J. clin. Path. (1956), 9, 333.

ALKALINE PHOSPHATASE ACTIVITY OFTHE SERUM IN KWASHIORKOR

BY

RUTH SCHWARTZFrom the Medical Research Council Group for Research in Infantile Malnutrition, Mulago Hospital,

Kampala, Uganda

(RECEIVED FOR PUBLICATION SEPTEMBER 9, 1955)

There are few reports of alkaline phosphataseactivity in the serum of children suffering fromkwashiorkor. The values quoted are somewhatconflicting: Waterlow (1948) found an averagelevel of 19.7 .King-Armstrong units in eight casesof "fatty liver disease " in Jamaica, and Jaya-sekera, de Mel, and Cullumbine (1951) reportedan average value of 15.8 units in 40 children withkwashiorkor investigated in Ceylon. Both valuesfall within the normal range of 15 to 20 King-Armstrong units quoted by Behrendt (1949). InKampala the average of 23 estimations in childrennewly admitted for treatment of acute kwashiorkorwas 11.8 units with a range of 4.7 to 23.1 units(Dean and Schwartz, 1953). Workers in Cape-town, Johannesburg, and Gautemala City havealso found low values (Hansen, Bersohn, andBehar, personal communications).Low activities have been reported for most of

the blood and tissue enzymes investigated in kwash-iorkor. In the Kampala investigation alreadymentioned the activity of three serum enzymes(amylase, non-specific esterase, and pseudocholin-esterase) was low when the children were ad-mitted, and rose after two to three weeks of treat-ment to levels approaching, and often exceeding,those that were considered normal for healthychildren. Enzymes in duodenal juice (amylase,lipase, and trypsin) showed similar alterations.The pattern for alkaline phosphatase was different:in about half the children investigated the levelsfell so that by the eighth day the average valuewas 9.5 units. Subsequently there was a steadyrise in activity up to the time when the childrenwere discharged, usually about 30 days after ad-mission. Bersohn (personal communication) alsofrequently found a fall in alkaline phosphataseactivity in Johannesburg children. As at Kampala,the fall did not occur in all cases, but it was fre-quent enough to merit attention.

Liver biopsy tissue taken from Indian childrenwho had kwashiorkor was examined by histo-chemical methods by Sriramachari and Rama-lingaswami (1953) and by Ramalingaswami,Sriramachari, and Patwardhan (1954) and wasfound to show an increased concentration of alka-line phosphatase. It was pointed out that thisfinding was difficult to reconcile with the lowvalues reported in Kampala for the serumenzyme.

Several workers have observed increased con-centrations of alkaline phosphatase in the liversof adult rats fed on diets deficient in protein(Miller, 1950; Ely and Ross, 1951 ; Rosenthal,Fahl and Vars, 1952). Rosenthal et al. found thatthe increased activity in liver tissue was accom-panied by a change in its response to magnesiuLmactivation and cyanide inhibition. In normallyfed rats the liver phosphatase was stronglyactivated by Mg +± and only slightly inhibited byCN-. In the plasma, inhibition by CN- wasalmost complete, but the degree of activation byMg+ + was considerably less than in the livertissue. Fasting lowered the total level of enzymein plasma and in the liver, but there was no changein the response of the liver enzyme to Mg++ andCN-. After the rats had been given a protein-free diet for two weeks the concentration of alka-line phosphatase in the liver was increased, theresponse to Mg+ + was less and the response toCN- greater than in normal rats. The enzymein plasma was apparently equally affected by fast-ing and protein depletion: the level was loweredto about half the normal value. The figures givensuggest that the response to Mg++ was slightlygreater than normal and the sensitivity to CN-somewhat less, but the authors presumably didnot think these variations significant as they didnot comment on them. They suggested that thechanges in the liver tissue might be explained by

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postulating that the liver normally disposed ofextrahepatic alkaline phosphatase, and that thisfunction was impaired by protein depletion.The aetiology of kwashiorkor is not yet fully

understood, but it is generally agreed that a de-ficiency of protein in the diet is an importantfactor. It was thought, therefore, that some

changes which have been found in protein-depleted rats might also occur in acute kwashior-kor. Much work has been done on serum alkalinephosphatase in pathological and normal conditionsin man, but little is known of its origins.Bodansky and Jaffe (1933-34), from a considera-tion of high values found in jaundice, suggestedthat part of the serum alkaline phosphatase mightbe produced in the liver. Previously the enzyme

was thought to originate in bone. Roche andSarles (1950, 1951) determined the affinity con-

stants of alkaline phosphatases found in differentorgans, and came to the conclusion that theenzyme of human plasma resembled that of liver;they found no difference in the nature of the alka-line phosphatase in plasma from normal adultsand from patients suffering from various abnormalconditions of liver or bone.The fall in phosphatase activity found in children

at the beginning of treatment has not been re-

ported in the blood of rats repleted after proteindepletion, and there is evidence that the bloodenzyme of rats is different in origin and generalproperties from that of humans. It seemedpossible, however, that an investigation on the linesfollowed by Rosenthal et al. (1952) might explainthe fall. In the children serum, and not plasma,was used, and for various reasons liver tissuecould not be analysed.

Material and MethodsThe Children.-Three groups of children were in-

vestigated, all between the ages of 6 months and3 years.

Group (1): Children without Signs of Kwashiorkor.-All the 13 children in this group had been broughtto the Out-patient Department of Mulago Hospital forthe treatment of upper respiratory infections or mildmalaria. There are no " normal " values for Ugandachildren, and uncertainties regarding ages and otherreasons make the collection of " normal " data difficult(Dean, 1954). The selection was therefore to some

extent empirical, but none of the children in this groupshowed any detectable degree of growth failure, or

signs of kwashiorkor or of any other severe illness.

Group (2): Children with Slight Kwashiorkor.-This group, which numbered 15, consisted chiefly ofchildren who had been brought to Mulago Hospitalas out-patients to be treated for various mild infective

conditions, and were found to have in addition someof the slighter signs of kwashiorkor. Some of themwere admitted to the ward of the infantile malnutritiongroup for treatment. All the children had a markeddegree of growth failure and their hair was discolouredand of altered texture. Dry and lustreless skin wascommon in this group, and slight oedema was some-times present, but children with definite dermatosisor other symptoms of severe kwashiorkor were notincluded.Group (3): Children with Severe Kwashiorkor.

The 22 children in this group were admitted to theward of the infantile malnutrition group with the diag-nosis of kwashiorkor. The signs of the disease asthey are usually seen in Kampala have been describedin full elsewhere (Trowell, Davies, and Dean, 1954).They include growth failure, changes in the hair andskin, oedema, digestive failure, anorexia, and charac-teristic psychological changes. Severity is not easyto define because it is rare for a child to show simul-taneously all the signs of the advanced disease; fre-quently the condition cannot be judged with accuracyuntil the rate of response to treatment has been ob-served. Oedema with marked dermatosis always indi-cates severity, especially if there is much anorexia,but often the child who is thought to be acutely illat the beginning will make a rapid recovery. Inthe severest cases there is usually considerable wast-ing, although this may become apparent only whenthe oedema is lost after some days of treatment. Be-cause growth failure, wasting, and oedema usuallyoccur together, it is not possible to correlate the degreeof underweight for age with severity. The only objec-tive measurement of severity that could be used inthe present series with any degree of confidence wasthe level of the serum albumin: if it was under1.8 g./100 ml. the case was regarded as severe. Insuch cases the total serum protein was usually from3.5 to 4.5 g./100 ml.

All the children in this group were treated withdiets based on milk protein (Dean, 1952). They werebled from the superior sagittal sinus or the externaljugular vein on admission and, as far as possible, everyseven days afterwards. The blood was allowed toclot, was ringed, and centrifuged without delay, andthe serum was stored at -5° C.The blood from the children in the other groups

was taken and treated in exactly the same way, butonly one sample was obtained from each child, eitherin the Out-patient Department or in the hospitalwards.Chemical Methods.-The method employed for the

measurement of enzyme activity was that describedby Rosenthal and others (1952), but certain modifica-tions were made because the activity of the alkalinephosphatase is much lower in human serum than itis in rat serum and because it was necessary to usethe minimum amount of serum.

In four microcentrifuge tubes (0.8 cm. x 7.5 cm.)were placed 0.10 ml. of 0.1 M sodium-18-glycero-phosphate (containing less than 0.1% a-glycero-

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SERUM ALKALINE PHOSPHATASE ACTIVITY IN KWASHIORKOR

phosphate), and 0.10 ml. of 2-amino-2-methyl-1, 3-propanediol buffer, pH = 9.6 (Gomori, 1946). Addi-tions were made as follows:Tube 1, 0.10 ml. of distilled water and 0.05 ml. of

0.06 M magnesium chloride.Tube 2, 0.10 ml. of distilled water and 0.05 ml. of

0.08 M potassium cyanide which had been adjustedto pH 9.6 with N HCI.

Tubes 3 and 4, 0.15 ml. of distilled water.

Tubes 1, 2, and 3 were incubated with 0.05 ml.of undiluted serum for three hours at 37° C. Thereaction was then stopped by adding 0.10 ml. of 25%trichloracetic acid.Tube 4 was a blank to which 0.10 ml. of trichlor-

acetic acid was added before 0.05 ml. serum.

The tubes were centrifuged for five minutes at about8,000 r.p.m., then 0.40 ml. of the supernatant was takenoff with a Carlsberg constriction pipette and added to2.4 ml. of distilled water. The phosphate was esti-mated by the method of Allen (1940), but the finalvolume was reduced to 4.0 ml. : the colour was

developed by the addition of 0.5 ml. of 60% per-chloric acid, 0.2 ml. of 8.300 ammonium molybdate,and 0.5 ml. of Allen's amidol reagent (2.0 g. amidoland 40.0 g. sodium metabisulphite made up to200 ml.). A blank and standards containing 5 pxg.and 10 a'g. P were included. The standards wereprepared from potassium dihydrogen phosphate. Thecolour was read in an E.E.L. photo-electric colori-meter using an Ilford filter No. 608. A very goodlinear relationship of colour to concentration wasobtained from 1 to 20 psg. P. The calculation usedwas as follows:Mg. P released by 100 ml. serumn=

Reading of test 100 5 10Reading of 10 pg. standard 0.05 )X 4 X 1000

Reading of test X 25Reading of standard

To conform with convention an arbitrary unit hasbeen adopted: it is the amount of enzyme thatliberated I mg. P under the conditions described. Thecalculation therefore gives the answer in units. Theunit is approximately three times as great as theBodansky unit, but the comparison is not exactbecause the conditions of hydrolysis in Bodansky'smethod are somewhat different from those employedin this investigation. It should be slightly greater thanthe King-Armstrong unit, which is two and a halftimes the Bodansky unit.

Extending the reaction time from the one hour usedby Rosenthal et al. (1952) to three hours was not con-sidered ideal, but it was necessary to measure lowactivities, and the modified method was not suitablefor the estimation of the small amounts of phosphatereleased in one hour. The activities of some sera weremeasured at one hour and three hours; they showedno falling off of activity during the longer incuba-tion time.For the phosphatase estimations done earlier in

Kampala (Dean and Schwartz, 1953) the King-ArmstTong method was used which measured activityin serum without activation by added Mg++. In thepresent series the activity so found is referred to as" basic." Under the conditions of the experimentsMg++ caused an increase in activity and CN- a re-duction. The increase in value due to activation, andthe residual value found after inhibition, have beenexpressed as percentages of the basic value.

ResultsTable I shows the basic values of serum alkaline

phosphatase, and the effects of magnesium activa-tion and cyanide inhibition.

Basic Activity of Alkaline Phosphatase.-Theaverage value for the group of children in goodgeneral condition was 30.8 units. The values

TABLE IALKALINE PHOSPHATASE ACTIVITY AND RESPONSE TO MG++ AND CN- OF THE SERUM OF CHILDREN WITHOUTSIGNS OF KWASHIORKOR, CHILDREN WITH SLIGHT KWASHIORKOR, AND CHILDREN WITH ACUTE KWASHIORKOR

ON ADMISSION TO HOSPITAL AND DURING RECOVERY

Alkaline Phosphatase Percentage Increase in PercentageNo. of (units/ 100 ml. serum) Activity due to Mg++ Resistance to CN-

Group iChildrenAverage Range Average Range Average Range

(1) Children without signs of kwashiorkor .. .. 13 308 88-68-0 27 24-32 10 0-23S.D. 15-40 S.D. 2 22 S.D. 5.77

(2) Children with slight kwashiorkor.15 1952 118 311 30 22-38 7 0-27ajW lshurens wiso severe 1wasn:or,or S.D. 5-89 S.D. 6-91 S.D. 7-81ti) 9-Allaren with severe kwashiorkor(a) On admission

(b) After 7-14 days ..

(c) ,, 15-20 , ..

(d) ,, 21-42 , ..

(e) ,, 43-70 ,. ..

22 14 0 8-3-32-6 35*S.D. 03 S.D. 10 20

16 10-8 7-9-20-1 43S.D. 3.5 5S D, 5 49

15 15-8 9 1-27-6 27S.D. 5.49 S.D. 6-46

14 23-1 10-4-33-6 31S.D. 7-01 S.D. 6-14

8 34-9t 26-5-52-3 30S.D. 7-86 S.D. 3-20

20-53

20-68

18-38

18-40

23-32

S.D.

S.D.

S.D.

S.D.

S.D.

158-09126-18S

5-195

3-8084-21

0-30

0-27

0-19

0-13

1-14

Each value printed in italics is significantly different, by statistical analysis, from that immediately above in the same column.* Significantly different from value for Group 1 but not from that for Group 2.t Not significantly different from value for Group 1.

1i~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

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FIG. I.-Francis. Boy aged12 months. Severekwashiorkor withoutcomplications. Grosslyunderweight; severehair and skin changes;much oedema; verymiserable but notanorexic. Clinical re-covery was excellentdespite a bout ofdiarrhoea, with slightdehydration, about the40th day. The alkalinephosphatase activity,low at first,felland thenrose. The response toMg-+ was high on ad-mission (50%) and atnine days. It fell to aminimum value on the19th day, then settledto levels between 35 to45°/0, which were some-what higher than wasusually found duringrecovery. The per-centage resistant tocyanide fell from thehigh initial level (18%)to zero on the 19th day,and was rising at thetime of discharge.

TIME (doys)

reported by Stearns and Warweg (1933) forAmerican children of the same age were between8 and 15 Bodansky units, and by King (1953) forEuropean children 11-28 King-Armstrong units.As the unit used in the present investigation isapproximately three times the Bodansky unit, theaverage value of 30.8 units agrees with othernormal values.The average value for children with slight

kwashiorkor was 19.2 units, a little more than halfthe figure for the children without kwash,orkor.The children with severe kwashiorkor had an

average value of only 14.0 units. There was a

further drop in this group to 10.8 Linits after seven

to 14 days of treatment, which was followed by a

steady rise to 34.9 units after 43 to 70 days oftreatment. By that time in most cases the childwas putting on weight well and the general con-

dition was satisfactory. The phosphatase activity,having reached a value between 30 and 40 units,remained fairly constant. The final value was

approximately the same as that for the childrenwithout signs of kwashiorkor.

Activation by Magnesium.-The percentage ofenzyme activated by magnesium was virtually thesame in the group without signs of kwashiorkorand the group with slight kwashiorkor, but therange was somewhat greater in the latter group.

In the group with severe, untreated kwashiorkorthe mean value was 3500, which was significantly

different from the value for the children withoutsigns of kwashiorkor, but not from that for thegroup with slight kwashiorkor. After seven to 14days of treatment there was a rise to 43 /. It wasfollowed by a drop to 27% in 15 to 20 days. Themean values after 21 to 42 and 43 to 70 days oftreatment were 31 % and 300%. The small increasewas without statistical significance.

Resistance to Inhibition by Cyanide.-Therewas no difference of statistical significance betweenthe group without signs of kwashiorkor and thegroup with slight kwashiorkor in the resistance ofthe serum enzyme to inhibition by cyanide. Insevere kwashiorkor the percentage of enzyme resis-tant to inhibition was at first raised to 15 and afterseven to 14 days of treatment was still 12. A dropto 5 was found after seven more days. In thelast stages of treatment the figure was 8. The slightrise, although without statistical significance, wasprobably not fortuitous, and it is discussed later.The Range.-All the average values recorded in

Table 1 are statistically significant, but the saria-tions about the average were consider;tble. Thiswas especially marked in the group with severekwashlorkor before and during treatment. It wasfound that, when the results obtained in individualcases were related to length of treatment, a changein the response to magnesium and cyanide thatwas more marked than was shown by the averagevalues could often be recognized. Although there

336

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SERUM ALKALINE PHOSPHATASE ACTIVITY IN KWASHIORKOR

I.-.

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1~

zE

zii

0 -

I X

" zo

*0

,0Total protein

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8-0

70

60

50

0

20I5105

10

2015 -

104 __

lO

S

60 R-

5010 0 2

10 20 30 40 50TIME (days)

FIG. 2

had been careful selection of the severe cases, con-siderable variation in initial severity was unavoid-able, and anorexia, the degree of psychologicalupset, or the presence of complicating infectionscould affect the course of recovery profoundly.Four individual cases, chosen to be illustrative, arepresented in detail (Figs. 1 to 4). Three of themwere severe cases of kwashiorkor and the fourthwas a slight case that had been treated in the wardof the infantile malnutrition group. The bodyweight, the total serum protein and serum albumin,the alkaline phosphatase, and the percentages ofenzyme activated by magnesium and remainingafter cyanide inhibition, on admission and duringthe stay in hospital, are shown in the Figures.

The Relation of Clinical Condition to EnzymeChanges During Treatment

Changes in Basic Alkaline Phosphatase.-Thefour cases represented graphically in Figs. 1 to 4are examples of different patterns of change inalkaline phosphatase activity in response to high-protein treatment. In the children severely ill(Figs. 1 and 2) the activity was low in thebeginning and dropped in each case by about fourunits in the first stage of recovery. In the lesssevere case (Fig. 3) the alkaline phosphatase waslow initially; it was at the same level six dayslater and had risen by over five units by the thir-teenth day. If a drop did occur it must havebeen missed between the first and seventh day.Recovery, in this case, was unusually rapid. In

0I40302010

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20-15-10.504030-2010

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0 10 20 30 0 10 20TIME (days) TIME (days)

FIG. 3 FIG. 4FIG. 2.-Lukwago. Boy aged 16 months. Severe kwashiorkor with

complicating pneumonia and bilateral hypopean ulcer. Grosslyunderweight; severe hair and skin changes; much oedema; verymiserable; very anorexic. Clinical recovery was slow, but weightgain was fairly good after the infections had cleared up. Theserum alkaline phosphatase showed a fall only on the 15th day,then rose very slowly. The sensitivity to Mg++ was highest onthe seventh day, then fell. The percentage resistant to CN- fellfrom the initial high level (16%) to zero on the 14th day. Itshowed its first rise only on the 42nd day of treatment.

FIG. 3.-Namagesere. Girl aged about 30 months. Moderatelysevere kwashiorkor; very underweight, but plump; severe hairand skin changes; much oedema; no misery; good appetite.Clinical recovery was rapid with good weight gain. There was nofall after seven days in the activity of the basic alkaline phospha-tase. It had risen sharply by the 13th day and continued risinguntil the child was discharged on the 27th day. The sensitivity toMg++ fluctuated, but remained throughout in the range found inchildren without signs of kwashiorkor. Similarly, the percentageresistant to CN- was never very high. It fel I to zero on the seventhday, and then rose.

FIG. 4.-Sumani. Boy aged 20 months. Mild kwashiorkor; under-weight, but plump; scanty hair; slight skin changes; oedema oflegs; no misery; good appetite. Clinical recovery was good.The serum alkaline phosphatase was not especially low, and roseimmediately with treatment. The sensitivity to Mg++ fluctuatedslightly about the value considered normal for children witlhoutkwashiorkor. The percentage resistant to CN- was lowest whenthe first sample was taken (on the third day) and rose duringtreatment. The value on discharge was a little higher than wasusual in such cases.

the mild case (Fig. 4) the initial level was a littlehigher than in the other cases and there was animmediate steady rise from the beginning of treat-ment.

Further investigation has shown that the drop inactivity during the early part of treatment occursvery seldom except in severe cases. As previouslyexplained, the level of serum albumin on admis-sion was used as an objective measure of severity,and it was found that in nearly every case in which

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the level was 1.8 g./ 100 ml. or less the fall in thephosphatase activity occurred: when the level wasabove 1.8 g./100 ml. a rise was more usual. InKampala cases 1.8 g. albumin is usually found inabout 4.0 g. total serum proteins, and this figureis equally valid as an index.

Sensitivity to Magnesium and Cyanide.-Therewas no direct correlation between the sensitivityto magnesium and the resistance to cyanide;especially during the first 14 days of treatment theyoften showed independent changes. The highestsensitivity to magnesium occurred almost entirelyin the severest cases. In Group 3 (a) in Table Ithere were four instances of sensitivities of over45% , and in Group 3 (b) there were nine. Innearly every case in which the sensitivity to mag-nesium was elevated above 45%, either on admis-sion or after treatment had begun, the drop inphosphatase occurred.On the other hand, no relationship could be

found between the severity and resistance tocyanide inhibition. The general trend in severe andmild cases of kwashiorkor was for high values onadmission, and for a decrease to a minimum valueafter two or three weeks of treatment. Fourchildren in Group 3 (a) had low percentages (under10) of enzyme resistant to cyanide on admission.Two of these children died after a few days, andthe remaining two, one a very severe case and theother moderately severe (Fig. 3), both showed zerosensitivities after seven days of treatment.Although it is not evident from Table I, almost

all children passed through a period at which thesensitivity to magnesium and resistance to cyanidewere at very low levels. It usually occurred after14 to 20 days of treatment and sometimes con-tinued for two or three weeks. A rise occurredwhen the basic enzyme level was approaching thatfinally reached.

DiscussionFew conditions are known in which the activity

of serum alkaline phosphatase is markedly low.All occur in association with growth failure ordepressed osteoblastic activity: low values havebeen reported in children with hypothyroidism(Albright, Sulkowitch, and Bloomberg, 1937;Talbot, Hoeffel, Shwachman, and Tuohy, 1941)and with scurvy (Shwachman and Gould, 1942).In other conditions in which the activity isabnormal, it is raised in association with patho-logical bone development or impairment of liverfunction. In acute kwashiorkor the lowered levelsof serum alkaline phosphatase are probably relatedto the growth failLire which is a constant feature.

In addition the liver is always involved: theamount of fat is usually increased, and the lowalbumin/globulin ratio and the high proportion ofunbound cholesterol in the serum probably indi-cate impaired liver function.The Indian workers who demonstrated increased

concentrations of alkaline phosphatase in the livertissue of children with acute kwashiorkor sug-gested that the accumulation was due to theinability of the liver to dispose of the excessenzyme in the normal way. It may be reasonableto expect the increased level in the tissue to beparalleled by a high level in the blood, but, as wxehave already shown, that does not occur. Someattempt at explanation seems to be called for. Itmay be assumed that in the severely ill childenzyme production in general is greatly reduced;the total amount of alkaline phosphatase which isproduced is probably small, and the amount thatcollects in the liver may also be small, but the livermay be unable to deal satisfactorily even withthat amount. As a result the enzyme accumulatesin the liver, and the concentration in the serum isslightly raised. With treatment liver function isimproved, the excess enzyme is removed from theliver, and there is a parallel fall in the serum level.At the same time enzyme production, probably atseveral different sites in the body, increases, andeventually the result of the opposing factors is arise in the serum level. The rise will becomeapparent at different times, or the fall may not beseen at all, according to the extent of the influenceof one factor or another.

N

30 -

L~~~~~~~~~~~~~~

MK

20 -

10

0 10

20

FIG. 5.-Phosphatase activity in units. Above horizontal line showsbasic activity, with residual activity after CN- (shaded portion).Below horizontal line shows enhanced activity due to Mg++.Columns a-e: severe kwashiorkor on admission and duringtreatment (as in Table I). Column N: children with no signs ofkwashiorkor. Column MK: children with mild kwashiorkor.

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SERUM ALKALINE PHOSPHATASE ACTIVITY IN KWASHIORKOR

There are obvious objections to this hypothesis,but the findings reported here on the changes insensitivity to magnesium and cyanide during treat-ment give it some support. So far, the sensitivityto magnesium and resistance to cyanide have beenrelated to the activity in the absence of added ions,on a percentage basis. The sensitivity to mag-nesium and resistance to cyanide have been repre-sented in units of activity in Fig. 5. In the initialstages of recovery there are reductions in the acti-vity that is not sensitive to magnesium, and in theactivity that is resistant to cyanide. The formerthen increases, but the latter continues to decrease(until about the 20th day of treatment). Up to thistime the activity sensitive to magnesium has re-mained virtually constant. After the 20th day theactivity of all three kinds increases until a constantproportionality is reached resembling that found inchildren without signs of kwashiorkor.

It is difficult to explain this course of eventswithout postulating the presence of at least threealkaline phosphatases in the serum, each with itsown pattern of response to magnesium andcyanide. If this is so, they must be present inunusual proportions in the acute stage of kwash-iorkor. An alternative theory, somewhat lessattractive, is that in kwashiorkor the serum con-tains enzymes that normally do not appear in it,or substances that modify the response of phos-phatase to added activators and inhibitors. Thereis plainly much more work to be done: it shouldinclude the investigation-difficult or impossible atKampala-of children with hypothyroidism, withscurvy, and with various forms of growth failure.Useful information might also be gained from astudy of rickets and other conditions in which thephosphatase level is high. The group of Africanchildren without signs of kwashiorkor all hadinfections, and children in the wards of the infantilemalnutrition group have been observed when theycontracted chickenpox, measles, and other commonillnesses. In some of those children it was foundthat the level of basic alkaline phosphatase ap-peared to be depressed, to the extent of a fewunits, as a result of the infection, but in none ofthem was the pattern of response to magnesiumand cyanide abnormal. In the children who wereconsidered to have slight kwashiorkor the basicphosphatase activity was considerably lower thanit was in the children with none of the signs, butthere were no obvious differences in the sensitivityto magnesium and cyanide in the two groups.Slight kwashiorkor differed from severe kwashior-kor, however, in that the severe condition not onlyshowed an even lower level of basic phosphatase

activity but also increased in sensitivity to mag-nesium and resistance to cyanide. The extent towhich these changes are peculiar to severe kwash-iorkor must await further investigation.At the moment there is little exact information

regarding the source of serum alkaline phospha-tase in man. It is generally supposed that bonemakes a large contribution to the level of enzyme,and the great increase found towards the end oftreatment of the cases described here may reflecta resumption of bony development. This possi-bility is being investigated with the help of serialradiographs of the bones, and the results will bereported at a later stage. It is obvious, however,that bony development is only a part of growth,and other developing tissues may make consider-able contributions to the high levels of the serumenzyme found in children who are rapidly grow-ing.

SummaryThe alkaline phosphatase and its response to

Mg+ + as activator and CN- as inhibitor weremeasured in the serum of African children whohad (1) no signs of kwashiorkor, (2) slight kwash-iorkor, and (3) severe kwashiorkor, before andduring treatment with milk protein.The enzyme level was considerably lower in the

children with slight kwashiorkor than in the child-ren without signs of the disease.

In the majority of cases of severe kwashiorkor:(a) The enzyme level was even lower than in

slight kwashiorkor, fell after seven to 14 days oftreatment and afterwards rose steadily towardsthe level usually considered normal. The falloccurred most consistently in the cases in whichthe serum albumin was 1.8 g./100 ml. or less.

(b) The percentage activated by Mg++ washigh at first, increased after seven to 14 days oftreatment and afterwards fell.

(c) The percentage resistant to CN- was highat first, and decreased during treatment, reach-ing a minimum after 15 to 20 days.

(d) After prolonged treatment (40-70 days)the percentages activated by Mg++ and resis-tant to CN- reached values similar to thosefound in the children without signs of kwash-iorkor.The causes of the fall in enzyme level are dis-

cussed. The fall, and the other changes foundduring treatment, suggest that the serum may con-tain a series of alkaline phosphatases.The clinical grading of the cases investigated was

made by Dr. R. F. A. Dean, and I am grateful to himfor his help in the presentation of the results.

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REFERENCES

Albright, F., Sulkowitch, H. W., and Bloomberg, E. (1937). Amer.J. med. Sci., 193, 800.

Allen, R. J. L. (1940). Biochem. J., 34, 858.Behar, M. (personal communication). I.N.C.A.P., Guatemala City,

Guatemala, Central America.Behrendt, H. (1949). Diagnostic Tests for Infants and Children.

Interscience Publishers, New York.Bersohn, I. (personal communication). South African Institute for

Medical Research, Johannesburg, South Africa.Bodansky, A., and Jaffe, H. L. (1933-34). Proc. Soc. exp. Biol.

(N.Y.', 31, 107, 1179.Dean, R. F. A. (1952). Brit. med. J., 2, 791.--(1954). J. trop. Med. Hyg., 57, 283.

and Schwartz, R. (1953). Brit. J. Nutr., 7, 131.Ely, J. O., and Ross, M. H. (1951). Nature (Lond.), 168, 323.Gomori, G. (1946). Proc. Soc. exp. Biol. (N.Y.), 62, 33.Hansen, J. D. L. (personal communication). Department of Paedia-

trics, Groote Schuur Hospital, Observatory, Capetown, SouthAfrica.

Jayasekera, H. T. W., Mel, B. V. de, and Cullumbine, H. (1951Ceylon J. med. Sci., 8, 1.

King, E. J. (1953). Brit. med. Bull., 9, 160.Miller, L. L. (1950). J. biol. Chem. 186, 253.Ramalingaswami, V., Sriramachari, S., and Patwardhan, V. N. (1954).

Indian J. med. Sci., 8, 433.Roche, J., and Sarles, H. (1950). Biochim. biophys. Acta, 5, 275.- (1951). Bull. soc. Chim. biol. (Paris), 33, 667.Rosenthal, O., Fahl, J. C., and Vars, H. M. (1952). J. biol. Chem.,

194, 299.Shwachman, H., and Gould, B. S. (1942). Amter. J. Dis. Child., 64,

949.Sriramachari, S., and Ramalingaswami, V. (1953). Indian J. Pediat.

20, 1.Stearns, G., and Warweg, E. (1933). J. biol. Chem., 102, 749.Talbot, N. B., Hoeffel, G., Shwachman, H., and Tuohy, E. L.

(1941). Amer. J. Dis. Child., 62, 273.Trowell, H. C., Davies, J. N. P., and Dean, R. F. A. (1954). Kwashior-

kor. Arnold, London.Waterlow, J. C. (1948). Spec. Rep. Ser. nied. Res. Couni. (Lotid.),

No. 263.

340

on July 8, 2020 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.9.4.333 on 1 N

ovember 1956. D

ownloaded from