SMAIL MEHENNAOUIA DELACRO1X-BUCHET, A. DUCHE, B.ENRIQUEZ, M KOLF-CLAUW , G. MILHAUD

Comparative Study of Cadmium Transfer in Ewe and Cow Milks During Rennetand Lactic Curds Preparation.

Archives of Environmental Contamination and Toxicology, 1999, 37, 389-395

Arch. Environ. Contain. Toxicol. 37, 389-395 (1999) A R C H I V E S OF

EnvironmentalContamination

Toxicologya n dC 1999 Springer-Verlug New York Inc.

Comparative Study of Cadmium Transfer in Ewe and Cow Milks During Rennetand Lactic Curds Preparation

S, Mehennaoui,1 A. Delacroix-Buchet,2 A. Duche,3 B. Enriquez,3 M. Kolf-Clauw,3 G. Milhaud3

1 Institut lies Sciences VeieYinaires, University de Batna, ()5(XX) Batna, Algerie2 Unite de Recherches Laitieres et de Gcneiique Appliquee, iNRA, Dornaine de Vilvert, 78352 Jouy-en-Josas Cedex, France3 Laboratoire de Toxicologie, Ecole Nationale V£terimiire d' Alfort, 7 Avenue de General de Gaulle, 94704 Maisons-Alfort, France

Received; 27 October 1998/Accepled: I April 1999

Abstract. Cadmium transfer from whole milk to cream, rennet,or lactic curds was studied before and following a repealed oralcadmium administration to three lactating ewes and one cow.Before cadmium administration, the cadmium levels in milkwere around 0.4 ug/L in ewes and less than 0.2 ug/L in cow.Throughout cadmium administration the mean cadmium levelsin milk were 3.3 ± 1.4 ug/L in ewes and 2.5 ± I jjg/L in cow.During cadmium administration, 86% of cadmium in ewe milkwas dispersed in the skimmed milk and 17% in the cream,whereas only 72% was dispersed in (he cow skimmed milk and27% in (he cow cream. Most of milk cadmium was associatedwith casein fractions. About 70% of milk cadmium wastransferred to the rennet or lactic curds of ewe. The remainingcadmium present in whole milk, about 9%, was transferred tothe rennet or lactic curd whey. In cow, the proportion ofcadmium associated with rennet or lactic curds, rennet curdwhey, and lactic curd whey was, respectively, 60%, 56%, 14%and 12% of total milk cadmium. The fraction of total cadmiumtransferred from milk to its milk products, whatever the species,ranged from 94% to 103%. The factor of concentration ofcadmium from whole milk to milk products ranged from threeto six. We suggest that the excretion of cadmium inio milk ismainly achieved via the milk casein secretion. This is, to ourknowledge, the first in vivo study where the cadmium transferfrom milk to its milk products after repeated cadmium oraladministration to ewe and cow has been studied.

Cadmium toxicity was first revealed in occupationally exposedworkers and then in people living in polluted areas. The kidneyis considered the critical target organ for ingested cadmium.Long-term inhalation studies in rats exposed io cadmium oxidefumes demonstrated a high incidence of primary lung cancer.The first clinical manifestation of ingested cadmium from foodor inhaled cadmium from cigarette fume is an increased urinaryexcretion of low molecular weight proteins (fl2-niicroglobulin,retinol-binding proteins) and of calcium and cadmium. Accept-

Correspondence to: G. Milhaud

able daily intake (ADI) for consumers was evaluated on thebasis of these effects. It is presently I ug/kg/day. In pollutedareas, cadmium intake from food has been found to range from140 to 260 ug/day. In nonpolluted areas, it is generally between10 and 40 ug/day; uptake from heavy smoking may equalcadmium intake from food (WHO 1992).

Contamination of agricultural products by cadmium is due toindustrial pollutants, but also to the use of phosphates andsewage sludge (Andersen and Hahlin 1981; WHO 1992).Herbivores ingest more or less contaminated forage; cadmiumiiccLimulaies in the organism (mainly in ihe liver and kidneys)and is eliminated in small quantity into milk. We havepreviously studied the transfer of cadmium from feed to ewemilk (Houpert el al, 1997b) and the kinetics of cadmium inlacialing ewes (Houpert et al. 1997a). Spivey Fox (1987) hasproved that milk is a significant source of cadmium andcontributes to about 54.5% of the FAO/WHO provisionaltolerable intake for children. More recently, Mullerefa/. (1996)and Rydzewska and Krol (1996) reported that the milk and milkproducts are still contributing to cadmium intake in humans. InFrance, the milk products provides for 18.5% of cadmiumintakes as reported by the CSHPF (1996).

Cadmium distribution in milk fractions was mainly studied invitro (Roh el al. 1976; Mata et al 1995). In order to get data invivo, we had conducted a previous sludy on cadmium transfer toewe milk (Milhaud el til. 1998), which showed that cadmiumlevels in cream were three times higher than in milk and thatcadmium levels in lactic and rennet curds were, respectively,three and five times higher. As the milks used in this 1998 studywere very rich in cadmium (from 4.8 to 21 pg/L). the presentstudy was performed wilh a less cadmium-contaminated ewemilk (concentration about 3 ug/L), and the results werecompared with those obtained in a slightly contamined cowmilk .

Material and Methods

Animals

Three Lacaune Prcalps lacialing ewes and one Prim' Holstein laclatingcow were used. The ewes were kept in (lie sheepfold of the experimen-

390 S. Mchermaoui ei at.

tal farm al Brouessy (INRA, France). Throughout the study, ihe ewesmean weights were 60 kg. They received daily I kg of a granulatedfeed, which contained 50% barley, 26% corn, 20% soya oil, and 4%mineral additive. The cow was kept in Bressonvillicrs center ( INRA.France). It weighed 780 kg. The daily feed consisted in 23 kg cornsilage, 7 kg brewery by-product, 5 kg potatoes, 4 kg citrus pulp, 3 kg"boviluz" lucern, 1.3 kg wheat straw and 1.7 kg "syniapro" rich inphosphorus.

The ewes were administered cadmium daily as cadmium chloride ala dose of 2 nig/kg/day via granulated feed supplementation from 7October 1997 to 16 December 1997. The laclating cow was adminis-tered cadmium daily as cadmium chloride at a dose of 4 mg/kg/day for4 weeks (from 9 March 1998 to 6 April 1998) and 6 mg/kg/day for thelast 7 weeks (from 7 April 1998 to 25 May 1998). The cadmium, mixedwith lactose, was administered by gavage to the cow. The amount ofcadmium ingested from the feed was very low, between 5 and 8Mg/kg/day, as it was shown in two previous studies conducted atBrouessy experimental farm (Houpeil ei al. I997a, 1997b).

were sliced in big chunks and put in a cheese mold to allow wheyflowing. The vessel used for preparing curds was made of polypropyl-ene. The vessel and the lools (curd knife , agitator) used for preparingcurds or for preserving Ihe different milk products pending analysiswere cleaned with Vapro detergent (1%) (Turco S.A, Alhis Mons,France), rinsed under running water, then immersed in HNOj (2%) andcarefully rinsed with deioni/ed water (the deionized water wascollected in a plastic container that was previously treated with nitricacid and then rinsed). Handling of the material before and after curdmaking was performed using single-use v inyl gloves.

The whole milk and its products were weighed with an O1IAUSbalance (capacity 6 kg with a precision of 1 g). Fat, pH, and totalnitrogen contents were determined al ihe URLGA. The fat content wasmeasured using the Gerberacido butyromelric method (AFNOR 1971)afler milk and cream aliquols had been diluted, respectively, to !/> and'/in wi th Mil l i -Q waler. The concentration of lolal nitrogen wasdclermined by Kjeldhal method using a Vapodesl 6 apparatus (Ger-hardt, Bonn, Germany).

Sampling

Blood samples (5 ml) were collected from the jugular vein into specialheparinized tubes under vacuum-guaranteed free of any heavy metals(ref. 367735 Vacutainer Tubes, Becton Dickinson, Maylan, France).The samples were collected weekly on Monday morning 4 weeksbefore cadmium administration and 9 weeks in ewes and 11 weeks inthe cow during cadmium administration. They were stored at 4°C un i i lanalysis.

Milk samples were collected in a polypropylene flask by hand-milking every week (on Monday evenings and Tuesday mornings) andwere delivered lo Ihe milk Research Unity (URLGA, INRA, Jouy-en-Josas, France). The Monday evening milk was refrigerated nl 4°C; theTuesday morning milk was kept at room temperature and mixed withihe milk sampled the evening before. For metal analysis, we used amilk aliquot (100 ml) sample. The remaining milk was treated to obtainmilk products.

Treatment of Milk

Milk products were processed as follows:

Whole milk

\. Centrifugation

Cream Skimmed mi lk

Lactic coagulation

Lactic curd

Rennet coagulation

Rennet curd

Lactic curd whey Rennet curd whey

The skimmed milk and the cream were separated using a slainlesssteel separator (Alpha Laval, 70 L/h) rinced with UNO, (2%) anddeionized water. The lactic curds were prepared by adding a Lurtm'tic-cus lactis strain culture (1% starter 4- rennet 0.05 ml/L) to skimmedmilk at 32°C and the rennet curds were prepared by adding 0.3 nil ofrennet/L of milk. Rennet curds were sliced in small chunks, lactic curds

Cadmium Determination

The cadmium levels in blood, whole milk, skimmed milk, cream, curdwhey, and curds were measured at the EN VA Toxicology Laboratory bygraphite furnace atomic absorption spectiopholometry (AAS) with aPeikin-Elmer Zeeman 4100 speclrophoiomeler (PE 4100 ZL).

The blood and whole milk samples were first diluted 1:10 for bloodand 1:5 lor mi lk in deionized water within 0.1% Triton-X and doseddirectly by AAS as described by Federspiel (1996). The decompositiontemperature was 550°C for blood and 600°C for milk; the atomizationtemperature was 1,450°C. The limit of detection was estimated to be0.2 ug/L. Ihe l imit of quantification 0.8 ug/L. The intraday coefficientof variation was 25% at 0.8 ug/L, 13% al 2 ug/L. 2% at 5 (Jg/L in blood,and 6% at 1 ug/L. 5% at 5 Mg/L in milk. The accuracy of the methodwas controlled with certif ied material: BCR no. 194: 0.5 ± 0.4 versus0.5 ± O.I ng/L, BCR no. 195: 5.4 ± 0.5 versus 5.37 ± 0.24 ug/L forblood; BCR no. 063: 2.8 ± 1.2 versus 2.9 ± 1.2 ug/L, BCR no. 150:21.8 ± 1.6 versus 21.8 ± 1.4 ug/L for milk (Commission of EuropeanCommunities, Community Bureau of Reference, Brussels, Belgium).The same procedures used for whole milk were applied for cadmiumdetermination in skimmed milk and curd whey. All these samples wereanalyzed in duplicate.

About 3 g of cream or 3 g of curds made with unconlamined milkwere weighed to wilhin about I mg and dry-ashed with 1 ml d'H2SO4(9 N) for 21 h up to 700°C. The while ashes were dissolved in I mlnitr ic acid (5 N) and transferred inlo a 50-ml volumetric flask, followedby addition of 25 nil of deionized water and 5 ml of cilrate buffer(pll = 8.5f. Subsequently I ml of 2% APDC (ammoniumpyrrolidin-d i lh ioca i tK i rna l e ) and 4 ml M I I J K (mdhylisobulylkelone) were added.The solution was shaken for 1 min. After 15 min, ihe absorption of theorganic upper phase was measured by graphite furnace AAS, using anormal calibration program and the following operating procedures:drying al 130°C, ashing at 700°C, and atomizing al 1,500°C.

The rennet or laclic curds made from contamined milk weredry-ashed in Ihe same way. The while ashes were dissolved in I mlnitric acid (5 N) and diluted with deionized water to a final volume of50 ml. Cadmium was measured by graphile furnace AAS using an addcalibration program and the following operating procedures: drying at130°C. ashing al 7WC. and alomixing at 1,500°C.

The curd samples wcic analy/.ed in six replicates, the cream samplesin four replicates. Certified material for cream, curds, arid curd wheywere not available from Ihe Community Bureau of Reference. In creamand in ctii'its obtained from unconlamined milk Ihe l imit of detectionwas estimated io he 0.2 ug/kg. (he l imit of quantification 0.8 ug/kg. Incurds obtained from conlaminaled milk, the values were 10 timeshtoher.

Cadmium Transfer in Ewe and Cow Milks 391

Table 1. Milk fat, cream fat, milk total nitrogen, and milk pi I beforeand throughout the cadmium exposure

Ewes(13 Samples)

Milk fat (g/L)Cream fat (g/L)Milk total nitrogen (%)Milk pH

56 ±561 ±

5.4 ±6.56 ±

14***37***0.8***0.04**

Cow(15 Samples)

45 ±494 ±

3.2 ±6.69 ±

4***

26***0.1***0.03**

**p

392 S. Mehetmaom etat.

7 i

14 .' I , - i i 35 42 49 56 63 70 77

• Whole blood (meanvalues)

O Whole milk

• - Whole bloodO- Whole milk

14 21 28 35 42 49 56 63 70 77

Time (days)

Fig. 1. Cadmium kinetics in blood andmilk after repeated cadmium oral adminis-tration to ewes and cow

Table 3. Mean cadmium levels in whole milk and milk products(pg/kg fresh matter) during the cadmium administration

Whole milkSkimmed milkCreamRennet curdRennet curd wheyLactic curdLactic curd whey

Ewe (9 Samples)

3.3 ± 1.43.1 ± 1.46.0 ± 2.3

17.1 ±7.90.4 ± 0.38.9 ± 4.20.4 ± 0.3* ,

Cow ( 1 1 Samples)

2.5 ± 12. 1 ± 0.98.0 ± 3.0

16.6± 8.00.4 ± 0.28.7 i 3.90.5 ± 0.3*

* Values below the quantification limit

from fivefold to sixfold in rennet curds, and approximatelythreefold higher in lactic curd (Table 3, Figure 2).

It is of interest to study the transfer of cadmium to milkproducts and to take into account not only the levels but also theweight of each product obtained from a certain amount of milk.The mean values of cadmium transfer from whole milk were:104 ± 3% in ewe and 100 ± 9% in cow to skimmed milk and

cream; 103 ± 4% in ewe and 102 ± 12% in cow to cream,rennet curd, and rennet curd whey; 99 ± 8% in ewe and 96 ±10% in cow to cream, lactic curd and lactic curd whey. Themean percentage of cadmium was significantly higher(p < 0.001) in the ewe skimmed milk than in the cow: 86 ± 5%versus 72 ± 7% (Figure 3). The cadmium found ia cream wassignificantly higher in cow than in ewe (p < 0.005): 27 ± 8%versus 17 ± 6%. Cadmium found in rennet or lactic curd of ewewere 72 and 68%. It was lower in rennet or lactic curd of cow(60 and 56%). In the ewes, aboul 9% of the cadmium in wholemilk was transferred to rennel or lactic curd whey. In the cow,14% was found in rennet curd whey and 12% in lactic curdwhey.

Discussion

In Ihe milks used in the experiment, the fat and total nitrogenlevels were within the range of variations of the two species.Yet, the cow milk fat was high (45 ± 4 g/L).

Cadmium Transfer in Ewe and Cow Milks 393

Cream Rennet RennetSkimmedmilk curd

whey

Fig. 2. Mean ratio of milk productscadmium concentration to milk cad-mium concentration

100

h

Skimmed Cream Rennetmilk curd

'p

394 S. Mehennuoui el al.

ranging belween 2 and 5 pg/L. These levels were similar tothose observed in ewes in a contaminated area (1.6 to 12 pg/L)by Kirova (1993). In cream, our results showed that thecadmium levels were 3.2 times higher than in cow milk, whichrepresented 27% of milk cadmium and 1.8 times higher than inewe milk, which represented 17% of milk cadmium. Britain etal. (1973) observed a cadmium cream level twice as high as thecadmium cow milk level sampled in a farm. Marietta andFavretto (1983) found cadmium concentration 2.2 times greaterin cream than in cow milk. In a previous ewe study, with milksvery rich in cadmium (from 4.8 to 21 u.g/L), the mean cadmiumlevels in cream were three times higher in the mi lk , whichrepresented 24% of milk cadmium. It was suggested that thehigher the cadmium level was in the original milk, the higherthe percentage of cadmium was in the cream fraction. Thecream fat amount in ewe milk was greater than that in cow milk .This result shows that there was no relation between the amountof fat and the cadmium levels in the same fraction. Theadministration of radioactive cadmium to mice showed that themetal associated in the cream fraction was very high (49%)(Oskarsson et al. 1998). That result was the expression ofspecies variation and was probably in relation with the fact thatthe proteins bound in the phospholipids of the fat globulemembranes are probably different in each species. The metalswere transferred to the milk fraction in different ways. In thecream obtained from rats and mice milk, Oskarsson el al.(1998) found 2% of lead, 15% of inorganic mercury, and 39%of methylmercury of the amounts contained in whole milk. Inour study, the mean cadmium levels in rennet curd obtainedfrom ewe or cow milk were similar (17 ug/kg/fresh matter).They corresponded to approximately five to six times the wholemilk cadmium levels. Our results are not different from thoseobtained by Marietta and Favretto (1983) and Milhaud et al.(1998), who observed cadmium levels six times higher incheese than in whole milk.

Cadmium transfers from whole milk to cream and skimmedmilk or curds and whey were about 100% whatever the species.These results are an indirect proof of the precision and accuracyof the analytical method. Cadmium percentage recovered inrennet curd was 72% in ewe, 60% in cow. Cadmium percentagerecovered in lactic curd was 68% in ewe and 56% in cow. Thislower value of cadmium recovered in curds from cow milk isunderstandable on account of the higher level recovered in thecream. Besides, the level of cadmium transferred to cow lacticcurd whey was higher than that observed in ewe lactic curdwhey (12% versus 9%).

The distribution of cadmium in bovine milk fractions wasvery different when cadmium was added in vitro at 4°C; only2.4% of whole milk cadmium was associated with creamfraction, 89.3% in rennet curds and only 40.6% in acid curds(Mala et al. 1995). The in vitro study did not reflect the realityof cadmium excretion via the mammary gland and its milkdisxtikutifiiu Many (arjm affect dsufc uutspoift into mttk sathas milk pH, plasma protein binding, milk fat partitioning(Palminger Halle'n et al. 1996; Notarianni et al. 1995). Theelimination of trace elements into milk is a complex processthat requires carrier proteins. For instance, lead, cadmium, andcalcium are bound with casein micelles in the mammary gland.Percentages of groups of caseins can greatly vary from onespecies to another and even sometimes within a same species(Ribadeau-Dumas 1993); this could explain the different cad-

mium recovery percentages in caseins and curds in ewes andcow. In rats and mice, the percentage of labeled cadmiumdistributed in casein was 43%, whereas labeled lead was almostexclusively found in casein fraction (96%) (Oskarsson et al.1998). Lead and calcium interactions are well established(Simons 1986). Thus, ionic lead could replace ionic calcium incasein micelle whose levels vary from one species to another.For example, about 25% of milk calcium was associated withcasein in cow milk (Neville et al. 1994) and 77% of milkcalcium in rat milk (Beach and Henning 1998). Lead was foundassociated with casein micelle both inside the alveolar cell andin the milk lumen, indicating that lead was excreted into themilk, bound to casein via the Golgi secretory system (PalmingerHallen et al. 1996). Cadmium excretion via mammary glandcould follow [he same mechanism, as cadmium and calciumpresent physical and chemical similitudes (Jacobson and Turner1980).

Acknowledgments. We are grateful to M. llervieu (INRA, Brouessy)and M. Marchal (INRA, Bressonvilliers) and their teams who super-vised itie herd throughout the study period. We are very grateful to M.Piltel and M. Bach (URLGA, INRA, Jouy-En-Josas) who prepared themilk products.

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