction
In Popokabaka district (D. R. C.) where cases of konzo were first reported three generations
ago (Trolli, 1938), new cases were found and the diet has changed little. Cassava flour, the so
called “luku” or “fufu” is consumed together with a sauce prepared from cassava leaves. Meat
and fish are not eaten daily in the villages (Tshala-Katumbay, 2001a).
Protein-energy malnutrition which is a major health problem in the region has been attributed
to the combined effect of infections and inadequate diet. An unbalanced diet is suggested to
be the main risk factor for several diseases such as obesity, stroke, cancer (Thiele et al, 2004)
and a factor aggravating growth retardation in children in Bandundu Province, D. R. C.
(Banea-Mayambu et al, 2000).
The present paper reports the prevalence of konzo, the household risk factors associated and
the dietary pattern in cassava consuming populations. Dietary intake patterns and socio-
economic variables are well known indicators for assessing nutritional status of a community
(Agrahar-Murugkar and Pal, 2004).
II.1.2 Materials and Methods
II.1.2.1 Study area
The study was conducted in Popokabaka rural health zone (Prhz) (5°38’35” – 5°43’0” latitude
South, 16°34’60” – 16°37’8” longitude East), district of Kwango, province of Bandundu (1 -
8° S, 16 – 20° E), D. R. C. in February 2003 during the rainy season. Prhz covers an area of
7,949 km2 with a population of 149,227 inhabitants in 2002 (density of 19 inhabitants/ km2).
Prhz is divided into 38 health areas. The vegetation is bushy savannah with few forest
galleries where the climate is tropical with annual rainfall varying around 1200 mm and a
Konzo and cassava toxicity
44
long dry season of about 5 to 6 months (Kama, 1970). The poor soil makes the rural
population focus on the cultivation of cassava, the most important cash crop and the main
staple food for this region. Prhz faces various constraints for its development. Accessibility to
Prhz is difficult especially during the rainy season and limits the communication with
Kinshasa, the capital of D. R. C. where manufactured goods and other food products can be
purchased or excess of crop production can be sold. Recrudescence of endemic pathologies
(malaria, trypanosomiasis, tuberculosis, leprosy, etc), poverty of the population and limited
number of unsafe drinking water sources are main problems encountered by Prhz (Mwela,
2002).
II.1.2.2 Subject
Four health areas in Prhz with a total population of 12,416 inhabitants in 2,069 households
(national mean size of household members is 6, see R. D. C. 2001) were selected based on the
accessibility and the reported prevalence of konzo in the areas. The Epitable calculator of
Epiinfo version 6 was used to calculate the sample size for a single proportion of a limited
community study based on the size of the population, the desired precision (0.99), the
expected prevalence of konzo (4 %), the designed effect (2) and on the confidence level (95
%). After introduction of those required data, a sample size of 2,685 inhabitants in 448
households was obtained. This sampled population was attained through heads or delegates of
household* who were enrolled in this study in a random sampling after selection of the first
participant (household) as starting point, near the main entrance road to the village or near the
health centre (clinic). The sample size was adjusted upward to 487 participating households to
avoid other factors that could decrease the yield of usable responses. No refusal was observed
during the survey. Written authorisations were obtained from the administrative and health
* A household was defined as group of persons sharing the same meals since at least 3 months before the survey.
Konzo and cassava toxicity
45
authorities, oral consent and assistance were also obtained from the village leaders and from
the subjects.
The heads or delegates of household were interviewed by trained enumerators using a closed
and open-ended questionnaire designed to collect the following information: identification of
the household (name of the respondent, village, name, sex, marital status, education and
origin of head of household), socio- demographic characteristics: family size, family member
affected by konzo (name, birthday, onset, sex, degree of walking), owning land farm or
husbandry, list of all foods consumed during the previous day (morning, lunch and evening),
the origin of the cassava consumed the previous day as staple food and the duration of retting
(soaking), the composition of the “luku” and list of foods consumed often during the rainy
season and the dry season. The WHO criteria for konzo were applied to detect cases and to
confirm the diagnosis (WHO, 1996).
II.1.2.3 Statistics
Descriptive statistics were used for socio-demographic and other household related variables.
On the basis of the prevalence found, the areas were pooled into two groups: the low
prevalence area and the high prevalence area. Thus, the degree of konzo prevalence in the
health area was used to measure the risk of konzo and as a dependent variable in multivariate
analysis. Data were entered using EpiInfo (version 6.04) and analyses were performed with
SPSS (version 11.5 statistical packages for Windows). Excel 2003 for Windows was used to
plot graphs.
Konzo and cassava toxicity
46
Table II-1: Socio-demographic variables and 24hr recall food consumption of participants
among the high prevalence of konzo health area (n = 224) and the low prevalence of
konzo health area (n =263)
Health area Variables
High Low OR (CI 95 %) P
Sex Female Male
81 142
70 193
1.36 (1.05 – 1.78)
0.024
Age Under 35 35 +
49 145
63 155
0.87 (0.63 – 1.20) 0.438
Marital status Unmarried Married
37 183
26 236
1.69 (1.06 – 2.71)
0.030
Education Illiterate Literate
109 114
99 161
1.28 (1.05 – 1.58)
0.021
Occupation Farmer Other
196 26
178 76
1.26 (1.15 – 1.38)
< 0.005
Native No Yes
52 172
50 213
1.22 (0.86 – 1.72)
0.266
Origin of cassava consumed in the household
Own farm Other origin
215 7
240 19
1.04 (1.00 – 1.09)
0.045
Soaking time of the cassava consumed
Less than 3 nights 3 nights +
62 153
202 38
0.34 (0.28 – 0.43)
< 0.005
Konzo and cassava toxicity
47
Composition of luku consumed
Only cassava Cassava + cereal
Meat consumption Yes No
Sesame consumption Yes No
Cereals consumption Yes No
Cowpea consumption Yes No
Vegetables consumption Yes No
Cassava tuber consumption Yes No
Cassava leaves “Saka-Saka” consumption Yes No
Luku consumption Yes No
192 28
84 140
5
219
105 119
76 148
161 63
39 185
91 133
223 1
244 15
79 184
108 155
34 229
70 193
186 77
23 240
104 159
260 3
0.93 (0.87 – 0.98)
1.25 (0.97 – 1.60)
1.66 (1.50 – 1.84)
3.63 (2.57 – 5.11)
1.27 (0.97 – 1.67)
1.02 (0.91 – 1.14)
1.99 (1.23 – 3.23)
1.03 (0.83 -1.28)
1.01 (0.99 – 1.02)
0.010
0.084
< 0.005
< 0.005
0.092
0.84
0.006
0.853
0.628
II.1.3 Results
The majority (69 %) of the interviewed heads or delegates of household were male. The mean
age of respondents was 43.3 years (SD 12.0) with a range of 61 years (maximum 78 and
minimum 17). 79 % of the members of the households were native from the area. The
illiteracy rate among the heads or delegates of household was 43 % and among the literate,
Konzo and cassava toxicity
48
more than 50 % did not finish the primary school (sixth grade), only 1.6 % and 0.4 % have
reached respectively secondary school (at least ninth grade school) and high school or
university, respectively. Most of the heads of household were married (86%) and small-scale
farmers (78 %) as main occupation. Other main occupation of the participants encompassed
mainly sawyer (5.5 %), teacher (4.5 %), worker (2.3 %), retailer (2.1 %), roadman (1.8 %)
and hunter (0.4%). The mean family size was 6.2 ± 2.7 (range 14; max 15 and min 1). About
75 % of female respondents were illiterate and almost all of them were farmer except three
with one retailer and two house wives with no other occupation. Table II-1 shows the
distribution of some socio-demographic variables of participants among the high prevalence
of konzo health area (n = 224) and the low prevalence health area (n =263). Degree of konzo
prevalence was statistically significant and associated with female gender [OR (95 % CI) =
1.4 (1.1 -1.8), P = 0.024], unmarried status OR (95 % CI) = 1.7 (1.1 -2.7), P = 0.030],
illiteracy [OR (95 % CI) = 1.2 (1.1 -1.6), P = 0.021], farmer as main occupation [OR (95 %
CI) = 1.3 (1.2 -1.4), P< 0.05] and slightly with consumption of cassava originated from own
farm as opposed to cassava obtained elsewhere [OR (95 % CI) = 1.0 (1.0 – 1.1), P = 0.045].
The origin of 24 hour cassava flour consumed in 93.4 % of households was from their own
farmstead and the retting time of the processed cassava was less than three nights in 57,4 % of
those families. In the households from where cassava originated from their own farmstead,
90.6 % of them consumed luku that was composed only of cassava flour and 9.4 % mixed
their cassava flour with maize flour.
Among the 3,015 individuals in the 487 households selected, 43 konzo patients were detected
in 33 (6.8 %) of the households; thus, a prevalence of 1.4% and an incidence in 2002 of 1.3 ‰
(Figure II-1). The mean number of affected family members per household was 1.30 (SD
0.6) with a range of two (minimum 1 and maximum 3). The distribution of konzo cases per
Konzo and cassava toxicity
49
selected health areas is presented in Table II-2. 77 % of patients were female (male-to-female
ratio 1:3.3) and 64 % of the patients were under 15 year of age.
Figure II-1: Distribution of onset of konzo from 1980 to 2002
0
2
4
6
8
10
12
1975 1980 1985 1990 1995 2000 2005
year of onset
Case
s
Table II-2: Distribution of konzo cases per health area
Number of household
per number of konzo
cases
Health area
1 2 3
Number of konzo patients
High prevalence area
Masina
Mutsanga
22
14
8
4
2
2
1
1
0
33
21
12
Low prevalence area
Popo-Secteur
Imwela
3
3
0
2
1
1
1
1
0
10
8
2
Total 25 6 2 43
Konzo and cassava toxicity
50
The majority [25/ 33 (75.8 %)] of the affected households had a single affected family
member. The degree of disability on walking and the age distribution of konzo patients by
gender are shown in Table II-3. The mild form of the disease was the most common and
found in 74 % of patients followed by the crawler or severe form (16%) and the moderate
form (9 %). No male was found to be attained by the moderate form. The earliest year of
onset of paralysis reported is 1980 and the latest 2002. Figure II-1 shows the distribution of
konzo cases by year of onset. Around half of the cases occurred between 1998 and 2002.
Table II-3 : Degree of disability on walking and age distribution of konzo patients by gender
Variables Sex Total
Female Male
Degree of disability
a. Walk without stick
b. Walk with stick
c. Can’t walk (crawl)
Total
24
4
5
33
8
-
2
10
32
4
7
43
Age (Years)
a. Under 5
b. 5 – 9
c. 10 – 14
d. 15 – 19
e. 20
Total
5
10
4
2
8
29
2
3
1
2
2
10
7
13
5
4
10
39
From the food consumption patterns of the respondents, we observed that less than 60 % ate
three main meals a day as breakfast, lunch and dinner, and their dietary pattern was based
Konzo and cassava toxicity
51
basically on cassava stiff porridge, the so called fufu or luku. Table II-4 presents the
frequencies of 24 hour foods consumed; 97.9 % had their evening meal whereas 58.7 % of
households had lunch and 91.6 % had breakfast in the morning. Luku, the cassava flour stiff
porridge was the main staple food consumed at least once during the day in 99.2 % of
households. Rice was consumed as staple food in 0.6 %. Maize and other cassava roots
products (boiled or raw cassava roots) were the other staple foods consumed mainly during
lunch time as snacks.
Table II-4: 24-hour recall of household food intake frequencies (%)
Food Morning Lunch Evening
Roots and tubers
Luku (cassava flour stiff porridge)
Other cassava roots products
Sweet potato
Yam
85.1
1.6
-
-
31.2
5.6
0.2
4.3
95.4
1.2
--
Vegetables and fruits
Vegetables
Saka Saka (pounded cassava leaves)
Tomato
Mushrooms
Mbondi (Salacia pynaertii)
Mfumbwa (Gnetum africanum)
Spinach
Ngayi-Ngayi (Hibiscus sabandja)
Matembele (Sweet potato leaves)
Kikalakasa (Psophocarpus scandens leaves)
Amaranth
Other vegetables (unspecified)
Fruits
Safou
Banana
Pineapple
25.5
16.6
10.6
8.8
4.4
2.0
1.0
0.6
0.4
0.2
1.6
1.0
0.6
-
5.9
6.5
2.8
4.1
1.8
0.4
-
0.4
-
-
0.8
1.0
2
0.4
20.7
14.5
11.9
11.1
7.0
2.2
3.2
1.4
0.4
0.8
2.4
0.6
-
-
Konzo and cassava toxicity
52
Legumes
Cowpeas (Vigna ungiculata)
Beans (Phaseolus vulgaris)
Voandzou (Vigna subterranea)
16.6
1.0
0.2
8.0
0.2
2.0
17.1
0.8
0.6
Cereals
Maize
Rice
4.4
0.6
13.3
1
3.8
0.2
Oleaginous grains (seeds and nuts)
Sesame
Peanut
Soybean
Pumpkins seeds (Curcubitaceae sp.)
13.9
12.8
1.2
0.8
4.3
17.8
0.2
1.0
12.6
11.9
0.2
1.2
Flesh
Red meat (cow, pork, goat, lamb, wild animals)
Fish
Chicken
Insect (Caterpillar and Larva)
Milk
Egg
Wild bird (unspecified)
Grasshopper
8.7
4.7
1.2
1
0.6
0.4
-
-
4
3.2
0.8
-
-
-
-
-
11.1
9.4
1.6
1.6
-
0.4
0.2
0.2
No Food 8.4 41.3 2.1
Saka - saka (pounded cassava leaves) was the main condiment consumed as side-dish with
luku in 40 % of households followed by cowpeas (30 %) and sesame (23.2 %). Peanut and
tomato were used as ingredients to prepare their sauce. Peanuts also were consumed during
lunch time as snacks to accompany grains of maize or other cassava roots products.
Mushrooms and unconventional green leafy vegetables such as mbondi (Salacia pynaertii)
and mfumbwa (Gnetum africanum), which villagers gather from the nearby bush or forests
were popular. They were consumed as supplementary foods to the staple luku in 17.7 %, 18.1
Konzo and cassava toxicity
53
% and 11.3 % of households respectively. Only 15 % of households consumed garden
vegetables.
Table II-5: Seasonal food consumption availability (%) listed by the respondents
Foods Rainy season Dry season
Sweet potato
Yam
Saka Saka (pounded cassava leaves)
Tomato
Mushrooms
Mbondi (Salacia pynaertii)
Mfumbwa (Gnetum africanum)
Spinach
Ngayi-Ngayi (Hibiscus sabandja)
Matembele (Sweet potato leaves)
Amaranth
Maize
Rice
Banana
Pineapple
Cowpeas (Vigna ungiculata)
Beans (Phaseolus vulgaris)
Sesame
Peanut
Pumpkin seeds (Curcubitaceae sp.)
Red meat (cow, pork, goat, lamb)
Rats & wild animals
Fish
Chicken
Insect (Caterpillar and Larva)
Egg
Wild bird (unspecified)
Grasshopper
3.1
12.7
76
49.5
67
36.1
21.1
30.6
15.7
9.1
29.6
54.5
0.4
24.4
23.2
50.9
18.6
36.4
54.5
5
3.3
1
18.4
0.4
18
0.6
0.4
1.2
4.8
36.9
7
4.6
4.1
13.3
21.1
1.9
1.2
1.2
3.1
5.2
0
5.2
3.1
9.1
8.7
8.9
11
52.1
68.7
78
49.5
0.6
17.4
0.2
12.7
36.7
Konzo and cassava toxicity
54
Meat and fish were at lesser degree consumed to supplement the staple food. Usually they are
added in the preparation of vegetables. Consumption of fruits is less; safou, pineapple, banana
and orange were the only fruits listed and consumed by less than 1% of households the day
before the survey.
The comparison between degree of prevalence of konzo in health area and 24 hour recall food
(groups) consumption is summarised in Table II-1. There is a statistically significant
association between the prevalence of konzo with consumption of cereals ([OR (95 % CI) =
3.6 (2.6 -5.1)], [OR (95 % CI) = 1.2 (1.1 -1.4)], with consumption of tubers of cassava [OR
(95 % CI) = 2.0 (1.2 -3.2)] and with consumption of sesame [OR (95 % CI) = 1.7 (1.5 -1.8)].
No statistically significant association was found between the prevalence of konzo in health
area with consumption of meat, with consumption of cowpea, with consumption of
vegetables, with consumption of saka-saka and, with consumption of luku. The frequencies of
foods consumed by season (Table II-5) show a decrease of consumption of almost all the
foods listed by the participants from the rainy season to the dry season, except the
consumption of meat and pumpkin seeds, which increases consumption.
II.1.4 Discussion and conclusion
Konzo is still occurring in this area three generations after the first report (Trolli, 1938). The
prevalence of konzo (1.4 %) found in our study, is lower than the expected prevalence of 4 %
but in the range of that reported in the literature (Tylleskär et al, 1991; Tshala-Katumbay et al,
2001b). The overall sex and age distribution of patients in our study was similar to most of the
previous studies in the region and elsewhere (Howlett, 1994; Banea- Mayambu, 1997c,
Tshala-Katumbay et al, 2001a). Preponderance of female patients (female to male ratio 3.3: 1)
in our study is similar to almost all other studies except the ones carried out in Tanzania and
Mozambique where male cases were preponderant (Howlett, 1994; Tshala-Katumbay et al,
2001b).
Konzo and cassava toxicity
55
Households whose head was illiterate carried an increased risk of konzo. More than half of
the heads of household or representatives were illiterate or did not go beyond primary school
level. Illiteracy was also found to be a factor that carries an increased risk of paralysis in the
case of neurolathyrism, a spastic paraparesis with many similarities to konzo (Lambein et al,
2004). Education drives both individual and community development, and illiterates are likely
to have low socio-economic status even in such remote rural areas. Literate people probably
have better access to information than the illiterate on food processing especially home
detoxification methods (Getahun et al, 2002b). Women, who belong to the most susceptible
group to develop konzo and who also play the primary role in the household food security,
has been found in this study to be less educated and at higher risk of konzo. Three quarters of
them were illiterate and among those who were literate, half did not finish the primary school
education. In D. R. C., more than half of the women of rural areas are illiterate (R. D. C.,
2001). Unmarried status of the head of household as a risk factor can be explained by the
excessive workloads. The main occupation as a farmer for a head of household is associated
with increased risk of konzo. This can be explained by the fact that people are relying on their
own sole culture and consumption of cassava. Increased risk of konzo is found to be
associated with consumption of cassava originating from the household’s own farm. This may
be related to poverty, as the less poor have more access to marketed commodities, which
results in a more varied diet.
The food consumption pattern of the selected households in both high and low konzo
incidence areas is dominated by cassava diet. Fufu, the processed product of cassava roots is
the staple food of almost all the households and saka-saka is the main side-dish consumed
with fufu. Cassava roots are an excellent source of carbohydrate but contain extremely low
levels of protein and fat (Bradbury and Holloway, 1988). The protein is of poor quality,
leucine and lysine are limiting amino acids. The proportion of methionine is low and the
Konzo and cassava toxicity
56
chemical score of the protein is around 40 (chapter III-2). Cassava leaves have a high protein
content but also this protein is of poor quality limited in lysine, histidine and sulphur
containing amino acids (methionine and cysteine) (chapter III-1). High dietary exposure to
cyanogen was found in this area (chapter IV).
Consumption of cowpea with fufu is also popular and provided a high quantity of protein in
the diet but also in cowpeas the sulphur containing amino acids are low (Finetin, 2001).
Consumption of cereals and sesame is found in this study to be protective factors against
konzo. Similar protection by methionine rich cereals was also found for neurolathyrism
(Getahun et al, 2003). Mixing cassava with cereals and mixing legumes such as cowpeas with
cereals may thus increase the quality of the meal by optimising the balance of essential amino
acids. Consumption of the unconventional green leafy vegetable mbondi (Salacia pynaertii)
and pumpkin seeds that are rich in protein with a high content in methionine and cysteine
(Mbemba and Remacle, 1992; Finetin, 2001), should also be promoted. Consumption of fresh
or boiled cassava tubers is a risk factor for konzo. Cassava roots contain high level of
cyanogenic glucosides that should be removed by processing. Peeling and boiling cassava are
not enough to lower the toxin compounds to a safe level. The peeled roots need to be soaked
in slow running water (retting or “rouissage”) for at least three nights. Consumption of luku
made from roots soaked less than three nights was associated with increased incidence of
konzo.
Nutritional resources listed became scarce during times of dry season. Almost all crops are
rain-fed and cannot survive during the dry season unless watering or irrigation which is not
done in this area because of absence of inputs or of major river systems for irrigation,
electricity for irrigation pumps and other factors that limit irrigation. Cassava roots that
constitute the staple food resist the drought. Under drought conditions the linamarin content
of cassava roots is known to increase due to increased water stress on the cassava plant
Konzo and cassava toxicity
57
(Bokanga et al, 1994). Moreover, the weather of the dry season is mostly cloudy and the mean
temperature is 20 0C or less; which may be favourable for high exposure to cyanogen. The dry
season is the period of intensive cassava trade resulting in frequently shortcuts in cassava
processing with high residual cyanogens level in the product. As the duration of dry season in
this area of particular dry tropical climate is estimated to 5 or 6 months, serious drought may
increase the cyanide intake of individuals, if non- efficient processing techniques are used, to
such a degree as to precipitate the occurrence of konzo. High prevalence of konzo has been
reported in the dry season (Banea-Mayambu et al, 1997a; Cardoso et al, 2004).
In conclusion, konzo is still occurring in this area with an incidence in 2002 of 1.3 ‰, where
women who play the principal role in the household food security are in majority illiterate.
Although konzo was reported in this area in 1938, in this study we found no cases with onset
before 1980. We found no reports on the life expectancy of konzo patients. The diet is largely
dominated by cassava and major foods consumed are of poor quality in protein especially in
sulphur containing amino acids. Methionine and cysteine are required for the detoxification of
cyanide in the body. The results obtained in this study, confirm that low intake of sulphur
containing amino acids (methionine + cysteine) is associated with incidence of konzo, as well
as with dietary cyanide exposure. Therefore the emphasis should be placed on increasing
production and access to cereals, sesame and pumpkin seeds to increase the availability of
sulphur amino acids in the diet. Vegetable gardens should be promoted to encourage the
consumption of leafy vegetables in all seasons. Appropriate information, communication and
training in cassava processing and promotion of a better balanced diet may prevent this
irreversible crippling disease, konzo.
Konzo and cassava toxicity
58
CHAPTER III.
CASSAVA FOOD QUALITY AND SAFETY
Konzo and cassava toxicity
59
III Cassava food quality and safety
III.1 Food Safety and Amino Acid Balance in Processed Cassava
"cossettes"*
III.1.1 Introduction
Cassava (Manihot esculenta Crantz, Euphorbiaceae) is the major staple food consumed by the
population of D. R. C. (Goossens, 1996). Processed cassava roots provide more than 60 % of the
daily energy intake (FAO, 1990).
Sweet varieties of cassava roots may be consumed directly while bitter varieties with high
content of cyanogenic glycosides are traditionally processed to reduce toxicity and to improve
palatability and storability. Many varieties of processed cassava roots with different local names
are known: cossettes, chikwangue, fufu, malemba, luku, ntuka, etc.
Cossettes, which is the most popular cassava product in D. R. C., is obtained by soaking or
immersing fresh bitter cassava roots (whole or peeled) in a stream or stationary water (near a
stream) for at least 3 days to allow them to ferment until they become soft. The fermented roots
are then taken out, peeled and sundried on mats, racks, roofs of houses, etc. Depending on the
weather, sundrying takes 2-5 days (Hahn, 1989). The dried fermented cassava root is the so-
called "cossettes" (Figure III-1). This form of cassava product is preferred because it can be
stored for a long period and can be traded over much longer distances (Goossens, 1996; Minten
and Kyle, 1999).
* This sub- chapter has been published as: Delphin Diasolua Ngudi, Yu – Haey Kuo and Fernand Lambein (2002). Food safety and amino acid balance in processed cassava roots “cossettes”. Journal of Agricultural and Food Chemistry 50, 3042 – 3049.
Konzo and cassava toxicity
60
Figure III-1: Flow diagram of cassava cossettes processing
Steps of cossettes production Location
Harvesting of whole fresh bitter cassava
roots
Fields
Peeling and chopping
or not
River, stream or village
Soaking and natural fermentation in water
for 3-5 days (and peeling)
River, stream or village
Sun-drying or fire-drying
for 3-5 days
Village
Cossettes Village
Pounding or
milling
Storage and packaging in
jute or propyl-ethylene
sacks
Cassava flour
Village
Trade Local markets or city markets
Konzo and cassava toxicity
61
When the roots are soaked and dried for a shorter period because of insufficient food supply or
poor agro-ecological conditions, the remaining cyanogen content can be much higher than that
after normal process. High intakes of dietary cyanogens from poorly processed cassava roots in a
diet deficient in sulphur amino acids have been implicated in the causation of konzo (Tylleskär,
1994b).
D. R. C. is the most affected country where konzo has been reported from remote rural areas of
Bulungu, Kahemba, Masi-Manimba and Popokabaka in Bandundu province (Howlett, 1994;
Tylleskär et al, 1995).
Besides the high content of cyanogens, cassava roots are also known to be poor in protein content
(Hahn, 1989). Proteins are a necessary part of the daily diet because the human body does not
store protein as it does with carbohydrate and fats. Furthermore, 9 of the 20 protein amino acids
are either not synthesized at all by our body or can only be synthesized in insufficient amounts.
Humans must obtain them from dietary sources. These are known as the dietary essential amino
acids that include histidine, isoleucine, leucine, tryptophan, lysine, methionine, phenylalanine,
threonine and valine. Failure to receive an adequate dietary supply of essential amino acids leads
to retarded growth and development in children and to disease and body deterioration in adults
(McMury and Castellion, 1996).
The objective of this study is to determine residual cyanogen in different samples of cossettes to
check the safety, to quantify the daily intake of cyanogen and to estimate the amount of sulphur
amino acids required for their detoxification. Free and total protein amino acids profiles of
cossettes are determined to evaluate the dietary protein quality and to compare with the amino
acid requirements of children and adults. Nonprotein amino acids have been reported to be
present in many commonly eaten foods and these compounds have the ability to interfere with a
wide range of fundamental biochemical processes and cause disease (Rozan et al, 2000;
Konzo and cassava toxicity
62
Rubenstein, 2000). Neurolathyrism, which shares clinical similarities with konzo, has been
associated with the overconsumption of grass pea (Lathyrus sativus L., Fabaceae) which contains
a neurotoxic nonprotein amino acid, BOAA or its synonym ODAP (Howlett, 1994; Tylleskär et
al, 1994c; Getahun et al, 1999). Therefore the presence of any inherent potentially toxic
nonprotein amino acid in cossette samples is also examined.
III.1.2 Materials and methods
III.1.2.1 Plant Materials
Cossettes were purchased in five different markets (Ngaba, Lemba, Livulu, Rond Point and
Matete) of Kinshasa, capital of D. R. C. The cossettes in those markets are supplied by Bandundu
province where konzo has been reported and depending on the size, they are sold in bulk of about
10 pieces of roots. About 500g (2 or 3 pieces) of the cossettes from each market were finely
ground with an electric small laboratory grinder "Culatti" with 200 µm sieve prior to sampling
and analyses.
Cassava flour from Cameroon was purchased from an exotic food shop in Antwerp, Belgium for
comparison. Cameroon is a part of central Africa where cassava is processed like in D. R. C.
III.1.2.2 Determination of Cyanogens
A simple picrate paper kit developed by Egan et al (1997) and improved by Bradbury et al (1999)
was used for the determination of all forms of cyanogens in cassava products. Protocol B1 was
followed for the determination of total cyanogens and acetone cyanohydrin + HCN/ CN-.
Konzo and cassava toxicity
63
III.1.2.2.1 Total Cyanogens
100 mg of sample was placed on top of 21 mm diameter Whatman 3 MM filter paper disc
containing 1 M phosphate buffer at pH 8 and linamarase in a flat bottomed plastic bottle
(supplied in the kit). Millipore filtered deionised water (0.5 mL) was added and a yellow picrate
paper attached to a plastic strip was immediately inserted into the vial that was closed
immediately with a screw lid and allowed to stand at room temperature for 24 h. The plastic
backing sheet was removed carefully from the picrate paper. This latter paper was immersed in
5.0 ml of deionised water for about 30 min. The absorbance of the solution was measured at 510
nm, using cuvettes of 1 cm light path against a blank, which contained a yellow solution
produced by a picrate paper not exposed to HCN/ CN-.
The total cyanogens content (expressed in ppm) was calculated by the simple equation:
Total cyanogens content = 396 x Absorbance
Other samples were prepared as above but without cassava flour, using square linamarin papers
equivalent to 50 and 400 ppm, to serve as controls.
III.1.2.2.2 Acetone Cyanohydrin + HCN/ CN-
This analysis was done following the above procedure. However 200 mg of guanidine
hydrochloride was added after the addition of the phosphate buffer pH 8 filter paper disc. The
incubation time was 3 h.
The amount of linamarin was calculated through the following equation:
Linamarin content = Total cyanogens - (acetone cyanohydrin + HCN/ CN-)
Konzo and cassava toxicity
64
III.1.2.3 Determination of Amino acids
An HPLC gradient system with precolumn phenylisothiocyanate (PITC) derivatisation (Khan et
al, 1994) was used to analyse free amino acids. Total protein amino acids were determined after
sample hydrolysis.
III.1.2.3.1 Extraction of free amino acids
50 μL of DL- Allylglycine (100 nmol/ ml) was added to finely ground sample (5 g) as internal
standard. The samples were then extracted in 3 volumes of 70 % ethanol and stored overnight at
4o C. The extracts were centrifuged (34800g, 20 min) and the pellets were washed twice with 70
% ethanol. The supernatants were pooled and concentrated under vacuum and stored in a deep
freezer at - 20 o C.
III.1.2.3.2 Sample hydrolysis
The flour sample was hydrolysed under vacuum in 6 M HCl following the AOAC 982.30 E
procedure (18).
III.1.2.3.3 Amino acid analysis
Aliquots of extract or hydrolysate were concentrated and dried under vacuum (37 o C, 20 mm Hg)
then a coupling reagent (methanol: water: triethylamine; 2:2:1; v/v) was added, mixed and dried
immediately under vacuum during 10 min. After this, PITC reagent (methanol: triethylamine:
water: PITC; 7:1:1:1; v/v) was added and allowed to stand at room temperature for 20 min before
drying under vacuum. PITC derivatives were dissolved in buffer A (0.1 M ammonium acetate,
pH 6.5) and filtered through a 0.22 μm Millipore membrane.
Konzo and cassava toxicity
65
20 μl of sample was injected into an HPLC (Waters model 991 equipped with photodiode array
detector) using a gradient system of buffer A (100 - 0 %) and buffer B (0.1 M ammonium acetate
containing acetonitrile and methanol, 44:46:10; v/v, pH 6.5) (0 - 100 % after 50 min). The
operating temperature was 43 o C. A reverse phase column from Alltech (Alltima C 18, 5 μm,
250 x 4.6 mm) was used. The absorbance at 254 nm was recorded and used for calculations. A
standard protein amino acid mixture (food hydrolysate A 9656, Sigma) was derivatised as above
and used as standard for calculations. The results were analysed by Millennium software (Waters,
version 1.10)
III.1.2.3.4 Tryptophan Determination
A rapid and simple acid ninhydrin method described by Gaitonde and Dovey (1970) and adapted
for colorimetric determination of tryptophan by Sodek et al (1975) was used. Cossettes samples
were partially defatted by suspension in 20 volumes of acetone and stirring occasionally for 30’.
After filtration, the powder was left to air-dry. Portions (500 mg) of defatted cassava cossettes
were extracted in a centrifuge tube with 2.0 ml of 70 % ethanol for 30’ at room temperature. The
mixture was occasionally stirred and homogenized with a glass rod. 5 mL of NaOH (0.5 %) were
then added and extraction continued for another 1 h. After centrifugation, a clear supernatant was
collected and 0.2 ml of it was taken for tryptophan assay. The acid ninhydrin method using
reagent b (250 mg of ninhydrin dissolved in 10 ml of formic acid- hydrochloric acid; 3:2; v/v)
was followed for the determination of tryptophan in the samples. Readings were made against a
reagent blank in a spectrophotometer (Shidmazu, UV-1601) at 390 nm using cuvettes of 1 cm
light path. Sample blanks contained a similar aliquot of extract together with reagent b without
ninhydrin.
Konzo and cassava toxicity
66
After subtracting the absorbance value of the sample blank, the tryptophan content was read off a
standard curve. Lysozyme (Grade I from egg white; Sigma Chemical Co.) was used to construct
the standard curve. Tryptophan values obtained from this graph were then corrected for tyrosine
interference according to Zahnley and Davis (1973).
III.1.2.4 Statistics
The results were computed and compared by analysis of variance using the software package
SPSS 9.0 for Windows. Significant differences amongst means were confirmed using the Tukey
Honestly Significant Differences set at 95 % confidence interval (P < 0.05). Data are expressed
as means ± standard deviation.
III.1.3 Results and discussion
III.1.3.1 Cyanogens
The six samples of cassava cossettes had residual cyanogens below 10 mg HCN equivalent kg-1
as shown in Table III-1. This is the recommended safe limit by the Codex alimentarius (FAO/
WHO, 1991). The highest level was found in samples from Cameroon (9.37 mg HCN equivalent
kg-1) and the lowest level in samples from Rond Point (1.45 mg HCN equivalent kg-1), showing a
6.5 fold variation with a significant difference between samples from Cameroon and all other
samples (P < 0.05). No significant differences was found between samples from Matete and
Ngaba, and among samples from Rond Point, Livulu and Lemba but those last samples were
significantly different from those from Matete and Ngaba (P < 0.05).
Enzymatic determination of the cyanogenic glycoside linamarin, the major source of cyanide in
cassava, showed a variation of almost ten fold between 0.924 and 8.58 mg HCN equivalent kg-1.
Konzo and cassava toxicity
67
Table III-1: Cyanogens content in cassava cossettes (mg HCN equivalent kg - 1 dry weight)*
Cossettes Total Cyanogens Acetone Cyanohydrin + HCN/
CN-
Linamarin
Matete
(N = 3)
2.772 b ± 0.396 0.264 a ±0.280 2.508 c ±0.457
Cameroon
(N = 3)
9.372 c ± 0.229 0.792 a ± 0.280 8.580 d ± 0.229
Lemba
(N = 3)
1.716 a ± 0.229 0.396 a ± 0.457 1.320 a, b ± 0.229
RondPoint
(N = 3)
1.452 a ± 0.229 0.528 a ± 0.229 0.924 a ± 0.229
Livulu
(N = 3)
1.584 a ± 0.396 0.132 a ± 0.229 1.452 a, b, c± 0.229
Ngaba
(N = 3)
2.904 b ± 0.457 0.792 a ± 0.280 2.112 b, c ± 0.243
Again the samples from Cameroon were significantly higher than all other samples (P < 0.05)
while the linamarin content of Rond Point was significantly different from Ngaba and Matete,
and also Lemba was different from Matete (P < 0.05). No significant difference was found for the
content of acetone cyanohydrin + HCN/ CN - between samples (P > 0.05), this varied 6 fold
between 0.13 and 0.79 mg HCN kg-1 in the cassava cossettes examined.
The fresh bitter cassava roots typically used in the region have total cyanogen levels of 100 to
500 mg HCN equivalent kg-1 root, even up to 1500 mg HCN kg-1 (Bradbury and Holloway, 1988;
O’Brien et al, 1992; Padmaja, 1996). Although the original content of the fresh roots from which
the cossettes were prepared is not known, it is obvious that the processing and handling of the
material resulted in a reduction of total cyanogen of at least 10-30 fold, up to 150-500 fold,
* Values are means ± standard deviation a, b, c same superscript within a column means no significant difference (P> 0.05)
Konzo and cassava toxicity
68
giving a final result within the recommended safe limit set at 10 mg HCN equivalent per kg of
dry weight. The processing and handling included soaking, sundrying, storage and transportation
to the open markets in Kinshasa where the cossettes are sold in jute sack or in bulk. The samples
from Cameroon bought in Europe were packed in a plastic foil of low permeability.
The low levels of glycosides in the flour from cossettes can be explained by continued cell
desintegration and enzymatic activity of the linamarase from the cytoplasm hydrolysing the
cyanogens from the disrupted vacuoles during soaking and throughout the four days of drying
before moisture fell to low levels in these big root pieces (Banea-Mayambu, 1997). When
considering time and temperature factor, it can be assumed that even in short soaked cossettes
cyanohydrins might be lost during storage and transportation over much longer distances from
Bandundu to the markets in Kinshasa than when consumed locally in Bandundu area. The low
cyanogen exposure from cassava might explain the absence of cases of konzo in urban
consumers, while the crippling disease konzo is prevalent in remote rural areas of Bandundu
Province (Minten and Kyle, 1999; Formunyam et al, 1985; Banea-Mayambu et al, 1998). Even
the shortcut processed cassava products from Bandundu area sold in Kinshasa do not cause
clinical symptoms of cyanide exposure (Banea-Mayambu et al, 1998).
Oke (1968) reported HCN contents of 1.0 mg/ 100 g in cossettes from D. R. C. and O'Brien et al
(1992) found a variation in cyanogens content of fermented cassava roots ranging between 0 to
11.3 mg kg-1 in villages of Cameroon. In populations with cassava roots as their main staple food,
a basic daily energy need of 6276 kJ (1500 kcal) can be satisfied with 500 g dry weight cassava
root products. Adult consumers would then be exposed to approximately less than 5 mg HCN
equivalent per day comparing to the Codex alimentarius safe level of 10 mg HCN equivalent per
kg dry weight (Rosling, 1988). If cossettes as staple food provide 60 % of dietary daily energy
Konzo and cassava toxicity
69
intake in D. R. C., calculated from the FAO/ WHO energy requirements (FAO/WHO/UNU,
1985), it means that for the samples from Matete about 0.7 mg HCN equivalent is present in 241
g of cassava cossettes to be consumed daily by children (1 to 3 years old) and about 1.5 mg HCN
equivalent is present in 532 g of cassava cossettes to be consumed daily by a moderately active
adult man to meet energy requirements of 3414 (816) and 7531 (1800) kJ (kcal) respectively
(Table III-2).
Table III-2: Estimated daily cossettes and total cyanogens intake
Daily cyanogens from
cossettes‡ (mg)
Subjects Daily
energy
required*
in kJ (kcal)
60 % daily
energy
required in
kJ (kcal)
Daily
cossettes
intake† (g) Matete
samples
Livulu
samples
Children 1 – 3 yr
Children 7 – 9 yr
Adult female
(moderately
active)
Adult male
(moderately
active)
5690
(1360)
9162
(2190)
9204
(2200)
12552
(3000)
3414
(816)
5497
(1314)
5523
(1320)
7531
(1800)
241
389
390
532
0.7
1.1
1.1
1.5
0.4
0.6
0.6
0.8
* from FAO/ WHO/ UNU (1985) † 100 g cassava provides 338 kcal (FAO/WHO/UNU, 1985) ‡ Total cyanogens (Table III-1) x daily cossettes intake (Table III-2)
Konzo and cassava toxicity
70
III.1.3.2 Total protein amino acids
Table III-3 represents the total protein amino acids profiles in cassava cossette samples. The
overall average of the total protein amino acids is 23.7 mg/ g dry weight cassava cossettes in
which essential amino acids represent 54.6 % and the sulphur containing amino acids 9.7 %.
Alanine was the major protein amino acid in all samples except in the samples from Rond Point
where glutamic acid was the most important. This finding is in agreement with some studies done
with fresh cassava roots (Bradbury and Holloway, 1988; Firmin and Kamenan, 1996; Glew et al,
1997). This suggests that during the post-harvest processing practised, the loss of protein is
negligeable while the loss of cyanogens is considerable.
The samples from Livulu had the highest total protein amino acid content (27.3 mg/ g of dry
weight cassava cossettes) with 54.2 % of essential amino acids. The samples from Lemba
contained the highest essential amino acids proportion (63.5 %) and those from Rond Point were
the lowest (50.4 %). Leucine and lysine, the purely ketogenic amino acids, were the limiting
amino acids in our samples (Table III-4). Leucine was the first limiting amino acid in the samples
from Livulu and Ngaba with an amino acid score of 0.36 and 0.45 respectively. Lysine was the
first limiting amino acid in the other samples with an amino acid score varying from 0.35 to 0.47.
Results of Firmin and Kamenan (1996) showed sulphur amino acids (methionine + cysteine) in
fresh cassava roots and leucine in fermented pulp of cassava roots as first limiting amino acid,
respectively. Yeoh and Truong (1996) found sulphur amino acids, leucine and lysine to be
limiting amino acid in different cultivars of cassava roots studied. Bradbury and Holloway (1988)
reported large differences in amino acid composition between different cultivars of cassava roots
examined and there was no essential amino acid, which was clearly the first limiting amino acid.
Nevertheless, on the average histidine was the first and leucine the second limiting amino acid.
Konzo and cassava toxicity
71
Although sulphur amino acids were not the limiting amino acids in our samples, we should notice
that the proportion of methionine represented in average only 13 % (4.7 - 16.2 %) and cysteine 87
% (83.8 - 95.3 %) of total sulphur amino acids. Methionine normally supplies part of the body's
needs for cysteine. With cysteine-free diets, methionine can supply all of the body's needs for
cysteine. Cysteine can spare methionine and a certain proportion of dietary methionine is
converted to cysteine (Brody, 1994).
III.1.3.3 Free amino acids
The free amino acid pattern of cossettes samples is summarised in Table III-5. The
concentrations of free amino acids were in general very low. Arginine and sulphur amino acids
(methionine and cysteine) were not found. Histidine was not found in in the samples from
Matete, Rond Point and Ngaba. No asparagine was detected in the samples fromMatete and
Livulu.
The samples from Livulu showed the highest amount in total free amino acids (6.2 mg/ g of dry
weight cossettes) and those from Rond Point, the lowest (0.27 mg/ g of dry weight cossettes).
This represents about 23 fold variation among the few samples examined; duration and flow rate
of water during soaking leading to leaching out can probably explain this finding. Threonine was
quantitatively the most important free amino acid in five of the samples examined, while in the
samples from Rond Point sample asparagine was the most abundant (Figure III-2). Alanine
ranked the second place except in the samples from Livulu, Rond Point and Cameroon. No
known potentially toxic nonprotein amino acids were detected in our samples.
Konzo and cassava toxicity
72
Table III-3: Total protein amino acids content in cassava cossettes (mg g - 1dry weight)*
Cossettes Total
protein
amino acids
Matete
(N = 4)
Cameroon
(N = 4)
Lemba
(N = 4)
RondPoint
(N = 4)
Livulu
(N = 3)
Ngaba
(N = 4)
Asp 00.200a ± 0.022 00.234 a ± 0.044 00.351 a b ± 0.074 00.480 b c ± 0.120 00.403 b ± 0.049 00.564 c ± 0.041
Glu 1.074a ± 0.257 1.255 a ± 0.079 1.343 a ± 0.209 22.016 b ± 0.186 1.702 a b ± 0.589 22.270 b ± 0.291
Ser 00.700a ± 0.157 00.688 a ± 0.058 00.749 a ± 0.112 1.053 b ±0.023 00.970 a b ± 0.272 00.691 a ± 0.066
Gly 1.342 a ± 0.175 1.254 a ± 0.231` 1.423 a b ± 0.164 1.921 b ± 0.173 1.388 a ± 0.413 1.217 a ± 0.111
His 1.218 a b ± 0.177 1.744 b ± 0.140 1.082 a ± 0.038 1.203 a ± 0.113 1.111 a ± 0.580 1.060 a ± 0.121
Arg 00.796 a ± 0.073 00.715 a ± 0.024 00.744 a ± 0.085 1.029 b ± 0.071 1.034 b ± 0.089 00.796 a ± 0.092
Thr 1.787a b ± 0.638 22.239 a b c ± 0.71 3.177 b c ± 0.807 00.945 a ± 0.335 3.508 c ± 0.907 3.493 c ± 0.594
Ala 5.124 c ± 0.869 44.309 b c ± 0.675 3.349 b ± 0.334 00.962 a ± 0.059 5.329 c ± 0.968 5.628 c ± 0.316
Pro 1.494 a b ± 0.072 1.345 a ± 0.154 1.664a b ± 0.269 1.930 b ± 0.264 1.679 a b ± 0.269 1.498 a b ± 0.110
Tyr 1.410 a ± 0.064 1.272 a ± 0.190 1.035 a ± 0.506 1.343 a ± 0.186 1.035 a ± 0.506 1.191 a ± 0.346
Val 00.961 a ± 0.287 1.153 a ± 0.485 3.306 b ± 0.363 1.104 a ± 0.449 1.733 a ± 0.783 1.374 a ± 0.343
Met 00.162 a ± 0.028 00.108 a ± 0.029 00.399 a ± 0.079 00.372 a ± 0.110 00.302 a ± 0.368 00.398 a ± 0.243
Cys 1.828 a ± 0.070 22.183 a ± 0.231 22.157 a ± 0.168 1.975 a ± 0.440 1.853 a ± 0.250 22.061 a ± 0.999
Ile 00.740 a b ± 0.148 00.554 a ± 0.237 1.604 b± 0.556 00.609 a ± 0.303 00.612 a ±0.241 00.914 a b ± 0.539
Leu 00.660 a ± 0.108 00.648 a ± 0.261 1.717 b ± 0.692 00.699 a ± 0.527 00.652 a ± 0.112 00.760 a ± 0.321
Phe 00.900a ± 0.104 00.783 a ±0.234 00.842 a ± 0.063 00.708 a ± 0.202 22.314 b ± 0.568 00.523 a ± 0.363
Lys 00.497 a ± 0.123 00.527 a ± 0.092 00.531 a ± 0.256 00.530 a ± 0.108 00.751 a ± 0.124 00.691 a ± 0.164
Try 00.741 a ± 0.004 00.754 a ± 0.022 00.877 a ± 0.188 00.780 a ± 0.026 00.907 a ± 0.018 00.740 a ± 0.012
* Values are means ± standard deviation a, b, c same superscript within a row means no significant difference (P> 0.05)
Konzo and cassava toxicity
73
Table III-4: Amino acid scoring pattern of different cossette samples
FAO/ WHO1* Amino acid Scores2† Essential Amino Acid
(EAA) Children
(2-5 years)
Matete Lemba Livulu Ngaba RondPoint Cameroon
Threonine 34 2.43 3.54 3.78 3.97 1.42 3.05
Cysteine + Methionine 25 3.68 3.88 3.16 3.80 4.80 4.24
Valine 35 1.27 3.58 1.81 1.52 1.61 1.66
Isoleucine 28 1.22 2.17 0.80 1.26 1.11 0.91
Leucine 66 0.46 0.99 0.36 0.45 0.54 0.45
Tyrosine + Phenylalanine 63 1.69 1.14 1.95 1.05 1.66 1.37
Histidine 19 2.97 2.16 2.14 2.16 3.24 4.25
Lysine 58 0.40 0.35 0.47 0.47 0.47 0.37
Tryptophan 11 3.11 3.02 3.02 2.60 3.62 3.17
First limiting Amino Acid Lysine Lysine Leucine Leucine Lysine Lysine
Second limiting Amino Acid Leucine Leucine Lysine Lysine Leucine Leucine
* Recommended amino acid scoring pattern from FAO/ WHO/ UNU (1985) † Amino acid score = mg of amino acid in 1g of test protein per mg of amino acid in 1 g of reference Protein (FAO/WHO, 1991)
Konzo and cassava toxicity
74
Figure III-2: Free amino acids in cassava cossette samples
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Phe Lys Asn Gln Trp
Free amino acids
% o
f tot
al fr
ee a
min
o ac
ids
in d
iffer
ent c
osse
tte s
ampl
es
MateteCameroonLemba RondPoint Livulu Ngaba
Konzo and cassava toxicity
75
Table III-5. Free protein amino acids content in cassava cossettes (mg g - 1 dry weight)*
Cossettes Free protein
amino acids Matete
(N = 4)
Cameroon
(N = 3)
Lemba
(N = 3)
RondPoint
(N = 3)
Livulu
(N = 3)
Ngaba
(N = 3)
Aspartic acid 0.005a ± 0.003 0.012 b ± 0.000 0.030 c ± 0.001 ND 0.051 d ± 0.005 0.044 d ± 0.007
Glutamic acid 0.013a ± 0.000 0.079 d ± 0.003 0.048 c ± 0.001 0.012 a ± 0.001 0.082 d ± 0.006 0.025 b ± 0.001
Serine 0.002a ± 0.000 0.008 a b ± 0.005 0.015 b c ± 0.003 0.003a ±0.002 0.029 d ± 0.001 0.023 c d ± 0.007
Glycine 0.007 a b ± 0.000 0.016 b c ± 0.003` 0.025 c ± 0.003 0.006 a ± 0.003 0.051 d ± 0.002 0.005 a b ± 0.009
Histidine ND∗ 0.017 a ± 0.000 0.048 a ± 0.050 ND 0.245 b ± 0.015 ND
Arginine ND ND ND ND ND ND
Threonine 0.370 a ± 0.119 1.106 b ± 0.056 1.135 b ± 0.212 0.072 a ± 0.005 2.171 c ± 0.344 1.523 b ± 0.264
Alanine 0.046 a ± 0.029 0.151 a b ± 0.016 0.223 c ± 0.047 0.025 a ± 0.000 0.284 b ± 0.116 0.270 b ± 0.065
Proline 0.039 b ± 0.002 0.064 c ± 0.007 0.098 e ± 0.004 0.017 a ± 0.000 0.298 f ± 0.008 0.081 d ± 0.005
Tyrosine 0.014 b ± 0.002 0.031 c ± 0.002 0.077 d ± 0.001 0.004 a ± 0.000 0.145 e ± 0.005 0.087 d ± 0.007
Valine 0.025a b± 0.000 0.043a b ± 0.001 0.076 b c ± 0.043 0.010 a ± 0.001 0.124 c± 0.010 0.056 a b ± 0.000
Methionine ND ND ND ND ND ND
Cysteine ND ND ND ND ND ND
Isoleucine 0.012 b ± 0.000 0.016 b ± 0.001 0.070 d ± 0.005 0.003 a ± 0.002 0.107 e ±0.006 0.027 c ± 0.003
Leucine 0.023 a ± 0.001 0.044 a ± 0.001 0.139 a b ± 0.002 0.007 a ± 0.000 0.541 b ± 0.000 0.081 a b ± 0.003
Phenylalanine 0.015 a b ± 0.000 0.043 a b ±0.000 0.089 c ± 0.004 0.006 a ± 0.004 1.925 d ± 0.060 0.075 b c ± 0.003
Lysine 0.010 b ± 0.000 0.024 c ± 0.002 0.036 d ±0.003 0.005 a ± 0.003 0.112 e ± 0.003 0.018 c ± 0.002
Asparagine ND 0.003 a ± 0.000 0.002 a ± 0.003 0.096 a ± 0.083 ND 0.010 b ± 0.001
Glutamine 0.006 a b ± 0.000 0.024 c d ± 0.000 0.015 b c ± 0.003 0.002 a ± 0.001 0.032 d ± 0.002 0.160 e ± 0.010
Tryptophan 0.027 a b ± 0.000 0.221 c ± 0.011 0.038 a b ± 0.026 0.006 a ± 0.003 0.005 a ± 0.004 0.063 b ± 0.008
* Values are means ± standard deviation a, b, c , d, e same superscript within a row means no significant difference (P> 0.05) ∗ ND: not detected
Konzo and cassava toxicity
76
III.1.3.4 Essential Amino Acid (EAA) requirements and estimated daily
intake
Rose and Wixon (1955) demonstrated the influence of cysteine on the methionine requirement
for an adult man by determining the conditions that supported a zero or slightly positive nitrogen
(N) balance. They observed that cysteine alone without methionine resulted in a negative N
balance. A near zero N balance was observed with a diet containing 0.8 g of methionine, while N
balance was negative with 0.7 g methionine diet. Higher levels of methionine resulted in a
positive N balance. They concluded that oversupply of cysteine could give a positive N balance
with lower intake of methionine, but even then the intake of methionine remains essential. This
statement illustrates the limiting of the ability of cysteine to spare methionine. Although cysteine
can fulfill a large fraction of our requirement for sulphur amino acids, according to Altman and
Dittmer (1974) in the combination cysteine + methionine, 30 - 50 % of total requirement for
adults may be furnished by cysteine and 50 - 70 % furnished by methionine.
The expected daily methionine and sulphur amino acids intake provided by cassava cossettes
consumption, which in the case of D. R. C. represents 60 % of daily energy intake, are compared
with the suggested amino acid patterns requirement (Table III-6). It can be concluded that
children of 1 to 9 years old cannot expect to meet methionine requirement whereas adults can
meet sulphur amino acid requirement. Sulphur amino acids are required for cyanide
detoxification in the human body (Bradbury and Holloway, 1988; Rosling, 1994). A daily supply
of about 1.2 mg of dietary sulfur from S-containing amino acids is needed by the human body to
detoxify 1.0 mg of HCN (Padmaja, 1996). When the body is regularly exposed to cassava
cyanogens the increased synthesis of rhodanese, enzyme responsible for cyanide detoxification in
Konzo and cassava toxicity
77
the human body by forming thiocyanate, makes extra demands on the body's reserves of sulphur
amino acids. If this demand is prolonged as in the regular consumption of cassava root
insufficiently processed, and the diet is inadequate, the synthesis of many proteins vital for bodily
Table III-6. Essential Amino Acid (EAA) requirements and estimated daily intake
EAA suggested patterns of
requirement*
Estimated daily EAA (from cossettes) intake† (mg)
(mg AA/ day) Matete samples Livulu samples
Child Child Child Child Child
> 1yr
Adult
female
Adult
male
1-3 yr 7-9 yr
Adult
female
Adult
male
1-3 yr 7-9yr
Adult
female
Adult
male
Thr 1000 305 500 431 695 697 951 845 1365 1368 1867
Cys + Met - 550 1100 - - 776 1059 - - 840 1146
Met 800 - - 39 63 - - 73 117 - -
Val 900 650 800 232 374 375 511 418 674 676 922
Ile 1000 450 700 178 288 289 394 147 238 239 326
Leu 1500 620 1100 159 257 257 351 157 254 254 347
Tyr+ Phe - 1120 1100 - - 901 1229 - - 1306 1782
Phe 800 - - 217 350 - - 558 900 - -
Lys 1600 500 800 120 193 194 264 181 292 293 400
Trp 250 157 250 179 288 289 394 219 353 354 483
functions may be impaired and lead to the development of protein deficiencies and other diseases
(Padmaja, 1996; Onwuka et al, 1992; Tor-Agbidye et al, 1998). Other food components of the
diet should contribute to a better balanced amino acid composition of the diet, especially the level
of sulphur amino acids. In the case of lathyrism, a neurodegenerative disease with similar clinical
* From Altman and Dittmer (1974) † Daily cossettes intake (Table III-2) x Amino acid (Table III-3)
Konzo and cassava toxicity
78
symptoms as konzo, Lambein et al (2001) have suggested that the ratio of cereals (rich in
methionine) to Lathyrus seeds (rich in lysine and low in sulphur amino acid) may be a
determining factor in the etiology. In the regions neighbouring the konzo-affected areas in
Bandundu where traditionally corn or millet flour is mixed with cassava as staple food, no cases
of konzo have been reported. This may corroborate our views as to the importance of methionine
for a healthy balanced diet.
Hence, the recommended daily methionine allowance should be reconsidered and given
separately from total S-amino acid requirement.
III.1.3.5 Conclusion
The processed cassava roots available on the markets in Kinshasa have cyanogens content within
the safe limit recommended by FAO/ WHO. Proper processing, time and storage conditions and
traditional transport in jute sacks appear to contribute to reduce residual cyanogens in the
cossettes whereas insufficient processing and transport in airtight wrapping which prevents the
release of cyanide can probably explain the level of cyanogen found in the cossettes from
Cameroon samples.
No potentially toxic nonprotein amino acids were detected in this study.
The dietary requirements for sulphur amino acids need to be adjusted for the loss caused by
cyanide detoxification. The total sulphur amino acids availability does not give a correct value for
the requirement of the essential amino acid methionine. In the case when cassava is taken as
staple food, the low methionine content may aggravate the risk for cyanide toxicity and konzo
disease, even when the cysteine present covers the dietary requirement for sulphur amino acids.
Konzo and cassava toxicity
79
III.2 Residual cyanogens, free and total amino acid profiles of cooked
cassava leaves "saka- saka” *†
III.2.1 Introduction
In the konzo-affected areas of D. R. C., processed cassava roots are prepared as described before
(chapter III-1), while cassava leaves or "saka-saka" are prepared as follows: the hard petioles are
removed, the tender leaves and the shoots are selected and may be blanched in warm/ boiled water
for a few minutes or partially dried on a pan or a pot over fire and then squeezed to remove liquid
before pounding. The spinach-like mass obtained after pounding with a traditional wooden mortar
and pestle is then cooked with some water added. Usually palm oil and salt are added and
sometimes also traditional spices and onion. (CEPLANUT, 1988; Almazan and Theberge, 1989;
Hahn, 1989).
Reports on the nutritional quality of cassava leaf protein as food are scanty and conflicting
(Bokanga, 1994). The majority of studies considered cassava leaf as animal feed and focused
mainly on cyanogen removal. Residual cyanogens and the presence of inherent potentially toxic
nonprotein amino acids were examined in this study before and after cooking pounded cassava
leaves to check their safety. The aim of this paper is also to assess the amino acid profiles and the
protein quality of cooked pounded cassava leaves as food, which is the most common daily side
dish as sauce and as main source of protein in a diet consisting of processed cassava roots as the
exclusive staple food in konzo affected areas of DRC, especially in Bandundu province.
* This sub-chapter has been published as:
Delphin Diasolua Ngudi, Yu – Haey Kuo and Fernand Lambein (2003). Cassava cyanogens and free amino acids in raw and cooked leaves. Food and Chemical Toxicology 41, 1193 – 1197.
† Delphin Diasolua Ngudi, Hu – Haey Kuo and Fernand Lambein (2003). Amino acid profiles and protein quality of cooked cassava leaves or “saka saka”. Journal of the Science of Food and Agriculture 83, 529 – 534.
Konzo and cassava toxicity
80
III.2.2 Materials and methods
III.2.2.1 Sample acquisition
Deep-frozen pounded raw cassava leaves from D. R. C., about 500 to 600 g packed in plastic foil,
were purchased in five different exotic food shops in Ghent, Belgium (Dampoort, Foreign and
Ghana) and in Paris, France (Congo and Chateau).
III.2.2.2 Sample handling and culinary processing
Each packet of raw sample was divided into two parts. One part was kept as such for analysis and
the other part was subjected to the following culinary treatment on a hot plate:
About 250 ml of water was added to 100 g of raw pounded cassava leaves and allowed to boil. 10
ml of palm oil and about 1 g of salt were added when boiling started and the dish was stirred with
a wooden spoon for mixing of the ingredients. The cooked pounded cassava leaves or "saka-
saka" were ready to eat after 30 minutes of boiling (CEPLANUT, 1988). The samples were
analysed after cooling down to room temperature.
III.2.2.3 Determination of cyanogens
See section III.1.2.2
III.2.2.4 Determination of protein
The samples were extracted in 3 vol of physiological solution (NaCl 0.15 M; pH 5.96 at room
temperature) and stored overnight at 4 oC. The extracts were centrifuged (34800g, 20 min) and
the pellets were washed twice with physiological solution. The supernatants were pooled and
used for protein analyses.
Konzo and cassava toxicity
81
The Bio-Rad® Protein assay kit, consisting of dye reagent concentrate and lyophilised bovine
albumin as protein standard was used to determine protein content in our samples. A standard
curve was made using several dilutions of protein standard containing 0.2 to 1.4 mg ml –1.
Analyses of protein were done as follows: 0.1 ml of sample was placed in a test tube and then 5
ml of diluted dye reagent was added and mixed several times. The absorbance was measured at
595 nm versus reagent blank within a period of 5 minutes to one hour after mixing. The
absorbance was converted to protein content using the standard curve.
III.2.2.5 Determination of amino acids
See section III.1.2.3
III.2.2.6 Tryptophan determination
See sub-section III.1.2.3.4
III.2.2.7 Protein quality evaluation
The amino acid scoring pattern recommended by FAO/WHO/UNU (1985) was used for the
evaluation of dietary protein quality as follows:
Amino acid score = mg of amino acid in 1 g of test protein 18 mg of amino acid in 1g of reference protein
The essential amino acid showing a score less than 1 was a limiting amino acid. The lowest
amino acid score (the most limiting amino acid) indicates the quality of the protein.
Konzo and cassava toxicity
82
III.2.2.8 Statistics
The software package SPSS 10.0 for windows was used for the analysis of variance of the data.
The statistically significant differences among means were confirmed using the Tukey Honestly
significant differences at 95 % confidence interval (P<0.05).
III.2.3 Results and discussion
III.2.3.1 Total cyanogens
The total cyanogens content of the raw and the cooked cassava leaves samples are summarised in
Table III-9. The initial levels of total cyanogens in the raw (pounded) cassava leaves samples
ranged from 35.9 ± 0.4 to 107.5 ± 0.8 mg HCN equivalent kg–1, acetone cyanohydrin + HCN/
CN- from 5.7 ± 1.9 to 24.1 ± 4.5 mg HCN equivalent kg–1 and the linamarin from 30.2 ± 2.4 to
83.4 ±5.3 mg HCN equivalent kg–1 dry weight. Those values are up to 10- fold higher than what
we detected in the processed cassava roots (Chapter III-1). A Significant reduction (P< 0.05) in
total cyanogens was observed when the raw samples were cooked; 96 - 99 % of the total
cyanogens were removed after cooking the cassava leaves. Bokanga (1994) observed that
pounding alone reduced the cyanogenic potential by about 60 - 70 %.
After cooking, the total cyanogens varied from 0.30 ± 0.04 to 1.9 ± 0.2 mg HCN equivalent kg-1
dry weight and the acetone cyanohydrin + HCN/CN- were not detected. The residual cyanogens
were below the recommended safe limit set at 10 mg HCN equivalent kg-1 by the Codex
alimentarius (FAO/ WHO, 1991).
Although the original content and the varieties of the fresh leaves from which the samples were
pounded are not known, this decrease can be explained by the following considerations. Besides
the genetic differences of the plant varieties, the variation in cyanogen content between samples
Konzo and cassava toxicity
83
can also be explained by the maturity of the leaves. Padmaja (1989) reported lower contents of
cyanide in the older leaves compared with young leaves. Cyanogen content may also depend on
the heat treatment during preparation of the leaves before pounding. The leaves may be washed
with tap water or blanched in warm water for a few minutes or partially dried over fire or
grinding before pounding (Almazan and Theberge, 1989). The heat treatment can be favorable
for cyanide reduction or can destroy the endogenous hydrolysing enzyme linamarase. Finally, the
consistency of pounding can also play a role in the reduction of cyanogens during pounding of
cassava leaves. Destruction of the cells leads to contact between the cyanogenic glucosides and
the endogenous enzyme with subsequent release of HCN. Our finding is in agreement with the
fact that the rapid removal of cyanogens from cassava can be attributed to the heat applied during
boiling which accelerates the evaporation of HCN and cyanohydrin produced by the linamarin
hydrolysis (Almazan and Theberge, 1989; Essers, 1989; Bokanga, 1994).
Konzo and cassava toxicity
84
Table III-7: Cyanogen content in raw and cooked cassava leaves (mg HCN equivalent kg-1 dry weight)
Dampoort Foreign Congo Chateau Ghana
Raw
(n=4)
Cooked
(n=4)
Raw
(n=4)
Cooked
(n=4)
Raw
(n=4)
Cooked
(n=4)
Raw
(n=4)
Cooked
(n=4)
Raw
(n=4)
Cooked
(n=4)
Total
cyanogens
Acetone
cyanhydrin
+HCN/CN-
Linamarin
35.9b±0.4
5.7ab±1.9
30.2b±2.4
1.3a±0.3
NDb
1.3a±0.3
107.5d±0.8
24.2d±4.5
83.4d±5.3
0.8a±1.1
ND
0.8a±1.1
87.9c±4.5
15.3c±0.3
72.7c±4.2
0.30a±0.04
ND
0.30a±0.04
83.7c±2.4
10.5bc±2.5
73.2c±4.9
0.7a±0.1
ND
00.7a±0.1
86.1c±3.1
15.0c±3.9
71.1c±0.8
1.9a±0.2
ND
1.9a±0.2
Same letter within a row means no significant difference (P>0.05)
a Values are means ± standard deviation
b Not detected.
Konzo and cassava toxicity
85
III.2.3.2 Total protein and amino acid profiles
The total protein content and the amino acids composition (g kg-1 dry weight) of five different
samples of cassava leaves before and after cooking are listed in Table III-7. The total protein
content of the raw pounded cassava leaves samples ranged from 235.8 g in the Foreign sample to
351.8 g in the Dampoort sample. Those values are within the ranges reported in the literature
(Almazan and Theberge, 1989; Hahn, 1989; Bokanga, 1994; Yeoh and Chew, 1976; Ravindran
and Ravindran, 1988). Ravindran and Ravindran (1988) observed a decrease of protein content in
cassava leaves with ageing: from 381 g kg-1 in very young cassava leaves to 286 g kg-1 in young
leaves and 197 g kg-1 in mature leaves. The total protein content in the cooked samples ranged
from 111.8 g kg-1 dry weight in the Chateau sample to 144.6 g kg-1 dry weight in the Dampoort
sample. The results showed a significant (P < 0.05) decrease by an average of 58% in protein
content after cooking pounded cassava leaves. The large volume of water added and the
prolonged cooking time (at least 30 min of boiling) necessary for this culinary treatment to
remove the bitter taste, which might lead to losses of amino acids by diffusion and by thermal
degradation, can explain this decrease (Clemente and al, 1998; De la Cruz, 1999). Cooking of
green beans in a covered pot or pressure cooker was observed to cause important losses of amino
acids compared to the raw sample (De la Cruz et al, 1999). Other authors also observed a
significant reduction of amino acids in chickpea seeds after cooking with distilled water (Attia et
al, 1994; Clemente et al, 1998). During preparation of food, the side chains of some protein-
bound amino acids can react chemically with each other or with other molecules present in the
food and those reactions can result in a reduction of nutritive value (Sherr et al, 1989).
Konzo and cassava toxicity
86
Table III-8: Protein content and amino acid composition of raw and cooked pounded cassava leaves (g kg-1 dry weight)*
Amino acids Foreign (n= 5) Chateau (n= 5) Congo (n= 4) Dampoort (n= 5) Ghana (n= 4)
Raw Cooked Raw cooked Raw Cooked Raw cooked Raw cooked
Aspartic acid 40.8c±0.1 12.1a±1.0 33.9b±0.4 17.0a±2.5 33.9b±0.4 16.3a±3.9 37.1bc±3.2 16.0a±2.6 38.6bc±2.8 13.6a±0.4
Glutamic Acid 36.8c±0.3 12.8a±2.4 36.2c±0.7 16.7ab±3.2 37.9c±4.1 16.9ab±4.0 44.4d±4.1 19.6b±3.0 41.1cd±0.2 15.5ab±0.6
Serine 11.8b±2.1 3.9a±1.0 12.9bc±2.3 5.3a±1.6 15.1bc±2.5 4.6a±0.7 16.8c±1.7 6.0a±0.3 15.0bc±0.3 4.7a ±1.1
Glycine 11.0c±0.2 5.2a±1.2 11.5cd±0.5 5.9a±0.8 13.7de±1.3 5.5a±1.0 18.0f±1.7 8.2b±1.1 13.9e±0.5 6.3ab±0.3
Histidine 5.4b±0.9 1.2a±0.2 6.7cd±0.5 1.3a±0.7 6.6bc±0.6 1.6a±0.2 7.9de±0.7 2.0a±0.2 8.1e±0.8 1.6a±0.3
Arginine 16.8b±0.6 8.5a±1.2 16.1b±0.5 9.3a±1.8 18.4bc±2.2 7.9a±1.0 24.0d±2.4 10.6a±1.5 21.4cd±0.8 8.8a±0.6
Threonine 8.8c±0.6 4.2a±0.6 9.6c±0.4 4.7ab±1.1 11.7d±1.1 4.4ab±0.1 17.5e±0.7 6.3b±0.6 12.8d±0.6 4.5ab±0.3
Alanine 21.7bc±1.9 13.6a±3.4 20.7bc±1.9 13.9a±1.9 25.1cd±3.0 14.4a±1.1 30.9d±4.2 15.9ab±3.7 25.4cd±1.7 15.8ab±3.2
Proline 11.3b±0.4 6.4a±1.3 11.6b±0.5 6.7a±1.0 14.4c ±1.8 6.3a±0.4 17.9d±2.1 8.1a±1.1 14.9c±0.3 6.3a±0.4
Tyrosine 9.4c±0.3 4.3a±0.5 10.1c±0.4 4.7a±0.8 12.1d±1.4 4.9a±0.1 15.4e±1.5 6.6b±0.5 12.2d±0.4 4.3a±0.3
Valine 10.6c±0.2 4.9ab±1.1 11.5cd±0.4 4.8ab±0.9 12.9de±1.5 4.6a±0.5 16.8f±1.4 6.6b±0.4 14.8e±1.0 4.7ab±0.2
Methionine 3.2c ±0.4 0.3a±0.1 3.1c±0.5 ND† 3.4c±0.2 0.8ab±0.3 4.7d±0.6 1.5b±0.4 2.8c±0.4 0.2a±0.2
Cysteine ND ND ND ND ND ND ND ND ND ND
Isoleucine 7.5b±0.4 2.3a±0.9 7.8bc±0.3 2.6a±1.0 9.0bc±1.1 2.5a±0.8 11.8d±1.2 4.1a±0.8 9.8c±0.8 2.5a±1.0
Leucine 14.3c±0.3 6.3a±1.4 15.9cd±0.4 7.1ab±1.1 17.6de±1.8 5.9a±0.7 24.0f±2.3 9.7b±0.3 18.6e±0.9 7.0a±0.2
Phenylalanine 12.4b±1.8 5.4a±1.2 13.4b±0.3 5.9a±2.7 15.0b±2.0 4.3a±0.8 18.7c±1.1 6.9a±0.6 14.9b±0.8 6.0a±1.1
Tryptophan 3.5 c±0.0 1.4a±0.0 3.5 c±0.0 1.4 a±0.1 3.8 d±0.0 1.3 a±0.0 4.1 e±0.2 1.7 b±0.0 3.6 cd±0.1 1.6 b±0.0
Lysine 9.6c±0.9 4.1a±1.1 15.3d±0.7 3.7a±1.3 15.5d±1.1 5.6ab±0.3 20.5de±2.0 7.1bc±0.6 18.3e±2.2 5.1ab±0.7
Total Protein 235.8c±5.6 114.1 a±2.4 256.7 d±7.3 111.8 a±2.4 293.3e±2.7 113.1a±3.2 351.8f±7.3 144.6b±3.5 291.7e±5.3 112.2a±1.1
* Values are means ± standard deviation a,b,c,d,e,f same superscript within a row means no significant difference (P>0.05) † ND= not detected
Konzo and cassava toxicity
87
Aspartic acid, glutamic acid and alanine were the major amino acids found in all the samples
studied. They represented together an overall average of 101 g and 46 g kg-1 dry weight in the
raw samples and in the cooked samples respectively. Histidine, tryptophan and methionine were
the amino acid found in lowest concentration with together an overall average of 14 g and 3.6 g
kg-1 dry weight in the raw and in the cooked samples respectively. Other authors obtained similar
profiles in all the varieties of raw cassava leaf studied (Yeoh and Chew, 1976; Ravindran and
Ravindran, 1998). Cysteine was not detected in any sample.
Figure III-3: Protein amino acids profile of the raw and cooked cassava leaves
Figure III-3 shows the profile of the individual amino acid per total protein, which is almost
similar for all the samples. No marked differences can be observed between the raw and the
cooked samples when considering individual amino acid, except alanine which increased
significantly after cooking and methionine showing the highest decrease after cooking.
0
20
40
60
80
100
120
140
160
Asp Glu Ser G ly His Arg Thr Ala Pro Tyr Val Met Ile Leu Phe Try Lys
am ino acids
g am
ino
acid
per
kg
tota
l pro
tein
Raw
cooked
Konzo and cassava toxicity
88
Methionine is highly required in the konzo-affected areas for dietary cyanide detoxification. The
reactive thioether group in methionine that is involved in oxido-reduction reactions and the
thermal breakdown of methionine can explain this finding (Clemente et al, 1998; De la Cruz et al,
1999). Excessive heat treatment causes considerable nutritional damage to methionine (Geervani
and Theophilus, 1980; Shemer and Perkins, 1975).
A comparison of the total essential amino acid profiles with the FAO/WHO reference pattern
(Table III-8) showed that the raw cassava leaves samples contained 357 g to 401 g of total
essential amino acids per kg of cassava leaves protein. This is higher than the 339 g of total
essential amino acids in the recommended FAO/WHO-reference protein. The cooked samples
contained less total essential amino acids than the FAO/WHO reference ranging from 299.3 g kg-
1 total protein content in the Foreign samples to 330.2 g kg-1 total protein content in the Ghana
samples
Konzo and cassava toxicity
89
Table III-9: Comparison of the essential amino acid contents of different raw and cooked pounded cassava leaves samples and their
amino acid score with the recommended FAO reference
Essential AA FAO
Ref.*
Foreign Chateau Congo Dampoort Ghana
Raw Cooked Raw cooked Raw Cooked Raw cooked Raw cooked
Histidine 19 (1.0) 23 † (1.2) 11 (0.6) 26 (1.4) 12 (0.6) 23 (1.3) 14 (0.8) 22 (1.2) 14 (0.7) 28 (1.5) 14 (0.7)
Threonine 34 (1.0) 37 (1.1) 37 (1.1) 37 (1.1) 42 (1.2) 40 (1.2) 40 (1.2) 50 (1.5) 44 (1.3) 44 (1.3) 40 (1.2)
AAA‡ 63 (1.0) 92 (1.5) 85 (1.3) 91 (1.4) 95 (1.5) 92 (1.5) 81 (1.3) 97 (1.5) 94 (1.5) 93 (1.5) 91 (1.4)
Valine 35 (1.0) 44 (1.3) 43 (1.2) 45 (1.3) 43 (1.2) 44(1.3) 41 (1.2) 48 (1.4) 46 (1.3) 51 (1.5) 42 (1.2)
SAA 25 (1.0) 13(0.5) 0.3 (0.01) 12 (0.5) ND§ 12 (0.5) 7 (0.3) 13 (0.5) 10 (0.4) 10 (0.4) 0.2 (0.01)
Isoleucine 28 (1.0) 31 (1.1) 20 (0.7) 30 (1.1) 23 (0.8) 31 (1.1) 22 (0.8) 33 (1.2) 28 (1.0) 34 (1.2) 22 (0.8)
Leucine 66 (1.0) 61 (0.9) 55 (0.8) 62 (0.9) 63 (0.9) 60 (0.9) 52 (0.8) 68 (1.0) 67 (1.0) 64 (1.0) 62 (0.9)
Tryptophan 11(1.0) 15(1.4) 12(1.1) 14 (1.3) 12(1.1) 13(1.2) 12(1.1) 12(1.1) 12(1.1) 12(1.1) 14(1.3)
Lysine 58 (1.0) 41 (0.7) 36 (0.6) 60 (1.0) 33 (0.6) 53 (0.9) 50 (0.9) 58 (1.0) 49 (0.8) 63 (1.1) 45 (0.8)
Total 339 357 299.3 377 323 368 319 401 364 399 330.2
1st limiting amino acid SAA SAA SAA SAA SAA SAA SAA SAA SAA SAA
2nd limiting amino acid
Other limiting amino
acids
Lysine
Leucine
Histidine
Lysine
Isoleucine
Leucine
Leucine Lysine
Histidine
Isoleucine
Leucine
Leucine
Lysine
Histidine
Leucine
Isoleucine
Lysine
Histidine
Lysine
Histidine
Isoleucine
Lysine
Leucine
* FAO-protein reference from FAO/ WHO/ UNU (1985) † Calculated from Table III-3: Essential amino acid per total protein, the results are expressed in g kg -1 of the protein, the amino acid scores are indicated
between brackets ‡ AAA= aromatic amino acids (Phenylalanine +Tyrosine) § ND= not detected
Konzo and cassava toxicity
90
The scoring pattern in Table III-8 showed that sulphur-containing amino acids were the most
limiting amino acid in all the samples (raw and cooked) with an amino acid score varying from
0.5 to less. Other investigators obtained the same results from their raw cassava leaves samples
studied (Yeoh and Chew, 1976; Lancaster, 1983). Besides sulphur amino acids, lysine and
leucine were limiting amino acids in the raw Foreign and Congo samples and leucine in the raw
Chateau samples. The maturity of the cassava leaves (young or mature) used and preliminary
processing (blanching or partial grinding or not) before pounding were unknown and might have
an effect on lysine and leucine. In all the cooked samples except the Chateau sample, histidine
was the second limiting amino acid. Lysine, leucine and isoleucine were also second or third
limiting amino acids in some of the cooked samples.
III.2.3.3 Free amino acids
The free amino acids and trigonelline (N-methyl-nicotinic acid) pattern of the raw and cooked
cassava leaves samples are summarised in Table III-10. The total free amino acids detected and
trigonelline varied from 10.8 g kg-1 to 38.2 g kg-1 in the raw samples and from 7.4 g kg-1 to 25.6 g
kg-1 in the cooked samples. Thus, pounded cassava leaves showed a decrease in the total free
amino acids content after cooking. The highest decrease was observed in the Ghana samples
(45.4 %) followed by the Foreign samples (38.3%), the Dampoort samples (31.3 %), the Congo
samples (23.5%) and the Chateau samples (15 %). The concentration of total free amino acids
including free protein amino acids and free nonprotein amino acids was at least 6-fold higher in
the leaves than what we found in the cassava roots (Chapter III-1). Aspartic acid, glutamic acid
and alanine are the major free protein amino acids found in the samples. Methionine and cysteine
were not detected as free amino acid in any samples. γ-Amino butyric acid (GABA) and α-amino
butyric acid (α- ABA) are the free nonprotein amino acids detected in all the samples.
Konzo and cassava toxicity
91
Table III-10: Free amino acid and trigonelline content in raw and cooked cassava leaves (g kg-1 dry weight)*† Amino acids Dampoort (n= 4) Foreign (n= 4) Congo (n= 4) Chateau (n= 4) Ghana (n= 4)
Raw Cooked Raw cooked Raw Cooked Raw cooked Raw cooked Aspartic acid 1.16b±0.44 0.48a±0.07 4.57e±0.24 2.23 c±0.01 1.34b±0.23 0.94a,b±0.16 3.53d±0.38 3.16d±0.23 1.98c±0.15 1.09 b±0.08 Glutamic Acid 0.21a±0.02 0.38a±0.18 4.44e±0.22 2.1 0 b,c±0.02 2.51c,d±0.13 1.92 b±0.27 5.53f,g±0.21 5.10 f±0.30 5.72f,g±0.39 2.73d±0.24 Serine 0.18a±0.01 0.13a±0.01 1.77e±0.11 1.02c±0.01 0.50 b±0.01 0.43b±0.00 1.75e±.0.08 1.87e±0.19 3.02f±0.16 1.39d±0.02 Glycine 0.28d±0.02 0.13b,c±0.02 0.18c±0.02 0.10a,b±0.00 0.07a,b±0.02 0.05a,b±0.03 0.10a,b±0.00 0.09a,b±0.06 0.52e±0.01 0.26d±0.00 Histidine ND‡ ND 0.87e±0.11 0.51c±0.03 0.19 b±0.01 0.21 b±0.03 0.72 d±0.11 0.87e±0.07 1.07f±0.06 0.53c±0.01 Arginine 0.40a±0.03 0.17a±0.02 2.55 d±0.28 1.52c±0.06 0.25a±0.01 0.26a±0.01 1.14b±0.19 1.32b,c±0.16 2.75d±0.16 1.25b,c±0.00 Threonine ND ND 0.15a±0.09 0.02a±0.00 0.24a±0.00 0.25a±0.02 0.89b±0.19 1.08b±0.19 1.01b±0.03 0.26a±0.26 Alanine 1.64a,b±0.15 1.05a±0.12 1.65a,b±0.10 0.70a±0.01 2.09b±1.47 0.79a±0.09 1.73a,b±0.12 2.59 b,c±1.22 3.67c±0.14 1.54a,b±0.01 Proline 0.51d±0.06 0.41b,c±0.02 0.48c,d±0.04 0.24a±0.00 0.37 b,c±0.06 0.31a,b±0.01 0.72e±0.05 0.64e±0.04 2.46g±0.09 1.01f±0.00 Tyrosine 0.42 b±0.03 0.27a±0.01 0.85c,d±0.09 0.45b±0.00 0.42b±0.06 0.39a,b±0.00 0.97d±0.06 0.89d±0.02 1.46e±0.10 0.72c±0.03 Valine 0.86b±0.10 0.44a±0.01 1.48c±0.08 0.85b±0.04 0.77b±0.05 0.73b±0.01 1.88d±0.08 1.74d±0.10 2.81e±0.06 1.40c±0.03 Methionine ND ND ND ND ND ND ND ND ND ND Cysteine ND ND ND ND ND ND ND ND ND ND Isoleucine 0.44±0.04 0.26±0.00 0.69±0.03 0.35±0.00 0.39±0.38 0.21±0.00 0.53±0.02 0.45±0.02 1.42±0.04 0.68±0.00 Leucine 0.65±0.04 0.37±0.01 0.75±0.04 0.43±0.00 0.24±0.02 0.26±0.00 0.57±0.03 0.51±0.02 2.21±0.08 1.12±0.00 Phenylalanine 0.51±0.05 0.39±0.01 2.09±0.21 1.10±0.01 0.71±0.14 0.67±0.10 1.44±0.33 1.38±0.15 2.39±0.07 1.16±0.00 Tryptophan 0.19±0.022 0.21±0.02 1.46±0.08 0.84±0.02 0.59±0.03 0.57±0.07 1.37±0.25 1.35±0.13 1.67±0.07 1.07±0.01 Lysine 0.28±0.02 0.07±0.00 0.55±0.07 0.45±0.00 0.12±0.01 0.18±0.02 0.33±0.04 0.41±0.07 0.98±0.06 0.67±0.02 Trigonelline 0.81±0.10 0.94±0.13 0.29±0.02 0.41±0.03 0.37±0.03 0.35±0.00 1.33±0.13 0.44±0.05 0.32±0.02 0.49±0.02 Asparagine 0.18±0.02 0.16±0.00 1.49±0.10 2.25±0.02 0.32±0.01 0.27±0.03 3.51±0.20 1.14±0.11 1.39±0.08 1.81±0.00 Glutamine 0.23±0.02 0.39±0.02 0.78±0.05 0.71±0.00 0.22±0.01 0.11±0.01 1.75±0.11 0.44±0.03 0.67±0.03 0.54±0.00 GABA 1.69±0.17 1.07±0.08 0.18±0.05 0.40±0.09 0.05±0.00 0.05±0.00 0.03±0.07 0.01±0.00 0.52±0.03 0.91±0.18 α-ABA 0.19±0.02 0.09±0.01 0.10±0.01 0.20±0.00 0.06±0.00 0.07±0.00 0.28±0.02 0.09±0.00 0.14±0.00 0.20±0.00
* Values are means ± standard deviation, a,b,c,d,e Same superscript within a row means no significant difference (P>0.05) ND= Not detected; GABA = γ-Amino butyric acid; α- ABA= α-Amino butyric acid
Konzo and cassava toxicity
92
GABA is a major constituent in higher plants and its physiological function in the plant is
suggested to be involved in pH regulation, nitrogen storage, plant development and defense, as
well as a compatible osmolyte and an alternative pathway for glutamate utilisation (Shelp et al.,
1999). GABA is a major inhibitory neurotransmitter in mammalian brain and alterations in
GABAergic function have been postulated to underlie seizure pathogenesis (Goldsmith et al.,
1990). Trigonelline, which is not an amino acid but a multifunctional natural plant hormone, was
also found in all the samples. The toxicological effects of trigonelline have not been studied but
considering its multiple effects in the plant, there is a need to study its potential effect on human
health (Rozan et al., 2000).
III.2.4 Conclusions
In konzo affected areas, cassava leaves can contribute to the total uptake of cyanide in the diet
besides the cassava roots. There is no electricity or gas available and in general cooking is done
in the evening when the mothers are tired after hard work and long walking from the field. The
availability of firewood, time to cook and duration of cooking can contribute as factors to higher
dietary exposure to cyanogens in those regions.
All the raw samples had high protein content and high essential amino acids compared to the
recommended FAO/ WHO pattern but limiting in sulphur amino acids, in lysine and leucine.
Cooking lowered the protein content of the raw pounded cassava leaves studied from 285.9 g kg-1
dry weight to 119.2 g kg-1 dry weight on average, but it is still relatively high comparing to other
vegetables. Quantitatively, the cooked cassava leaves can almost fulfil the recommended daily
protein intake (FAO/WHO/UNU, 1985). This can be illustrated by the comparison between the
average daily consumption of cassava leaves in DRC estimated at 500 g, thus an average of about
60 g of protein (from our samples), and the recommended safe level of daily protein intake in
Konzo and cassava toxicity
93
terms of protein qualities which is 48 g or 62 g for food protein quality of score 0.6 for adult
woman and adult man, respectively (Lancaster, 1983; FAO/WHO/UNU, 1985). Unfortunately,
our results showed that the cooked-pounded cassava leaves were deficient in at least 3 essential
amino acids (sulphur amino acids, histidine and lysine) and thus of poor quality (Friedman,
1996). Therefore the consumption of cassava leaves as the main source of dietary protein cannot
compensate the methionine deficiency in konzo-affected areas where the dietary requirement for
methionine needs to be adjusted for the loss caused by cyanide detoxification (Diasolua Ngudi,
2002). This dietary methionine requirement may be further increased if the leaves are also not
properly cooked because of high level of cyanide in the fresh leaves (Lancaster, 1983). Cereals
and legumes should be promoted as sources of sulphur amino acids and lysine respectively to
improve protein quality of the diet of the poor population at risk for konzo and thus to prevent
konzo and malnutrition.
It has been suggested that the deficiency in sulphur amino acids in unbalanced diets could be a
contributing factor in the etiology of neuro-toxico-nutritional diseases such as konzo and
neurolathyrism (Lambein et al, 2001). Neither ODAP nor other known potentially toxic
nonprotein amino acid was detected in our samples. One major peak found in our samples with
elution time of 37.6 min in HPLC analysis and absorption maximum at 265.4 nm after PITC
derivatisation was not identified.
Better information and education especially of those preparing the food could be a relatively
cheap and sustainable intervention. Considering the level of socio-economic impact of such
diseases, such intervention would save resources to those communities.
Konzo and cassava toxicity
94
CHAPTER IV
DIETARY CYANOGEN AND SULPHUR METABOLITES
EXCRETION*
* This chapter has been submitted for publication in Food and Chemical Toxicology as: Delphin Diasolua Ngudi, Yu – Haey Kuo, Fernand Lambein and Patrick Kolsteren. High risk of dietary cyanogen exposure in a population living in a konzo – affected area of Democratic Republic of Congo.
Konzo and cassava toxicity
95
IV Dietary cyanogen and sulphur metabolites excretion
IV.1.1 Introduction
Urinary amino acid excretion is an important tool for the diagnosis and clinical management
of disturbances of amino acid metabolism. Common indications for urine amino acid testing
include clinical presentations such as neurological deterioration, hyperammonemia, kidney
stones, metabolic acidosis, failure to thrive, inborn errors of amino acid metabolism, etc
(Bezkorovainy & Rafelson, 1996, Venta, 2001). The alteration in urinary excretion is
principally a reflection of changes that occur in plasma amino acid composition since the
concentrations of the free amino acid in urine seem to be mainly related to protein intake
(Pavy et al, 1988, Brand et al. 1997).
Urinary thiocyanate is commonly used to check cyanogen overload in a population using
cassava roots and cassava products as staple food (Haque & Bradbury, 1999, Ernesto et al.
2002a). The level of thiocyanate normally present in body fluids is low but increases with
chronic exposure to cyanide and with smoking habits (Vesey et al. 1999, Kussendrager and
Van Hooijdonk, 2000). Thiocyanate remains the most useful chemical biomarker for dietary
cyanogen intake because it is a very stable metabolite that can be determined with relatively
cheap, specific and sensitive methods (Rosling, 1994, Ressler and Tatake, 2001).
Taurine (2-amino-ethyl sulphonic acid) is an ubiquitous free amino acid highly abundant in
excitable tissues, including the heart and brain. In addition to functioning as a
neuroprotectant, antioxidant, osmoregulator and Ca2+ modulator, taurine may function as an
inhibitory neuromodulator and neurotransmitter in the central nervous system. It is an end
product from the catabolism of sulphur amino acids methionine and cysteine, and it is
excreted almost entirely in urine (Laube et al. 2002, Olive, 2002, Hou et al. 2003). The
Konzo and cassava toxicity
96
urinary levels of taurine have been proposed as a potential biochemical marker of total body
protein status or of sulphur amino acids catabolism (Waterfield et al. 1995, Hou et al. 2003).
In this study, we compared the level of total cyanogen in the sampled cassava flour to the
recommended FAO/ WHO safe limit. We measured urinary thiocyanate to check cyanogen
overload in the selected community. The potential relationship between urinary taurine and
urinary thiocyanate, biomarker of daily cyanogen, was assessed and cases of konzo were
detected.
IV.1.2 Material and methods
IV.1.2.1 Subjects
Samples of cassava flour and urine were obtained and examined from about one tenth of the
participants selected randomly in an epidemiological study (Chapter I) we carried out in
February 2003 in Popokabaka rural health zone (Prhz), province of Bandundu (1° - 8° South;
16° – 20° East), Democratic Republic of Congo (D. R. C.). Three health areas were chosen in
cooperation with the chief medical doctor and the nurse supervisor of Prhz based on the
number of reported konzo cases: Popo-secteur (low prevalence area), Mutsanga (medium or
moderate prevalence area) and Masina (high prevalence area). After informed oral consent,
forty two heads of household or their delegates (11 females and 31 males; age 46 ± 12 yr,
range 20 – 76 yrs) among the participants of the above mentioned study were randomly
selected to provide samples. After the interview, each selected participant received two empty
plastic vials; one to fill up with cassava flour of the evening meal and the other with the first
morning urine of the next day. Konzo affected-households were registered and the patients
were checked for confirmation. Filled vials were collected without addition of any
preservative early in the morning of next day. Twelve participants did not return the vial with
cassava flour because they either had no evening meal or did not prepare cassava flour for the
Konzo and cassava toxicity
97
evening. The collected samples were transported from the field to the laboratory at 4° C.
Samples were stored at –20 °C until assayed.
IV.1.2.2 Analytical methods
IV.1.2.2.1 Total cyanogen in cassava flour
See chapter III 1.2.2
IV.1.2.2.2 Urine sample
Urinary thiocyanate
Protocol D1 of the picrate kit method developed by Haque & Bradbury (1999) was used to
determine thiocyanate in the urine. The thiocyanate content in ppm was calculated by the
equation:
thiocyanate content (ppm) = 78 x absorbance
The thiocyanate content in µmol/l was obtained by multiplying the thiocyanate content in
ppm by 17.2. Blank and controls were prepared as described using water instead of urine and
standard paper disc loaded with thiocyanate of 68.8 or 688 µmol/l solution (4 ppm or 40
ppm), respectively. The absorbance was measured at 510 nm, using a spectrophotometer
(Shidmazu, UV 1601).
Urinary taurine
Taurine was analysed by high performance liquid chromatography as described for the other
amino acids in chapter I.
IV.1.2.3 Statistics
The data showed skewed distributions, therefore median and intervals are presented as the
observed ranges of total cyanogens, urinary thiocyanate and taurine. The Spearman’s rho
statistics were used for bivariate correlations to measure the association between two
Konzo and cassava toxicity
98
variables; correlation was significant at the 0.01 level or at the 0.05 level (two-tailed). Results
were computed using Microsoft Windows Excel 2003 and statistical analyses of the data were
carried out using the software package SPSS 11.5 for Windows.
IV.1.3 Results
Out of 42 participants, 21 % were living in a household affected by at least one konzo case
(Table IV-1). No konzo cases were reported in the low prevalence area while in other areas,
13 cases of konzo were reported, from which 9 patients were in the high prevalence area and
4 other patients in the moderate prevalence area. Confirmation of the diagnosis was done by
applying the WHO criteria for konzo (WHO, 1996): a visible symmetric spastic abnormality
when walking and/or running, a history of abrupt onset (< 1 week), a non- progressive course
in a formerly healthy person, showing bilaterally exaggerated knee and/or ankle jerks without
signs of spinal disease.
Table IV-1: Distribution of konzo- affected households in each health area with the number
of konzo patients given in brackets
Number of affected household
(Number of konzo patient)
Health area
1 2 3 Total
Low prevalence
Moderate prevalence
High prevalence
0
2 (2)
4 (4)
0
1 (2)
1 (2)
0
0
1 (3)
0
3 (4)
6 (9)
Total 6 (6) 2 (4) 1(3) 9 (13)
Cyanogen content in cassava flour, thiocyanate and taurine content in the urine samples are
summarised in Table IV-2. Cyanogens were not detected in 26.7 % of samples and 46.7 % of
cassava flour samples had total cyanogens below 10 ppm (µg HCN equivalent/g cassava
Konzo and cassava toxicity
99
flour), the WHO/FAO recommended safe limit (FAO/ WHO, 1991). There is a large variation
in cyanogen content, values ranged from 2.90 to 169.75 ppm with a median of 16.50 ppm.
One sample had cyanogens content above 100 ppm. This highest concentration was found in
the low prevalence area where 60 % of cassava flour samples contained total cyanogens
within the WHO/FAO recommended safe limit. In the moderate area, only 22.2 % of cassava
samples had total cyanogens within the WHO/FAO recommended safe limit compare to 54.5
% found in the high prevalence area.
Table IV-2: Total cyanogens in cassava flour, thiocyanate and taurine in urine samples
collected in three konzo prevalence areas of Popokabaka (DRC).
Konzo prevalence area
Low Moderate High Total
Total Cyanogens
(µg HCN equivalent/ g cassava flour)
n
Median
Min
Max
10
19.00
9.11
169.75
9
22.44
2.90
62.83
11
10.69
4.75
54.12
30
16.50
2.90
169.75
Urinary thiocyanate (µmol/ l) n
Median
Min
Max
12
279.28
6.26
675.27
14
400.91
41.59
1037.06
16
287.10
21.02
1101.00
42
300.74
6.26
1101.00
Taurine (mmol/ mol creatinine) n
Median
Min
Max
12
6.47
0.00
23.90
14
4.29
0.00
97.59
16
13.57
0.00
41.55
42
8.84
0.00
97.59 The urinary thiocyanate content ranged from 6.26 to 1101 µmol/l urine. The lowest
concentration (6.26 µmol thiocyanate/l urine) was found in the low prevalence area while the
highest concentration (1101 µmol thiocyanate/l urine) was found in the high prevalence area
and was excreted by a participant from a konzo-affected household. 69 % of the urine samples
had thiocyanate content above 172 µmol/l urine (10 ppm) with 13.8 % of them above values
Konzo and cassava toxicity
100
of 900 µmol. All the konzo-affected household participants of the moderate area excreted
high urine concentration of thiocyanate (more than 500 µmol thiocyanate/l urine), while in the
high prevalence area only one participant had excessively high values.
The urine concentrations of taurine were low. Except the one sample with the highest
concentration (97.59 mmol/mol creatinine), the taurine concentrations of all other samples
ranged between 0 (or not detected) to 41.55 mmol/mol creatinine. More than half (61.9 %) of
the urine samples were below the reference limits (13 to 534 mmol taurine/mol creatinine)
calculated by Venta (2001). The highest concentration of taurine was found in a sample from
the moderate prevalence area. Taurine was detected in only 83.3 % of urine samples among
which 71.4 % excreted thiocyanate above 10 ppm or 172 μmol/ l urine. Urinary taurine was
slightly or not correlated to urinary thiocyanate (R2= 0.017, P = 0.415).
IV.1.4 Discussion
The identification of cases of konzo in the moderate and high prevalence areas in the present
study shows that this crippling neurodegenerative disease is still occurring in this part of
Bandundu province from where the first cases were reported three generations ago by Trolli
(1938). The study found that a high proportion of cassava flour samples contain total
cyanogens above the recommended safe limit set at 10 µg HCN equivalent/g by the Codex
alimentarius (FAO/ WHO, 1991). Even when compared to the higher acceptable limit (40
ppm) used in Indonesia, that is one of the highest cassava producer and consumer countries
worldwide (Djazuli & Bradbury, 1999), 16.6 % of our cassava flour samples are still above
this limit. The values in this study were higher than the ones we previously reported (Diasolua
Ngudi et al. 2002) on the processed cassava roots available on the markets of Kinshasa, the
capital of D. R. C., where important quantities of cassava cossettes coming from the study
areas are sold. Therefore, there is a risk of dietary exposure to cyanogen from consumption of
Konzo and cassava toxicity
101
cassava flour in the areas studied. Essers et al. (1998) stated that the cyanogen content in
cassava flour in rural areas of Africa usually grossly exceeds the safety limit set by the Codex
alimentarius but toxic effects are rare under normal conditions. Partly because the cyanogens
are mainly bound in glucosides which are relatively stable in the human body, and the form in
which the product is consumed (stiff paste) causes a slow release of the toxicant which can
then be detoxified gradually and more effectively by the body’s defence mechanism. Konzo
has been reported to occur most frequently when the mean cyanide content of cassava flour
exceeds 100 ppm (Lawrence, 1999). Shortcuts in the processing of cassava roots have been
reported to result in high residual levels of cyanogen substances and the consumption of such
roots leads to dietary cyanogen exposure (Banea et al. 1992).
Cyanogen exposure from cassava roots is the essential risk factor for konzo and thiocyanate
levels remains the best indicator of daily cyanide intake (Rosling, 1994, Banea-Mayambu et
al. 2000). An additional source of dietary cyanogen exposure can come from consumption of
cassava leaves which is the main source of protein in a diet consisting of processed cassava
roots in those konzo-affected areas (Chapter III). The preparation of this vegetable requires
prolonged boiling (at least 30 minutes) with additional water and firewood in order to reduce
the cyanogen content. Consumption of cassava leaves inadequately prepared and long-term
smoke inhalation from the firewood, the only fuel available, may be additional factors in the
overall cyanogen exposure. Low availability of water and firewood might be considered as
additional risk factor for cyanide exposure (Chapter III).
Half of the urine samples analysed contained levels of thiocyanate above 300 µmol/l while
Tshala-Katumbay et al. (2001b) reported that 75 % of the urine samples contained
thiocyanate levels above 300 µmol/l in the same “high prevalence area” of this study.
Tylleskär et al. (1992) also reported a high thiocyanate excretion in this region. When
comparing the study areas, we found higher cyanogen content in cassava flour from the low
Konzo and cassava toxicity
102
prevalence area than in cassava flour from the other two areas. However, this cyanide content
in the flour reflects only the safety of the food and the potential human exposure to cyanide,
as the excretion of thiocyanate is higher in the urine from moderate and high prevalence areas
than in the urine from the low prevalence area. The most affected population might be better
aware of a further konzo attack and take more precaution on improving the processing of
cassava, the sole staple food consumed (Cardoso et al. 2004).
The proportion of thiocyanate formed from a cyanide load will decrease if the subject is
malnourished. The conversion of cyanide to thiocyanate implies a reaction with sulphur
originating from dietary sulphur amino acids in the presence of rhodanese (thiosulphate-
sulphurtransferase, EC 2.8.1.1) as catalyst. The rate of detoxification is therefore limited by
the supply of a sulphur donor. Addition of different condiments containing sulphur amino
acids in the cassava – based diet might also reduce the risk for konzo.
The concentrations of taurine in our samples were lower than those reported as reference
limits from urine samples of participants on a normal diet (Venta, 2001) and from female
college students of Japan (Nakamura et al. 2002). More than half (61.9 %) of the urine
samples were below the reference limits of 13 to 534 mmol/ mol creatinine set by Venta
(2001). The average excretion of taurine from our samples (0.11 ± 0.15 mmol/g creatinine)
was 7 fold lower than the average excretion of taurine (0.78 ± 0.53 mmol/g creatinine) from
urine of 58 female college students of Japan (Nakamura, 2002). This may reflect a low intake
of dietary sulphur containing amino acids in the study areas. Taurine as well as thiocyanate is
an end product of the catabolism of sulphur containing amino acids metabolism involving
methionine and cysteine. Low sulphur amino acid intake can lead to low excretion of taurine
and thiocyanate. However, in this study the two metabolites were found to be slightly or not
correlated. Production of thiocyanate may affect the quantity of taurine excreted. In vivo
production of taurine in rats has been observed to be reduced preferentially over sulphate
Konzo and cassava toxicity
103
production when the supply of sulphur containing amino acids is limited (Tomozawa et al.
1998). Taurine is abundant in the brain where it has multiple functions as an anti-oxidant and
neuroprotectant. The physiological effects of taurine depletion from the brain are not well
documented.
It has been mentioned before that konzo occurs when the diet contains predominantly
insufficiently processed cassava (Tylleskär et al. 1992), which is also deficient in sulphur
containing amino acids (chapter III). Intake of sulphur amino acids from other components of
the diet needs to be known to allow correct evaluation.
In conclusion, this study reveals that konzo is still occurring in this area and that there is a risk
of dietary cyanogen exposure from cassava flour. Cassava flour samples from more than half
of the selected households contained total cyanogen above the WHO/ FAO recommended
safe limit. The urine analyses suggest an overload of cyanogen. The urine samples from more
than half of the participants excreted high amounts of thiocyanate and low amounts of taurine.
The low concentrations of taurine in the urine samples may suggest that more sulphur
metabolites be directed to detoxification of cyanide by formation of thiocyanate and can also
reflect the suboptimal intake of sulphur containing amino acids in the diet. No correlation was
found between taurine and thiocyanate; this might be due to the small number of urine
samples and the large variability in the data (taurine content of 61.9 % of the samples were
below the limit reference). More samples are needed to better evaluate the relationship
between urinary taurine and thiocyanate.
Food diversification and proper cassava processing combined with better and organised public
information can contribute to decrease the high dietary cyanogen exposure and the risk for
konzo. There is a need to adjust upwards the dietary requirements for sulphur amino acids to
compensate for the demand for cyanide detoxification in cassava consuming areas.
Konzo and cassava toxicity
104
CHAPTER V
GENERAL DISCUSSION AND CONCLUSIONS
Konzo and cassava toxicity
105
V General discussion and conclusions
This thesis summarises research on toxicological and dietary factors involved in konzo. The
investigations were observational and focussed on possible associations between the
occurrence of the disease and the exposure to cyanogen and the composition of the diet. Field
observations and measurements and laboratory analyses were done. Cross sectional and
ecological design were used to construct an updated epidemiological picture of differential
distribution of konzo among people with different risk profiles, to measure and to explore
both exposure and outcome. The design focussed on the incidence and distribution of the
disease in the community, and on the characteristics of the population groups rather than on
the individual members. Aggregate data based on surveys of groups of people were used to
assess diet-disease relationships. The unit in which the data were collected is the household.
Urine was collected on an individual basis. The household food consumption focussed on
nutritional aspects of the diet. Although individual based studies would allow a more direct
estimation of the risk of disease in relation to exposure, population based methods might
show that populations that have a higher exposure to cyanogen also have a higher rate of
konzo but it would not necessarily follow that konzo only occurred in areas with high dietary
exposure of the population to cyanogen.
Therefore, a limited community design was used to describe konzo and to identify possible
dietary and household associated factors. We subsequently sampled cassava foods and urine
to allow the evaluation of potential etiologic exposure as well as the interrelationships among
them.
Cases of konzo were unambiguously distinguished by their physical disability.
Misclassification was not an issue in our studies. High expert medical doctor and nurse, and
the author of this thesis confirmed, after examination, all konzo cases included and further
supervised the interviews conducted by trained enumerators. However since the studies rely
Konzo and cassava toxicity
106
on information obtained from the heads of household, recall bias cannot entirely be excluded.
Furthermore, in a typical rural setting like that of the study area where most of the heads of
household have a low educational level and food intake is based mainly on cassava as staple
food, the use of a food frequency questionnaire was not applicable. A qualitative 24 hour
recall was used to assess food consumption. The lack of quantitative measurements is a
limitation of the work. Estimation of food portion sizes and measurement of food intake per
kg of individual body weight could allow a better toxicological evaluation. However, the
study subjects share the same geographical environment, ethnicity and culture and we have
validated the data through repeated observation, using a set of open questions format to
investigate the 24 hour and seasonal food recall.
The studies in this thesis addressed new areas in konzo research ranging from the
identification of household factors to dietary risk and protective factors. The identification of
this wide array of associated factors led us to suggest possible measures to prevent the disease
and to recommend new directions for further research. We can not claim geographical
representation since we did not cover all konzo prone areas of the D. R. C. Underestimation
of the magnitude of the konzo problem is possible. The numbers of konzo cases in the study
area represent about 4.3 ‰ of the total number of cases in D. R. C., roughly estimated at
100,000 (R. D. C., 2000)
V.1 Occurrence of konzo
Although most of the cases reported in chapter 3 occurred in the 1990s. At present, konzo, a
preventable disease, is still occurring in Popokabaka Rural Health Zone areas, one of the
regions where cases were described in the first report on konzo in 1938 (Trolli, 1938). The
clinical picture of the affected subjects in our studies was similar to those previously
described in the same region (Trolli, 1938; Tylleskär et al 1992; Banea-Mayambu et al 2000;
Tshala-Katumbay et al, 2001; Bonmarin et al., 2002). The main symptom was non-
Konzo and cassava toxicity
107
progressive symmetrical spastic paraparesis (paralysis of both legs) with sudden onset. Konzo
was already described in other parts of D. R. C. and in other sub-Saharan African countries
such as Mozambique, Tanzania, Angola, the Republic of Central Africa and Cameroon.
However, konzo does not occur in all regions with cassava-based diets even in the same
country. No konzo case has been reported in Latin America from where cassava originated
and is consumed as staple food neither in Nigeria, the big producer and per capita consumer
of cassava nor in Indonesia and Thailand where cassava is a popular food. Therefore, high
consumption of cassava root by itself is not the only or exclusive cause for konzo (Tylleskär,
1994c). The occurrence of konzo in a community reflects a deterioration of socio-economic
conditions and those who are affected by konzo are trapped in a spiral of poverty, and
educational and political neglect. Poverty and lack of education affect people’s capacity to
prevent the disease as well as their ability to live in areas having less exposure to this risk.
There is a relationship between poverty (socio-economic as well as educational) and
vulnerability, and they are mutually reinforcing.
Age and sex distributions of konzo affected subjects in our study were similar to most of the
previous studies in the same region and elsewhere (Howlett, 1994; Banea-Mayambu, 1997,
Tshala-Katumbay et al, 2001b). Children less than 14 years and women at childbearing age
are particularly susceptible to konzo. In Chapter 4, our studies compared the expected daily
intake of the sulphur amino acids methionine and cysteine provided by cassava cossettes
consumption with the suggested essential amino acid requirement, and we concluded that
children of 1 to 9 years old cannot expect to meet methionine requirement whereas adults can
meet the minimal requirement for these sulphur amino acids. No child under two years was
affected by konzo. In Bandundu province, breastfeeding is general at birth and this practice
decreases slowly until two years old: 98.6 % of children aged from 12 to 15 month and 62.7
% of those aged from 20 to 23 month old are still breastfed (R. D. C., 2001). Mother milk is
Konzo and cassava toxicity
108
rich in amino acids containing sulphur. The dominance of female patients (female to male
ratio 3.3: 1) in our studies is similar to almost all other studies except the ones carried out in
Tanzania and Mozambique where male cases were preponderant (Howlett, 1994; Tshala-
Katumbay et al, 2001b, Bonmarin et al, 2002). The absence of a suitable animal model for
konzo makes it difficult to explain or to study the reasons for the high susceptibility of
females at reproductive age for konzo. The female hormones, especially 17 β-oestrogen have
been proposed as protective factor for neurolathyrism to explain the high susceptibility for
young men, but can these hormones be an aggravating factor for konzo (Lambein et al,
2004)? Rural depopulation resulting in migration of active males to bigger cities looking for
“welfare” and the primary socio-economic role played by women in the household food
security may expose women to higher workload and higher oxidative stress.
The socio-economic crisis that affected D. R. C. for several years has probably had a major
impact in this particular region and led to the persistent occurrence of konzo during several
generations. The socio-economic burden of this crippling disease on this impoverished region
is heavy (Bonmarin et al, 2002). Additional stresses such as military activity, political
conflicts and drought-provoked food shortage have been identified as factors leading to
inadequate diets which triggered konzo epidemics (Essers, 1995).
V.2 Cassava foods and sulphur metabolites
Cassava flour mixed or stirred in boiling water to obtain a stiff porridge, the so called “fufu”
or “luku” is the main daily staple food for almost all the households studied (Chapter 2).
Cassava flour is derived from the roots which in normal conditions are soaked (retted) for at
least three nights, then sun dried for 3 to 5 days, then pounded and finally sieved (Chapter 3-
1). Cassava flour is an excellent source of carbohydrate (Bradbury and Holloway, 1988).
However, we find that the protein of cassava roots is of poor quality, leucine and lysine are
limiting amino acids and also the proportion of methionine is low, giving a chemical score of
Konzo and cassava toxicity
109
the protein of around 40 (Chapter 3 - 1). Our studies (Chapter 4) also reveal that more than
half of the households were soaking cassava roots for less than three nights. As a
consequence, a high proportion of households had cassava flour containing total cyanogens
above the recommended safe limit set at 10 µg HCN equivalent/g by the Codex alimentarius
(FAO/ WHO, 1991). High cyanogen exposure from frequent and exclusive consumption of
insufficiently processed cassava roots is thought to be a major etiological factor in konzo.
In this region, saka-saka (pounded cassava leaves) constitutes the main condiment consumed
as side-dish with luku (Chapter 2). Although quantitatively the main source of protein in the
diet, this protein is also of poor quality with sulphur amino acids as the most limiting amino
acids (Chapter 3-2). Cassava leaves were also found to be a potential additional source of
dietary exposure to cyanogens, apart from the cassava roots. The cooking of saka-saka
requires prolonged boiling (at least 30 minutes) with additional water and firewood in order to
reduce the cyanogen content but the supply of both water and firewood is limited (Chapter 3-
2). There is no water source in the villages, neither is there electricity nor gas. Sources of
water are located at least at 15 minutes walking (RDC, 2001) and cooking is done exclusively
with firewood which nowadays is becoming scarce in this savannah area.
No potentially toxic nonprotein amino acid was detected in cassava roots as well as in cassava
leaves (Chapter 3). Neurolathyrism, which shares clinical similarities with konzo, has been
associated with β-ODAP, a neuro-excitatory nonprotein amino acid present in the grass pea
(Lathyrus sativus) (Getahun et al, 1999).
High thiocyanate content was found in more than half of the urine samples analysed. This
suggested a high exposure to cyanide (Chapter 4). Luku and saka-saka are the known and the
main sources of dietary cyanide, but smoke inhalation from wood fires inside the primitive
housing and probably also inhalation of HCN escaped during soaking and sun drying of
cassava roots might be considered as additional sources of cyanide exposure that need further
Konzo and cassava toxicity
110
study. The conversion of cyanide to thiocyanate in the human body requires sulphur
originating from dietary sulphur amino acids. The rate of detoxification is therefore limited by
the supply of a sulphur donor. When the body is regularly exposed to cyanogens, the
increased synthesis of rhodanese, enzyme responsible for cyanide detoxification in the human
body by forming thiocyanate, makes extra demands on the body's reserves of sulphur amino
acids. If this demand is prolonged as in the regular consumption of cassava insufficiently
processed, and the diet is inadequate, the synthesis of taurine may be impaired (Chapter 4).
Taurine and thiocyanate are excreted in the urine as end products of the catabolism of sulphur
containing amino acids methionine and cysteine. Production of thiocyanate may affect the
quantity of taurine excreted. In the case when insufficiently processed cassava is consumed as
staple food, the low methionine content may aggravate the risk for cyanide toxicity and konzo
disease. Moreover, the consumption of cassava leaves as the main source of dietary protein
can not compensate for the methionine deficiency in konzo-affected areas where the dietary
requirement for methionine needs to be adjusted for the loss caused by cyanide detoxification
(Chapter 4).
This dietary imbalance can be corrected if other components of the diet contribute to a better
balanced amino acid composition, especially the level of sulphur amino acids. Consumption
of cereals and sesame is found in Chapter 2 to be protective factors against konzo. Similar
protection by methionine rich cereals was also found for neurolathyrism (Getahun et al,
2003). In the regions neighbouring the konzo-affected areas in Bandundu, where traditionally
corn or millet flour is mixed with cassava as staple food, no cases of konzo have been
reported. This may corroborate our views as to the importance of methionine for a healthier
balanced diet.
Konzo and cassava toxicity
111
V.3 Conclusions and recommendations
The high urinary thiocyanate levels found in our studies show that the population of
Popokabaka is still highly exposed to dietary and perhaps environmental cyanogens and to the
risk of konzo. A better balanced diet, especially richer in methionine is required to allow
efficient detoxification of cyanide in the body. Therefore, we recommend:
a) Supplementation of methionine to contribute to the detoxification of cyanide should
be done in parallel with the promotion of consumption of foods rich in methionine
locally available (cereals, sesame, soybean, pumpkin seeds, eggs, meat, etc). A study
of the effect of methionine administered on urinary levels of taurine and on
thiocyanate can help to better understand the role of taurine, as an antioxidant, a
neuroprotectant, and an inhibitory neuromodulator in the central nervous system, and
the relationship between taurine and thiocyanate in konzo.
b) Promotion of safe cassava processing to reduce significantly dietary cyanogen
exposure. Sufficient soaking (retting) combined with sun drying has been proven to be
effective in reducing cyanogen. Organised public information should be promoted.
Attention should be drawn on the duration of cassava processing especially during the
dry season which is the period of low ambient daily temperature and low water
supply. Days are cloudy and the temperatures are below 20°C; HCN is evaporated
more slowly at lower temperature as the boiling point of HCN is 25.7°C, thus removal
of HCN is not optimal during the cold dry season. The dry season is reported to be the
period of high incidence of konzo. We suggest the increase of the period of soaking
and drying during this season (at least 5 days each).
c) Promotion of consumption of luku obtained from the mixing of cassava with maize
flour or other cereals. Maize is locally available but pounding by hand to transform
grain to flour seems to be hard for the population. Therefore, an adapted milling
Konzo and cassava toxicity
112
technology should be implemented. Better information on the benefit of mixing
cassava and maize or other cereals should be promoted.
d) Food diversification should be promoted especially during the dry season. Nutritional
resources are scarce during the dry season. Almost all crops are rain-fed and cannot
survive during the dry season. Watering or an irrigation system is not practicable in
this area because of absence of inputs and of major river systems for irrigation. Roads
should be better maintained to open the region to the market.
Konzo is a very much neglected disease in D. R. C., where political instability and military
activity are factors attracting the international attention. The patients suffering from this
incurable disease become a socio-economic liability for their family. Prevention of this
disease and its dramatic socio-economic consequences can be attained by simple education of
the basics of nutrition and the supply of the simple means to produce a more varied diet. A
national campaign should be organised to identify isolated pockets of konzo affected
communities and to distribute preventive information.
Konzo and cassava toxicity
113
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Konzo and cassava toxicity
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CURRICULUM VITAE
Curriculum Vitae
Identity
Full name: Delphin DIASOLUA NGUDI
Place and date of birth: Kinshasa, May 2, 1961 Sex: Male
Nationality: Congolese (Democratic Republic of Congo)
Marital status: Married and 3 sons
Permanent address: Inga 57 Quartier 10, N’djilj/ Kinshasa, D R Congo
Present address: Filips Van Cleeflaan 386, 9000 Gent, Belgium
Email address: [email protected]
Education
Master of Science in Food Science and Technology, Universiteit Gent/ Katholieke
Universiteit Leuven, Belgium, 1999
Masters Thesis: Effect of sprouting and lactic acid fermentation on protein in finger millet
(Eleusine Coracan) and kidney beans (Phaseolus vulgaris
Promoter: Prof Dr Ir André Huyghebaert and Dr John Van Camp
Degree in Complementary Studies in Nutrition and Food Science, Universiteit Gent,
Belgium, 1997
Thematic study: Mise en place des activités de surveillance nutritionnelle à Kinshasa
Graduate in Medical Techniques: Nutrition and dietetics, Institut Supérieur de Techniques
Médicales, Kinshasa, R. D. Congo, 1983
Thematic study: Evaluation du critère de prise de poids ou du périmètre brachial pour la
récupération des enfants malnourris.
iii
Additional Training
Summer course on “biosafety assessment and regulation of agricultural biotechnology”,
Plant Biotechnology Institute for Developing Countries (IPBO), Ghent university,
Belgium, 2004.
International Training Course in Dairy Technology: “Dairy technology from rural to
industrial level”, B.A.D.C./ Universiteit Gent, Belgium, 1998
Socrates Intensive Course: Food Packaging, European Union/ Universiteit Gent, Belgium,
1998
International Training in Nutrition and Food Science, Target Program on food security:”
Micronutrients deficiencies, Université de Benin, Cotonou, Bénin,1994
Training in writing of health education school manuals, UNICEF/ Ministry of Primary
and Secondary Education, Kisantu, R. D. Congo, 1991
Training of Health Community Workers Trainers, (U.S.A.I.D./ SANRU Project),
Kimpese, R .D. Congo, 1985
Employement record
• 2000 to date: Research on cassava and konzo at Universiteit Gent, Belgium
• 1984 to date: Nutritionist and Food quality control Officer at the National Program of
Nutrition (PRONANUT/ D R. Congo) (former CEPLANUT)
• 1992 to 1996: Collaborating Assistant to the Director of CEPLANUT and Member of
the Technical Committee
• 1993 to 1995: Technical Assistant to the project TCP/ ZAI/ 2355(A) – FAO/
CEPLANUT) “Food vended street”
• 1989 to 1992: Assistant to the Activity coordinator of CEPLANUT Regional Office
of Bandundu Region in Kikwit
iv
• 1984 to 1989: Head of the Nutrition Education section and Contact for the regional
council for food and nutrition of Bandundu Province (CRANB), project
660-079 ( United States Agency for International .Development
(USAID) / CEPLANUT)
International meeting Attendance
The first International Conference on Food Systems, college of Food systems, United
Emirates University, Al- Ain (United Arab Emirates), October 19 – 21, 2003
World Health Organization (WHO) AFRO Regional awareness raising workshop on food
safety evaluation, Bamako, (Mali), December 4 – 6, 2002.
International Food Policy Research Institute (IFPRI) 2020 vision: Sustainable Food
Security for All by 2020, Bonn (Germany), September 4-6, 2001 (Participation sponsored
by GTZ- Echborn)
Publications
1. Diasolua Ngudi D., Kuo Y.H., Lambein F.: Cassava cyanogens and free amino acids
in raw and cooked leaves. Food and Chemical Toxicology 2003, 41, 1193-1197.
2. Diasolua Ngudi D., Kuo Y.H., Lambein F.: Amino acid profiles and protein quality of
cooked cassava leaves 'saka saka'. Journal of the Science of Food and Agriculture.
2003, 83, 529-534.
3. Diasolua Ngudi D., Kuo Y.-H., Lambein F. Cassava leaves, a non-negligible source of
dietary exposure to cyanogens. Cassava Cyanide Diseases Network (CCDN) News
2003, 2, 1-2.
4. Diasolua Ngudi D., Kuo Y.-H., Lambein F. : Food Safety and Amino Acid Balance in
Processed Cassava "cossettes" Journal of Agricultural and Food Chemistry 2002, 50,
3042-3049.
v
5. Lambein F., Diasolua Ngudi D., Kuo Y.-H.: Vapniarca revisited: Lessons from an
inhuman human experience. Lathyrus lathyrism newsletter 2001, 2, 5-7.(website URL:
http://go.to/lathyrus).
6. Mbithi-Mwikya S., Ooghe W., Van Camp, J., Ngudi, D., Huyghebaert A.: Amino acid
profiles after sprouting, autoclaving, and lactic acid fermentation of finger millet
(Eleusine coracan) and kidney beans (Phaseolus vulgaris L). J. Agr.Food Chem. 2000,
48, 3081- 3085.
7. Diasolua Ngudi D., Kuo Y.-H., Lambein F., Patrick Kolsteren. High risk of dietary
cyanogen exposure in a population living in a konzo – affected area of Democratic
Republic of Congo. Food and Chemical Toxicology (submitted for publication)
8. Diasolua Ngudi D., Banea-Mayambu J.-.P., Lambein F., Kolsteren P.:.Crippling konzo
in DRC, three generations later. The Lancet (submitted for publication)
Memberships
Member and country contact of cassava cyanide diseases network (CCDN)
Member of the Congolese Nutritionists and Dieticians Association and former Secretary
of the Council (1995-1996)
Member of Science Press, vzw
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