Breast milk as a source of vitamins, essential minerals and trace elements

Pergamon 0163-7258(94)E0018-W

Pharmac. Ther. Vol. 62, pp. 193-220, 1994 Copyright© 1994ElsevierScienceLtd Printed in Great Britain. All rights reserved 0163-7258/94$26.00

Associate Editor: P. N. BENNETT

BREAST MILK AS A SOURCE OF VITAMINS, ESSENTIAL MINERALS A N D TRACE ELEMENTS CHRISTOPHER J. BATES* a n d ANN PRENTICE M R C Dunn Nutrition Unit, Milton Road, Cambridge, CB4 I X J, U.K. Abstract--Human breast milk provides all of the vitamins and essential minerals and trace elements (micronutrients) that are required by the normal term infant, until weaning. With a few exceptions, excessive micronutrient supplies to the mother, or a moderate deficiency in her diet, do not greatly alter the supply to the infant. Thus, the infant is weU-protected by maternal homeostatic processes, although the mechanisms of these are not yet well understood. Considerable progressive changes in concentration occur for some of the micronutrients during the course of lactation. Because the concentration of these nutrients, and of other substances that modify their absorption by the infant, such as binding proteins, differs considerably between human milk, animal milk and, hence, commercial milk formulae, there is great interest in the quantitative significance of micronutrient supplies, and their variability in breast milk, in the quest for improvement of commercial formulations. The aim of this review is to summarize the available information about the factors that determine breast milk contents of micronutrients. Keywords--Human, milk, micronutrients, vitamins, minerals, trace elements.

CONTENTS 1. 2. 3. 4. 5.

Introduction Comments on Human Requirements for Micronutrients Comments on Toxicity of Excessive Intakes of Micronutrients Comments on Assay Methodology Fat-soluble Vitamins 5.1. Vitamin A and carotenoids 5.2. Vitamin D 5.3. Vitamin E 5.4. Vitamin K 6. Water-soluble Vitamins 6.1. Vitamin Bt (thiamin) 6.2. Vitamin B2 (riboflavin) 6.3. Vitamin B6 (pyridoxine) 6.4. Vitamin Bt2 (cobalamin) 6.5. Folate 6.6. Niacin 6.7. Biotin 6.8. Pantothenic acid 6.9. Vitamin C (ascorbate) 7. Minerals 7.1. Sodium and potassium 7.2. Calcium and phosphorus 7.3. Magnesium 7.4. Iron 7.5. Zinc 7.6. Copper 7.7. Chromium

*Corresponding author. Abbreviations: RDA, recommended dietary amount; RNI, reference nutrient intake. 193

194 195 196 197 197 197 198 199 200 200 200 201 201 202 202 203 203 203 203 204 204 205 206 206 207 207 208

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C.J. BATES and A. PRENTICE

7.8. Manganese 7.9. Molybdenumand nickel 7.10. Selenium 7.11. Iodine 7.12. Fluorine 8. Synopsis and Conclusions References

208 208 208 209 210 210 211

1. INTRODUCTION The need for essential micronutrients by the growing infant, after parturition, is frequently greater on a body weight basis than the corresponding needs by older children and adults: first, because of rapid growth and tissue-accretion, which demand specific nutrients for both structural and functional purposes, and second, because the high level of activity of metabolic pathways involved in growth, physical activity, the combating of infection, etc., places additional demands on certain essential catalysts, whose increased use may induce turnover, losses and, hence, the need for replacement. These demands must be met by maternal breast milk, until weaning occurs. In primitive societies, where human lactation originally evolved, it seems likely that breast milk provided all or most of the micronutrients received by the infant for the first few months of its life. Present day practices in Third World countries suggest that weaning foods often fail to provide adequate amounts of micronutrients, and partially-weaned children have to rely on the breast milk in their diets to provide these. Evolutionary pressures must have arisen for the mammary gland to secrete micronutrients in generous amounts, even when maternal intakes were low. Thus, it is not surprising to find that the breast milk concentration of micronutrients varies much less between populations than maternal intakes do; and that often there is no evidence of a relationship between breast milk concentrations and maternal intakes. However, the available information on this subject varies considerably between nutrients, some having been studied much more extensively than others, for example with respect to maternal deficiency and supplementation. Excessive intakes or exposures due to medication can also affect breast milk levels of some nutrients, and rare instances of toxicity for the infant have been recorded. This review will address only those substances that are currently recognized as vitamins or as essential minerals and trace elements for humans. Of the trace elements, those that have toxicity significance only (such as lead and mercury), will not be covered, nor will others (such as sulphate, chloride, etc.), which are of less interest from a nutritional viewpoint. Ultra-trace elements, whose nutritional significance for humans is uncertain, and whose measurement is currently fraught with difficulty, are only briefly mentioned. This is an evolving subject whose future development should have great relevance, not only to the fundamentals of human physiology and nutrition, but also to the practical questions of achieving improvements in milk formula design, and of improving feeding practices for sick and vulnerable infants, including those who require parenteral nutrition. In considering the possible effects of changes in maternal intake, and, in particular, of maternal supplementation, upon breast milk nutrient levels, it is important to consider maternal status (supplements may have a much greater effect on undernourished subjects than on well-nourished ones), stage of lactation, duration of supplementation, route of supplementation, etc. Because of methodological variations, it is often difficult to make meaningful comparisons between dissimilar population groups, unless they each have been studied by the same investigators, under the same strictly defined conditions of milk collection and storage, and at an identical stage of lactation. However, such interpopulation comparisons can sometimes yield very useful clues about responses to extremes of nutrient intake (e.g. selenium in different parts of China; Levander, 1989), which cannot be obtained by any other means. Studies on species other than humans have been of only limited applicability to human lactation. Factors, such as the stage of development at birth, numbers in litters, growth rates, nature of digestive systems and many other quantitative biochemical differences, can affect both the composition of milk and its responsiveness to maternal dietary variations. For instance, rat milk

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is very responsive to variations in maternal iron intake, whereas human milk is not (Keen et al., 1980; Murray et al., 1978). Because of the differences in bioavailability, particularly of the mineral elements, equal concentrations in different types of milk may have different nutritive values. Even vitamins may be affected by the milk matrix, if (like folate and vitamin A, for example), they need to be released from 'storage' forms before they can be absorbed and utilized by the infant. Some detailed studies of breast milk micronutrient concentrations were carried out during the period 1940-1980, and three that stand out were the studies of Macy and colleagues in the United States in the 1940s (e.g. Roderuck et al., 1945a,b); that of Kon and Mawson (1950) in the United Kingdom and the United Kingdom Department of Health and Social Security (1977) study entitled "The composition of mature human milk". Relevant recent reviews are those of L6nnerdal (1986), Bates and Prentice (1988), Jensen (1989) and Harzer and Haschke (1989). A recent paper by Parr et al. (1991), and earlier studies cited therein (e.g. WHO (1989)), have examined the concentration of many of the minor and trace elements in human milk from several contrasting populations.

2. COMMENTS ON HUMAN REQUIREMENTS FOR MICRONUTRIENTS Committees setting recommended dietary intakes of nutrients have had to grapple with the task of estimating, for each nutrient, the most relevant criterion, or combination of criteria, for defining adequate status and the daily dietary amount that will achieve adequate status in the majority (e.g. 97.5%, or mean plus 2SD) of normal, healthy individuals. During the latest revision of the United Kingdom tables (Department of Health, 1991), a single Recommended Dietary Amount (RDA) for each nutrient was felt not to be appropriate, partly because it failed to depict the wide range of individual requirements and partly because the use of any single value as a specific 'recommendation' seemed inappropriate. Therefore, an attempt was made to define three separate "dietary reference values" to describe the range of requirements for each nutrient. These were: the mean or "estimated average requirement", the mean minus 2SD to define the lower end of the requirement curve ("lower reference nutrient intake"), and the mean plus 2SD to define the upper end (reference nutrient intake or RNI). It was recognized that there might be a normal distribution of intakes to cover this range, or, more likely, a skewed distribution. Requirements by lactating women are likely to be greater than those of their nonlactating, nonpregnant counterparts, and in general, it is assumed that this is equivalent to the amount (of each nutrient) that represents the breast milk content, plus an increment that represents inevitable losses during absorption, and utilization for breast milk production. For many nutrients, the efficiency of utilization appears remarkably high, especially in women who are marginally malnourished, but direct measurement of requirements during lactation and of possible adjustments in absorption, utilization and conservation are incomplete, and more research is needed in this area. In this review, it is assumed that the daily milk volume produced is 750 mL during the mature lactation period, for the purpose of calculation of nutrient intakes by breast-fed infants. Requirements by breast-fed infants are even less well-defined. Fully breast-fed infants, even from somewhat nutrient-deficient mothers, usually do not exhibit overt micronutrient deficiencies, and breast milk nutrient concentrations vary less than maternal intakes do, both within and between subjects. It is believed that breast milk usually contains adequate amounts of micronutrients for infants and young children, and, thus, can be taken as the main yardstick of dietary recommendations, or reference values. However, certain exceptions to this rule should be noted. Individual mothers may secrete milk that is consistently at the extreme end of a wide individual range of composition, apparently unrelated to the offspring's specific requirements. For instance, some breast-fed infants actually develop overt signs of zinc deficiency, which can be corrected by zinc supplements; others may develop blood coagulation abnormalities, which are curable by giving extra vitamin K. Preterm breast milk composition does not reflect the (probably) increased nutrient requirements of preterm infants. Sick infants, and those with special needs--either genetically or environmentally determined--may require intakes that are not easily provided by breast milk

196

C. J. BATESand A. PRENTICE TABLE1. Recommended, "Safe and Adequate" or Reference Intakes of Mieronutrients by Lactating Women and Young Infants: Three Recent Compilations Lactating women

0-6 month infants 2

Nutrient

WHO 3

U.S.A. a

U.K. 5

WHO 3

Vitamin .4

1200

1300

950

350

10

10

(#g retinol equiv) Vitamin D (#g) Vitamin E (mg) Vitamin K (/~g) Thiamin (mg) Riboflavin (mg) Pyridoxine (mg) Vitamin B~2 (/~g) Folate (/ag) Niacin equivs (mg) Biotin (/~g) Pantothenate (mg) Vitamin C (mg) Sodium (mg) Calcium (mg) Magnesium (mg) Iron (mg) Zinc (mg) Copper (mg) Chromium (/~g) Selenium (/~g) Fluorine (/~g) Iodine (#g)

10

1.1 1.7 2.5 600 18 60 1100 14-28

10 12 65 1.6 1.8 2.1 2.6 280 20 30-100 4-7 95 1200 355 15 19 1.5-3.0 50-200 75 1500-40,000 200

1 1.6 1.2 2.0 260 15 70 1600 1280 312 15 13 1.5 79 140

0.3 0.5 0.1 20 5.4 20 550 5-20

U.S.A. 4

375 7.5 3 5 0.3 0.4 0.3 0.3 25 6 10 2 30 400 40 6 5 0.4-0.6 10-40 10 100-500 40

U.K)

350 8.5

0.2 0.4 0.2 0.3 50 3 25 200 524 53 1.7 4 0.3 8 50

~First trimester of lactation, if specified. 2Mean value, if different between age groups (assumes formula feeding). 3Most recent publication available (Committee 1/5 of IUNS, 1983; FAO, 1988). 4National Research Council (1989). 5Department of Health (1991). The figures quoted are the RNI, which are analogous to the RDA from the other two sources.

alone. An important medical challenge is that of estimating the additional requirements of 'protective' nutrients, especially antioxidant ones, needed to minimize damage caused by infectious agents, high oxygen tensions, mutagens, trauma, etc. Table 1 shows the United States' and World Health Organization's recommended daily amounts and the United Kingdom's reference nutrient intakes for lactating women and for 0- to 3-month-old infants. The values assume that the infants are receiving cows' milk formulae, whose nutrients are sometimes less available than those of breast milk. Parr et al. (1991) have shown that for many of the minor and trace elements, the current recommendations for infants are much greater than could be provided by breast milk, and they suggest that these recommendations may be inappropriately high.

3. C O M M E N T S ON T O X I C I T Y OF EXCESSIVE I N T A K E S OF M I C R O N U T R I E N T S For most micronutrients, there appears to be a very effective barrier against excessive amounts entering the breast milk from the maternal diet: first, by limited absorption into the maternal circulation, and rapid excretion once the renal threshold is exceeded, and second, by limited transfer through the mammary gland into the breast milk. Although several micronutrients (notably vitamins A, D and B 6 (in very large doses), iron, zinc, fluoride and selenium) can produce toxic effects when given directly, e.g. by mouth, to adults or infants, the only recorded cases of toxicity to breast-fed infants through excessive accumulation in breast milk include one case of vitamin D toxicity (Greer et al., 1984b), and possibly some cases of iodine and selenium toxicity (see Sections 7.10 and 7.11).

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197

Although the threshold of overdose toxicity for the micronutrients is poorly defined, especially for young children, it appears that the risk of overdosage from breast milk is very small. Conversely, the belief that nutrients can be supplied to the infant in raised amounts by supplementing the breast-feeding mother has proved to be largely unfounded. These questions will be discussed in Sections 5-7 for each of the individual nutrients.

4. COMMENTS ON ASSAY METHODOLOGY Accurate measurement of micronutrients in breast milk is not an easy task. First, a representative sample of milk must be obtained from the subject, or population, by carefully defined procedures. Since foremilk differs greatly in its composition from hindmilk, it is advisable either to strip a full breast completely or else to collect samples at measured representative intervals during a feed. The composition of the milk may vary between left and right breasts; for many nutrients there is an important diurnal variation, and there may also be a seasonal variation. Most importantly, the concentration often varies with stage of lactation, which, thus, needs to be defined. This variation may or may not parallel the changes in the fat content of the milk with stage of lactation. Traditionally, the time-course of lactation has been subdivided into the periods of colostrum production (0-5 days), transitional milk (6-10 days) and mature milk (15 days to 15 months). Vitamin C is unstable unless the sample is quickly acidified and then stored at a very low temperature (e.g. in 5% metaphosphoric acid, at -40°C or lower). Other vitamins (riboflavin, vitamin B6) are light sensitive. For trace element analysis, rigorous precautions against contamination with dust, sweat, creams and contamination from collecting pots must be taken. Following storage of a frozen sample, adequate mixing is essential because of inevitable phase separation. The analytical techniques available for each nutrient include microbiological analysis (e.g. for the B-vitamins), HPLC separation methods and competitive protein-binding assays; atomic absorption spectrometry (mineral elements) and mass spectrometry are frequently taken as the ultimate 'gold standard'. The choices of initial preparation techniques (extraction, chemical modification, etc.), separation procedures and detection methods can all affect the accuracy (and precision) of the assays. The reader is referred to recent reviews by Jensen (1989), Canfield and Hopkinson (1989), Neville et al. (1985), Casey et al. (1985b), WHO (1989) and Parr et al. (1991) for further information on this subject. Wherever possible, the accuracy of the chosen method should be verified by inclusion of quality controls with assigned values, covering the entire (minimum to maximum) range that is observed in the unknowns. If human milk standards are available, they should be used (Parr et al., 1991). The percentage recovery of spikes of the pure nutrient added to human milk samples should also be tested. Because the earlier studies on trace and ultra-trace element levels in breast milk were performed before the necessity and techniques for these precautions were realized, many of the earlier results are now having to be discarded.

5. FAT-SOLUBLE VITAMINS 5.1. VITAMINA AND CAROTENOIDS Vitamin A is required for vision, for reproduction and for the maintenance of epithelial structures. It has a specific and well-characterized role in the visual pigment cycle and a less well-understood role, via retinoic acid, in some aspects of genome regulation. In omnivore diets of developed countries, there is a considerable amount of preformed vitamin A, but in vegetarian diets, including the typical diets of developing countries, carotenoids provide the majority of the vitamin A supply, and are partly converted to vitamin A by an enzyme in the intestinal mucosa. Until recently, the carotenoids were thought to function primarily as precursors of vitamin A in animal tissues, but an independent protective role has been recognized recently (Ziegler, 1991).

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Deficiency of vitamin A is a serious health problem for young children in many developing countries, leading to blindness through damage to the corneal epithelium, and to increased morbidity and mortality from infectious diseases (measles, respiratory trace disease, diarrhoeal disease, etc.). Although still somewhat controversial, the present consensus of opinion tends to favor massive occasional oral dosage (e.g. 67-rag vitamin A every 3 months), for all preschool children in high-risk environments (Fawzi et al., 1993). The extent to which infantile vitamin A deficiency can arise, or be exacerbated, by inadequate breast milk vitamin A levels during suckling is uncertain. There is some evidence that breast milk vitamin A levels are lower in poorly nourished communities (Meulemans and de Haas, 1936; Belavady and Gopalan, 1959; Arroyave et al., 1974; Gebre-Medhin et al., 1976; Pereira and Begum, 1976; Thein, 1979; Boediman et al., 1979; Muhilal et al., 1988) than in well-nourished Western ones (Lesher et al., 1945; Kon and Mawson, 1950; Gebre-Medhin et al., 1976; Thomas et al., 1981; Kim et al., 1990), but methodological and nondietary influences cannot be excluded. Most maternal supplementation studies have employed relatively large single- or short-term supplements. Several of these have achieved a positive increment in breast milk vitamin A levels (Hrubetz et al., 1945; Kon and Mawson, 1950; Sobel et al., 1950; Belavady and Gopalan, 1959; Venkatachalam et al., 1962; Tarjan et al., 1963), but it might be of more practical relevance to study the efficacy of smaller doses, similar to the reference nutrient intake, over a more prolonged period. In one early study on Indian women (Belavady and Gopalan, 1960), daily doses of 0.7 to 3.0-mg vitamin A had no detectable effect on breast milk levels, whereas in a more recent study of Gambian women, 0.65-mg vitamin A given for periods of a few months up to 1.5 years produced a moderate (23%) increment in breast milk vitamin A, but only for those women who had been lactating for 5-9 weeks (Villard and Bates, 1987). In considering the implications of these supplementation studies, it should be noted that breast milk vitamin A (and carotenoid) levels typically decline ca. 10-fold between colostrum and late lactation, so that the stage of lactation is an overwhelming determinant of the vitamin A supply to the infant. The other major factor that controls the vitamin A supply is that most of the vitamin A that is delivered to the mammary gland is derived from the retinol:retinol-binding protein complex in plasma, whose concentration is determined, and is maintained at a homeostaticallycontrolled level, by the liver. This effectively buffers any variation in supplies, so that only a prolonged maternal deficiency, or massive supplements, can override this control and affect breast milk levels. Although it is, thus, unrealistic to expect that infant vitamin A deficiency can be corrected primarily by vitamin A supplementation of breast-feeding mothers, nevertheless, the prolongation of even partial breast-feeding could provide an important source of vitamin A to vulnerable weaning Third World children (Prentice and Paul, 1990). In Third World countries, typical weaning foods are notoriously deficient in vitamin A and its precursors, and it seems inherently better to deliver vitamin A in small, regular daily amounts, together with the other essential nutrients in breast milk, than to give periodic massive doses as prophylaxis.

5.2. VITAMIND Vitamin D is required as part of the complex regulatory system for transport and control of calcium, with major sites of action in the genomes of the intestinal mucosa, the kidney and bone. It is normally synthesized in skin, from 7-dehydrocholesterol, by a nonenzymic reaction that is catalyzed by U.V. light, and it is then converted in the liver to the 25-hydroxy-derivative, and thence, in the kidney to the 1,25-dihydroxy-derivative, in a strictly controlled manner. The 1,25-dihydroxy-derivative is the functionally active form, possessing hormone-like properties that allow it to control calcium transport. Attempts to measure vitamin D and its derivatives in human and cows' milk have had a chequered history. Studies since 1980, using HPLC, have confirmed that only fat-soluble derivatives of vitamin D are present, and that the 25-hydroxy form constitutes 30-75% of the vitamin D by weight, and is the major form there in terms of biological potency (Hollis et al., 1981; Reeve et al., 1982; Hollis, 1983; Kunz et al., 1984). This may be especially important for newborn infants,

Micronutrients in breast milk

199

whose enzyme system for the 25-hydroxylation of vitamin D is not well-developed (Hoff et al., 1979). There have been several reports of clinical (rachitic) or biochemical vitamin D deficiency developing in breast-fed infants (Markestad, 1983; Markestad et al., 1984; Anonymous, 1984). The vitamin D content of breast milk from a vitamin D-deficient subject was undetectable (Hollis et al., 1983). Thus, vitamin D perhaps constitutes an exception to the general rule that breast milk micronutrient levels are protected from the effects of maternal deficiency. A mean value for total breast milk vitamin D, from 14 studies published since 1980, was 0.71/~g/L (equivalent to 0.53/~g/day at 750mL milk/day) for unsupplemented, but apparently well-nourished, mothers. This compares with an RNI of 8.5 #g/day in the United Kingdom for children aged 0-6 months (Department of Health, 1991). Thus, it is difficult for breast-fed infants to obtain the reference amount without supplementation. For babies born in the autumn, plasma 25-hydroxy-derivative can decline to a very low level in the following winter, because winter milk contains very little vitamin D (Greer et al., 1981; Markestad et al., 1984). Therefore, it is considered prudent for infants and young children (up to at least 2 years of age) to receive supplementary vitamin D, at around 7~tg/day (Department of Health, 1991). Milk from dark-skinned mothers in the United States have a lower level of breast milk vitamin D than that from white-skinned mothers (Specker et al., 1985), presumably because the rate of synthesis is lower in dark skin. Hollis et al. (1983) found that supplements of 12.5- or 62-/~g vitamin D were able to increase breast milk vitamin D levels 5- and 9-fold, respectively, and supplements of 25- or 50-#g vitamin D to Finnish mothers significantly increased their breast milk vitamin D levels given in February and April, the seasonal low point (Ala-Houhala et al., 1988). Exposure of the mother to U.V. light also produced a useful increase in breast milk vitamin D (Hollis et al., 1982; Greer et al., 1984a; Hollis et al., 1986). Vitamin D is one of the few essential nutrients that sometimes may accumulate in excessive and toxic amounts in breast milk. This was well illustrated by a case report of a woman who was being treated with 2500-#g vitamin D/day for hypoparathyroidism (Greer et al., 1984b). Her breast milk vitamin D concentration was ca. 2 orders of magnitude above the normal range, and her breast-fed infant had a high serum calcium, which was indicative of pathological hypercalcemia. The authors recommended that, in such cases, possible symptoms of vitamin D toxicity be closely monitored. The small (25 and 50/~g/day) doses used by Ala-Houhala et al. (1988) produced responses that were "highly variable", and these authors also warned that "the safety of such a dose over prolonged periods should be examined". Clearly, the changes of both under- and overprovision of vitamin D and its derivatives in breast milk needs further study. Exposure of either the mother or the infant (or both) to moderate sunlight or equivalent U.V. light may be safer than oral supplementation, since the skin, at least, seems incapable of producing excessive, toxic amounts. However, in Western countries, oral supplements are considered more practical and are still being recommended. 5.3. VITAMINE Vitamin E (a mixture of tocopherols of varying biological activity) is considered to act primarily as a lipid-soluble antioxidant, protecting polyunsaturated fatty acids and related substances from peroxidation and, thus, from rancidity. There is still a great deal that needs to be learned about its nutritional significance. Although there is no well-defined deficiency syndrome that has been described in normal human subjects (apart from preterm babies), nevertheless, vitamin E is considered to be an essential and nutritionally important nutrient and an important component of breast milk. Because of the absence of a characteristic human deficiency syndrome, or of a 'target' biochemical or physiological value that could define adequate status, the United Kingdom Dietary Reference Values (Department of Health, 1991) do not list a reference intake, but instead, a "safe intake" for vitamin E derived from normal population intakes, namely 0.4-mg vitamin E/g polyunsaturated fatty acids, for young infants. Breast milk typically contains around 400 mg long-chain polyunsaturated fatty acids/L (Harzer et al., 1983) and 4-mg vitamin E/L (mean of 21 studies).

200

C.J. BATESand A. PRENTICE

The concentration of vitamin E (mainly a-tocopherol) is higher in human milk than in cows' milk, and is much higher in colostrum and transitional milk than in mature milk (Harzer et al., 1986; Haug et al., 1987). This pattern, of course, is similar to that for vitamin A and the carotenoids. The effects of maternal deprivation or supplementation are poorly documented. One normal mother received a single large dose (1.1 mg, ca. 100 times the Department of Health's "safe intake") during week 11 of lactation, and responded by a 6.6-fold increase in her breast milk vitamin E by the third day, falling to baseline again by the fifth day (Kanno et al., 1989). However, the peak value of 4. l mg/L was not particularly high, in comparison with normal values from other studies. In another study (Anderson and Pittard, 1985), continuous maternal supplementation at a similar level raised the breast milk concentration to 11 mg/L. These amounts seem very unlikely to be associated with any adverse effects in the infant; vitamin E seems to be nontoxic, even at very high intakes, across all age-groups. The vitamin E content of the milk from mothers of preterm infants does not appear to differ from that of term infants' mothers (Haug et al., 1987). Preterm infants frequently can benefit from increased vitamin E intakes, which may protect them from anemia and from oxidation damage by the high oxygen tensions that are required by their immature lungs. 5.4. VITAMINK Vitamin K is an essential component of the blood-clotting cascade, by virtue of its requirement as the cofactor for y-carboxylation of certain peptidyl-glutamate residues to enhance their ability to chelate calcium. It has a similar role in the modulation of glutamic acid residues in osteocalcin and in other peptides that control bone calcification, but these functions are less well understood. In a small proportion of young infants, a life-threatening hemorrhagic syndrome may develop (O'Connor et al., 1983; Motohara et al., 1984; yon Kries et al., 1984), which can be alleviated by pharmacological doses of vitamin K. This syndrome is confined almost wholly to breast-fed infants when it persists beyond the perinatal period, an observation that has drawn attention to the fact that breast milk contains only very small amounts of vitamin K (Canfield and Hopkinson, 1989). The amount of vitamin K in the breast milk of those mothers whose infants had suffered from intracranial bleeding was especially low (Motohara et al., 1984). Breast milk probably contains only phylloquinone (vitamin K~), and although bacterially synthesized menaquinones (vitamin K2) have been reported by some workers, these probably were an artifact (Canfield and Hopkinson, 1989). Maternal supplementation with 88-#g vitamin Kt/day produced no detectable effect on breast milk vitamin K levels (Pietsching et al., 1993), whereas daily doses of 0.5-3 mg produced an "order of magnitude" increase to 25-30 #g/L within 24 hr (von Kries et al., 1987). Haroon et al. (1982) also observed a 70-fold increase in breast milk vitamin K after a single massive (20 mg) dose to the mother. Thus, breast milk vitamin K can respond to maternal supplementation, and this may provide an alternative to prophylactic dosing of the infant, especially if prophylaxis can be associated with dangerous side effects (Golding et al., 1992). Because of the difficulty of measuring the low levels of vitamin K in milk and other body fluids, this subject is still at an early stage of investigation and further research effort is required.

6. WATER-SOLUBLE VITAMINS 6.1. VITAMINB~ (THIAMIN) Thiamin functions primarily as the precursor of cocarboxylase, or thiamin pyrophosphate, which is the essential cofactor for several enzymes of carbohydrate metabolism, notably pyruvate decarboxylase, 2-oxoglutarate decarboxylase and transketolase. Severe deficiency results in the syndrome of beriberi, characterized by oedema, cardiac failure and neurological symptoms. Beriberi has been recorded in breast-fed infants (Aykroyd and Krishnan, 1941; Debuse, 1992), but it is not clear whether reduced thiamin levels in the breast milk were primarily to blame.

Micronutrients in breast milk

201

A mean breast milk thiamin value for 8 studies in Western countries was 0.17 mg/L compared with a mean value of 0.15 mg/L for six studies in developing countries (India, Kenya and The Gambia), all on unsupplemented women. Maternal supplementation can increase the thiamin content of breast milk, either early in lactation (Kon and Mawson, 1950) or in malnourished women (Deodhar and Ramakrishnan, 1960), but mature milk from malnourished women seems resistant to the effects of supplements. There are progressively increasing amounts in the early stages of lactation (Roderuck et al., 1945b; Kon and Mawson, 1950; Ford et aL, 1983). Toxicity of high doses is not a serious problem, and there is negligible risk of toxicity to the infant via maternal supplementation.

6.2. VITAMIN B 2 (RIBOFLAVIN)

Riboflavin provides two essential coenzymes of lipid and general intermediary metabolism: riboflavin phosphate (flavin mononucleotide) and flavin adenine dinucleotide. It also gives rise to covalently bound flavin coenzymes for certain other enzymes. Clinical deficiency, which is common in developing countries, is characterized by lesions of mucocutaneous surfaces, and this is common among women in late pregnancy and early lactation. Breast-fed infants of deficient mothers are less deficient than their mothers are, by evidence of clinical signs and by the biochemical indices of status. Breast milk, therefore, is generally protective, despite the fact that human breast milk contains much less riboflavin than does the milk of species such as the cow and the rat. If the status of the mother is poor, then maternal supplementation has a prolonged beneficial effect on breast milk riboflavin levels (Deodhar and Ramakrishnan, 1960; Deodhar et aL, 1964; Bates et al., 1982b), but this effect is more transient and smaller for women who are already well-nourished (Thomas et aL, 1980; Nail et al., 1980). The peak of secretion into breast milk occurs ca. 4 hr after a supplement or enriched meal (Kon and Mawson, 1950; Funai, 1986). The riboflavin content of breast milk does not change very markedly during the course of lactation (Roderuck et al., 1984a; Kon and Mawson, 1950; Bamji et aL, 1986), but its response to supplementation may be greatest in the later stages (Kon and Mawson, 1950). The mean riboflavin content of breast milk from I0 studies in Western countries was 0.38 mg/L, and from 8 studies in developing countries, it was 0.20 mg/L for unsupplemented women.

6.3. VITAMINB6 (PYRIDOXINE) Vitamin B 6 gives rise to the cofactor forms pyridoxal phosphate and pyridoxamine phosphate in tissues which, in turn, control many group-transfer reactions, such as transamination and decarboxylation. Overt deficiency is rare and not well characterized in humans, although it was associated with convulsions in infants fed heat-damaged milk formulae during the 1950s. There is likely to be a heavy demand for vitamin B6 during pregnancy, lactation and early infant growth, but, unfortunately, the evidence from different biochemical status indices is conflicting and difficult to interpret. Requirements are linked to protein intakes because vitamin B 6 is heavily involved with amino acid metabolism. Vitamin B6concentration in early milk is generally lower than in mature milk (Karra et aL, 1986). It has been suggested that a very low concentration in milk during the first 2 weeks of life may be a cause for concern (Wilson and Davis, 1984). Following supplementation, peak levels occurred about 4 hr later (Styslinger and Kirksey, 1985). A number of studies in the United States have demonstrated a fairly marked response to supplementation even in women who were not severely deficient at the outset (Sneed et al., 1981; Styslinger and Kirksey, 1985; Morrison and Driskell, 1985; Reinken and Dockx, 1985; Borschel et al., 1986; Chang and Kirksey, 1990; Moser-Veillon and Reynolds, 1990; Kang-Yoon et al., 1992). Concern has been expressed over the fact that breast milk does not provide the United States' RDA for vitamin B6 for young infants (Reynolds, 1982). Seventeen studies of unsupplemented women in Western countries yielded a mean breast milk vitamin B 6 level of 0.15 mg/L compared with 0.09 mg/L from five studies of women in developing countries.

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C.J. BATESand A. PRENTICE 6.4. VITAMINBI2 (COBALAMIN)

Vitamin Bt: represents the only known function of cobalt in human metabolism. It gives rise to methyl cobalamin, which functions in conjunction with folic acid during methionine synthesis from homocysteine, and to adenosyl cobalamin, which functions in molecular rearrangement (malonyl CoA mutase). Deficiency arises most commonly as a result of pernicious anemia, a failure of B~2 absorption through loss of secretion of intrinsic factor in the stomach. It characteristically causes megaloblastosis (in blood cell precursors in bone marrow) and macrocytic anemia, plus progressive irreversible demyelination of some peripheral nerves. Although B~2 is not present in plant tissues, a simple dietary deficiency is not common because bacterial contamination frequently provides adequate amounts, unless hygiene is immaculate. However, vegetarians do have lower Bt2 intakes and run a greater risk of deficiency than omnivores do. Breast-fed infants of mothers consuming vegan or strict vegetarian diets have occasionally presented with B~2 deficiency (Jadhav et al., 1962; Higginbottom et al., 1978; Kuhne et al., 1991). Likewise, lactating mothers with untreated pernicious anemia have occasionally been responsible for B~2 deficiency in their suckling infants (Johnson and Roloff, 1982; Hoey et al., 1982). Specker et aL (1990) reported that low levels of breast milk vitamin B12 in vegetarians may be associated with methylmalonic aciduria in their infants, an observation that helps to define the B~2requirement of young infants. For l0 studies of breast milk B12 in unsupplemented women in Western countries, the mean breast milk B~2 level was 510 ng/L, whereas for 5 studies in developing countries, it was 460 ng/L; the latter recorded a much wider range than the former. Parenteral doses of vitamin Bj2, given to treat pernicious anemia, can increase breast milk BI2 levels considerably (Craft et ak, 1971). On the other hand, BI2 given by mouth has a relatively small effect on breast milk levels (Thomas et al., 1979, 1980). Toxicity from Bt2 is essentially unknown. There are avid binding proteins (transcobalamin and cobalophilin or R-type) in milk that may aid absorption and reduce potential losses caused by bacterial uptake. B~2 levels are much higher in colostrum than in mature milk (Samson and McClelland, 1980; Imamura, 1981; Hoey et al., 1982). 6.5. FOLATE The folates are a complex family of substances that function in single-carbon transfer between molecules; two central functions are in DNA synthesis and methionine formation, but many others also exist. By virtue of their synergistic function in DNA synthesis and, hence, cell division, either folate or Bi2 deficiency cause megaloblastosis and macrocytic anemia. There is recent evidence that periconceptional folate intake is important in determining the risk of fetal neural tube defects. Naturally occurring folates are easily destroyed by heat and oxidation, and various studies have reported reduced growth and hematological abnormalities in preterm or other high-risk infants fed milk formulae with inadequate folate. Breast milk folate levels seem relatively insensitive to variations in maternal folate intake, unless severe maternal depletion has occurred. Three supplementation studies (Thomas et al., 1980; Tamura et al., 1980; Sneed et al., 1981) failed to achieve substantial increments in breast milk folate in American women. A single study from India (Deodhar et al., 1964) recorded such low values, even after supplementation, that the methodology seems suspect. From 12 studies of unsupplemented women in Western countries, the mean breast milk folate concentration was 57 #g/L, and from 7 studies since 1980 in developing countries or populations, it was 33 #g/L. Studies from India have again yielded low values (Bijur and Desai, 1985; Bijur and Kumbhat, 1987), which is consistent with the observation of clinical folate deficiency in pregnant Indian women. Folate concentrations rise considerably during the course of lactation from colostrum to mature milk (Imamura, 1981; Cooperman et al., 1982; Ford et al., 1983; Ek, 1983; Eitenmiller et al., 1984). Recent studies of the different types of folate present in human milk samples (Selhub, 1989; O'Connor et al., 1991; Cooperman and Lopez, 1991) have not yet yielded a consistent picture;

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however, it is likely that human milk folate is more readily available than the folate from formula feeds. Folate-binding proteins in breast milk may protect the folate from bacteria and assist absorption. Folate appears essentially nontoxic, although very large doses may possibly interfere with zinc absorption (Fuller et al., 1992).

6.6. NIACIN Niacin is an essential component of the pyridine nucleotide coenzymes, nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, which are essential components of the electron transport chain. Severe deficiency of niacin causes pellagra, characterized by dermatological and neurological lesions, and it is used to be a common condition associated with maize diets, but is now comparatively rare, even in developing countries. Tryptophan provides another source of niacin in vivo. Two studies from developing countries (India and The Gambia) suggest that breast milk niacin levels are moderately responsive to maternal supplementation (Deodhar et al., 1964; Prentice et aL, 1983), and one early study from the United States (Pratt et al., 1951) gave the same impression. Amounts present in colostrum are less than those in mature milk (Ford et al., 1983). Niacin is considered generally nontoxic, although vasodilatation and anorectic effects have been observed after gram doses in adults, where it has been used as a lipid-lowering drug (Figge et al., 1988).

6.7. BIOTIN

Biotin is an essential cofactor for the carboxylation reactions of lipid metabolism: notably fatty acid synthesis. Deficiency is rare in humans, but it can be induced artificially by raw egg white diets because of the strong and specific affinity for the biotin-binding protein, avidin, which is not easily digested. A recent study (Mock et al., 1992a,b) has shown that nearly all biotin in human milk is water-soluble, and it does not change consistently in amount during the course of lactation. A single Indian study (Deodhar et al., 1964) recorded an increase in breast milk biotin after supplementation, but this requires verification. Mean breast milk biotin concentrations in studies from Western countries generally lie within the range of 5-11/~g/L for unsupplemented women. Toxicity risk is considered to be negligible.

6.8. PANTOTHENICACID Pantothenic acid is part of the coenzyme A molecule and is, thus, an essential component of the fatty acid utilization system in the mitochondria. It is widely distributed in food, it is stable, and a human deficiency syndrome is virtually unknown. Mean concentrations in human milk, from 10 studies in both Western and developing countries, have yielded a range from 1.0 to 6.7 mg/L, but some of this apparent variability may be methodological. There is an increase in level between early and mature milk (Coryell et al., 1945; Deodhar et al., 1964; Johnston et al., 1981), but little reliable information is available on the effects of maternal deficiency or supplementation. Toxicity risk is considered to be negligible.

6.9. VITAMINC (AscORBATE)

Vitamin C is essential for humans, for higher primates and a few other mammalian species (none of which can synthesize it de novo) in order to prevent the symptoms of scurvy. It is thought to have a wide variety of beneficial and protective functions in vivo, many of which remain poorly understood. Its activity as a reducing agent and free radical chain-breaking

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agent appears to be important. Mixed function oxidase enzymes, such as collagen prolyl and lysyl hydroxylases, require ascorbate to maintain their catalytic iron (or copper) in the active, reduced form. Ascorbate also reacts with potentially "toxic" species, such as nitrite, hypochlorite, etc., and with free radical pro-oxidant species, it may also be capable of recycling oxidized vitamin E. It chelates certain metal ions and, thus, modifies their transport and functional properties. Because many of its functions are essentially transient and respond to highly variable demands, e.g. during infections and other environmental insults, it has proved very difficult to define human requirements. The prevention of scurvy can be achieved by intakes of only 6-10 mg/day in adults, but the other less well-characterized functions appear to need larger amounts. In well-nourished lactating women, the mean concentration in breast milk varies between ca. 40 and ca. 100 mg/L and declines to a moderate extent as lactation progresses (Munks et al., 1945; Thomas et al., 1980). Deficient status in the mother, undoubtedly, can reduce this concentration, although not dramatically, and 20 mg/L is probably near the minimum (Deodhar and Ramakrishnan, 1960; Bates et al., 1982a; Squires, 1952; Walker et al., 1954; Rajalakshmi et al., 1965). When poorly nourished women receive supplementary vitamin C, their breast milk levels increase rapidly (Deodhar et al., 1964; Bates et al., 1983). This increment is much smaller in women who are already well-nourished (Thomas et al., 1979; Sneed et al., 1981; Byerley and Kirksey, 1985). Homeostatic limitations of maternal absorption, of mammary gland transfer, and a rapid urinary excretion of amounts that exceed the renal threshold, all combine to ensure that breast milk does not contain excessive amounts. Although vitamin C seems nontoxic over a very wide range of intakes for normal subjects, there is evidence that some types of abnormal physiology may increase the risk of toxic effects of large doses. The mean breast milk vitamin C level from 11 studies of unsupplemented women in Western countries was 55 mg/L, whereas the mean level from 9 studies in developing countries was 35 mg/L.

7. MINERALS 7.1. SODIUMAND POTASSIUM Movement and concentration of the electrolyte cations sodium and potassium are under hormonal control and are regulated by a sodium-potassium pump in the cell membranes of the mammary gland secretory cells (Peaker, 1976; Keenan et al., 1983). During normal mature lactation, the sodium and potassium concentrations in human milk vary little, if at all, with the stage of lactation (Keenan et al., 1983; Nagra, 1989). However, there may be an abrupt fall in sodium levels during the first 3 days (Koo and Gupta, 1982). In abnormal conditions, such as an infection of the breast (mastitis), the junctions between the secretory cells can become leaky and allow sodium to enter the milk directly from the extracellular fluid via a paracellular pathway (Prentice et al., 1985). In this condition, sodium concentrations in the milk may increase by as much as 25-fold. It has been suggested that a high sodium intake in early life may predispose towards hypertension (Guthrie, 1968), so the determinants of breast milk sodium concentrations are of potential medical interest. Few studies have yet addressed the response of breast milk sodium levels to maternal dietary changes, but those that have done so have observed little or no effect. In two studies (Keenan et al., 1983; de Filippi et al., 1981), a considerable reduction in maternal sodium intakes (and, hence, in urinary sodium levels) failed to alter breast milk sodium concentration. In a third study (Ereman et al., 1987), women received a lunch with high or low sodium content and their breast milk samples were analyzed at 15-min intervals over the following 2-hr postprandial period. Neither the sodium content of the meal, nor the postprandial duration, significantly affected breast milk sodium levels. Longer-term or more extreme maternal dietary changes or variations have not been examined yet. Because fetal accretion rates are much higher than extrauterine accretion rates, and there is also evidence that small preterm infants can develop hyponatremia on breast milk diets, it has been

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recommended that preterm infants should receive sodium supplements so as to provide at least 2.1 mmol/1000 kcal (Ziegler et aL, 1981; Tonz and Schubiger, 1985).

7.2. CALCIUM AND PHOSPHORUS Both calcium and phosphorus arc major components of thc milk of all animal species,because of thc major rcquiremcnt for bonc formation in the suckling infant. In human milk, calcium is present throughout lactation at around 300mg/L and phosphorus rcachcs a level of around 150 mg/L at thc end of the firstwcek postpartum. Both nutrientsincrcasc from lowcr levelsduring the firstwcck of lactation. In addition to bone matrix formation, calcium is also essentialfor musclc contraction, for thc intcgrity of membranes and for blood coagulation, and phosphorus is an essentialcornponcnt of nucleic acids, high cncrgy phosphates, phospholipids and many othcr metabolitcs and structural componcnts. Homcostatic mechanisms control plasma calcium Icvclsvcry prcciscly,if nccessary, at the cxpcnsc of bone mincral, so that plasma calcium is not a very useful index of calcium status. Diffcrcnt human populations worldwidc havc widcly diffcring dietary calcium intakes, yet thc prcvalcncc of diseases (such as ostcoporosis), which might bc affected by calcium supply, is not obviously correlated with this variation. Howcvcr, thcrc is some cvidcncc from studies of infantilc tctany (causcd by the use of nonhuman milk formulac with calcium and phosphorus contcnts and ratios that differ considerably from those of human milk), that thc intakc of thcsc nutrients is probably criticalin early lifc(Grccr, 1989). Bcforc measuring totalphosphorus as phosphatc, itis csscntialto ash thc samplcs (Laskcy et al., 1991a). Phosphorus Icvels in breast milk secm remarkably constant, bctwccn very dissimilar communities ( W H O , 1989). The pcrccntagc of protcin-bound phosphorus is high (45%) in colostrum, but declincs to 2 3 % in mature milk. A significantproportion of human brcast milk calcium (ca. 16%) is associatcd with the lipid fraction (Fransson and LSnncrdal, 1983, 1984). In the aqucous fraction, it is prcscnt in low molccular weight compounds or associated with whey proteins, but unlike cows' milk, vcry little is casein-bound (Fransson and L6nncrdal, 1983). The calcium concentration declines gradually in late lactation (Vaughan et al., 1979; Karra et al., 1986; Karra and Kirkscy, 1988; Laskcy et al., 1990; Prentice and Barclay, 199 I). Maternal age, parity and ratc of milk production appear to have littleor no effecton thc calcium concentration, but other unidentifiedfactors rcsult in at least a 2-fold differcnccbetween individuals (Laskcy et al., 1990; Prcnticc and Barclay, 1991). Maternal calcium intakes probably have littlcinfluence on brcast milk calcium Icvcls (Karmarkcr and Ramakrishnan, 1960; Kirkscy et al., 1979; Vaughan et al., 1979; Freclcy et al., 1983; Moscr et al., 1988). The Icvelsin differentpopulations oftcn appear similar(Karra et al., 1988; Moscr et al., 1988; Parr et al., 1991), although somc exceptions may occur. Thus, Bailey (1965) rcported vcry low calcium concentrations in milk from N e w Guinea mothers with low intakes,whcrcas Fransson et al. (1984) paradoxically observed high levels in the milk from Ethiopian mothers with low intakes. Grccr et al. (1982) reported a positive corrclation bctwccn matcrnal calcium intakes and milk calcium levels,and reccnt studies of African women (Laskcy et al., 1990, 1991b; Prentice and Barclay, 1991) havc dcmonstratcd lower milk calcium Icvcls,at an cquivalcnt stagc of lactation, than thosc of Unitcd Kingdom mothers, whose intakcs are charactcristicallymuch highcr. Thc Gambian mothers also had relativelylow calcium:phosphorus ratios. Howcvcr, it is not yct clear whether thcsc diffcrcnccs arc, indccd, dict rclatcd and supplementation trialsarc now in progrcss. Provided that thc vitamin D supply from thc dict,or from U.V.-lightcxposurc, is adcquatc, thcn thc calcium and phosphorus rcquiremcnts of term infants can bc providcd by breast milk for at least 26 weeks postpartum. Prctcrm infants fed cxclusivcly on human milk, howcver, have dcvclopcd scvcrc rickcts and osteopcnia (Rowe et al., 1979; Sagy et al., 1980; Carey et al., 1987), apparently because human milk docs not providc sufficicntcalcium and phosphorus to approach thc corresponding fetalaccrction rates.For this rcason calcium (ca. 600 rag/L) and phosphate (ca. 400 rag/L) supplements arc now rccommcndcd for prctcrm infants fed human milk (Zicglcret al., 1981; Sentcrrc et al., 1983; Tonz and Schubiger, 1985). JPT 62/I-2--N

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Abnormalities in magnesium status are generally associated with pathological conditions rather than with inappropriate dietary intakes. However, low blood magnesium levels in infants has been associated with convulsions and tremors (Wong and Teh, 1968). Around 60% of the body's magnesium is in bone. Breast milk contains around 30-mg magnesium/L, which is nearly all (98%) in the aqueous fraction and mostly in the whey proteins or low molecular weight fraction (Fransson and L6nnerdal, 1982). There appears to be some disagreement about changes in the milk content during the course of lactation: several studies recorded virtually no changes between early and mature milk (Vaughan et al., 1979; Gross et al., 1980; Sann et al., 1981; Lemons et al., 1982; Hibberd et al., 1982; Nagra, 1989); however, Karra and Kirksey (1988) and Karra et al. (1988) recorded a moderate increase between the first and sixth month of lactation in two dissimilar communities. Magnesium secretion into breast milk appeared not to be affected by maternal dietary intake or supplements, at least during early lactation and for generally well-nourished women (Kirksey et al., 1979; Vaughan et al., 1979; Greer et al., 1982; Feeley et al., 1983; Moser et al., 1988). Cows' milk formulae, which have a considerably higher magnesium content than human milk, do not appear to result in any evidence of magnesium toxicity (Greer, 1989). Atkinson (1985) found that the amount of magnesium retained by preterm infants from breast milk increased at a rate similar to the calculated intrauterine magnesium accumulation rates.

7.4. IRON The largest iron compartment and demand for dietary iron is that of hemoglobin, but iron plays an important structural and catalytic role at many other sites, notably in myoglobin and in the mitochondria. Whereas the body can tolerate moderate iron depletion by reducing its hemoglobin load, iron excess is a potentially greater threat, and there are efficient homeostatic mechanisms designed to avoid excessive absorption. Breast milk contains less iron than many other human foods, with the result that the rapidly growing infant exhibits a reduction in hematocrit during the first few months of life, which is considered to be normal event. Breast milk typically contains around 500-1000 #g/L in early lactation, falling to below 300 #g/L by the sixth month (Fransson and L6nnerdal, 1980; Freeley et al., 1983). Although lactoferrin in milk has a high affinity for iron (Aisen and Leibman, 1972), a significant amount of the iron in the aqueous fraction is in a low molecular weight form (Fransson and L6nnerdal, 1980). About one-third of the iron in milk is associated with the lipid fraction, possibly the fat globule membranes (L6nnerdal et al., 1981; Fransson and L6nnerdal, 1983). Several studies have suggested that iron in breast milk is unrelated to maternal iron intake and unaffected by iron supplements, at least in the short-term (Karmarker and Ramakrishnan, 1960; Murray et al., 1978; Vaughan et al., 1979; Kirksey et al., 1979; Vuori et al., 1980; Feeley et al., 1983; Fransson et al., 1984; Finley et al., 1985). Although in one study of Nigerian women a positive correlation between dietary iron and mature breast milk iron levels was observed, no causal relationship was demonstrated. Other studies found no relationship of breast milk iron levels with maternal hemoglobin, ferritin or transferrin saturation levels (Loh and Sinnathury, 1971; Murray et al., 1978; Celada et al., 1982). Paradoxically, one study observed high iron concentrations in breast milk from mothers with extremely low hemoglobin status (Fransson et al., 1985). Iron supplementation does not appear to alter the secretion of other trace elements into breast milk (Picciano and Guthrie, 1976). In order to mitigate the possible effects of iron-limitation by breast milk and unfortified cows' milk diets (especially in view of the reported effects of low iron status on developmental indices), the use of iron-rich foods from 6 months onwards has been advocated (Dallmann, 1986; Haschke et al., 1988). In large doses, however, iron can be toxic, and because of its pro-oxidative functions in the presence of oxygen, it can cause subtle tissue damage that can have long-term consequences. Clearly, further studies on these important topics are needed.

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7.5. ZINC Zinc has a wide variety of biochemical roles in vivo, but marginal zinc deficiency is not easy to recognize, either by biochemical or by physiological tests. It is characteristically associated with loss of appetite and a failure to grow, and possibly by abnormal immune function. Favorable responses to zinc supplements have been observed in certain vulnerable subgroups of young children, in a wide variety of countries and cultures. Because the amount of zinc that is secreted into breast milk varies very widely between mothers, there have arisen occasional instances, especially for preterm babies, where low breast milk zinc levels have apparently given rise to severe zinc deficiency in breast-fed infants (Aggett et al., 1980; Ahmed and Blair, 1981; Weymouth and Czarnecki, 1981; Zimmerman et al., 1982; Moore et al., 1984; Murphy et al., 1985; Roberts et al., 1987; Atkinson et al., 1989). However, even large doses of zinc sulphate given to the mother in such cases have failed to alter her breast milk zinc content (Weymouth and Czarnecki, 1981; Zimmerman et aL, 1982; Moore et al., 1984). Two-thirds of the zinc in milk is associated with iactoferrin, albumin and with low molecular weight substances, such as citrate and picolinate, in the whey fraction; about one-fifth is in the fat layer (Ainscough et al., 1980; L6nnerdal et al., 1981; Fransson and L6nnerdal, 1983). A large number of studies have demonstrated that there is always a steep decline in zinc levels during the first few weeks of lactation, followed by a slower, but continuing, decline through the period of mature milk production, so that the mean level in late lactation (usually less than 0.5 mg/L), is more than 10-fold lower than it is in the early stages of lactation (Vaughan et al., 1979; Casey et al., 1985a, 1989; Dorea et al., 1985, 1993; Harzer et al., 1986; Lamounier et al., 1989; Karra et al., 1989; Bates and Tsuchiya, 1990; Simmer et al., 1990). Milk from mothers of preterm infants was similar in its zinc content to that of milk from mothers of term infants (Mendelson et al., 1982; Moran et al., 1983; Trugo et al., 1988). In general (as noted above), breast milk zinc levels do not respond readily to maternal supplementation (Kirksey et al., 1979; Weymouth and Czarnecki, 1981; Zimmerman et al., 1982; Feeley et al., 1983; Moore et al., 1984; Krebs et al., 1985; Moser-Veillon and Reynolds, 1990). However, two groups of workers have achieved modest increases in breast milk zinc levels by long-term maternal supplementation with doses in the range of 15- to 50-mg zinc/day (Krebs et al., 1985; Karra et al., 1988, 1989). Two other similar studies (Shrimpton et al., 1985; Moser-Veillon and Reynolds, 1990) did not observe such an effect, however. Severe maternal zinc deficiency has not been studied in this respect. Concern has been expressed over the apparent inadequacy of breast milk to provide the infants' calculated requirements of zinc during the later stages of lactation (Picciano and Guthrie, 1976; Moser-Veillon and Reynolds, 1990; Krebs et al., 1985). However, the prevalence of zinc-responsive inadequacy is not yet clear, nor is the optimum public-health approach to its prevention. Like other metals, zinc can become toxic in very large doses, so that high-dose supplementation may carry some element of risk.

7.6. COPPER Copper, like zinc, is an essential component of a wide variety of enzymes, but as with zinc, the biochemical indices of copper status cannot define satisfactorily a marginal deficiency state. Humans rarely develop copper deficiency as a result of low dietary intakes. However, preterm infants fed cows' milk formulae have occasionally developed copper deficiency, characterized by anemia, neutropenia and bone lesions. Copper occurs mainly in the aqueous fraction of breast milk, with 15% in the lipid fraction (Fransson and L6nnerdal, 1983, 1984). Like zinc, its concentration is highest in colostrum and lowest in late, mature milk (Vuori and Kuitinen, 1979; Casey et al., 1985a, 1989; Dorea et al., 1985; Kirsten et al., 1985; Mbofung and Atinmo, 1986; Salmenpera et al., 1986; Burguera et al., 1988). Like zinc, copper appears to be generally unresponsive to maternal supplementation (Vuori et al., 1980; Salmenpera et al., 1986), although studies on deficient subjects and of long-term supplementation have not been performed yet. Even the intravenous administration of copper, which raised

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the serum levels considerably, failed to alter breast milk copper levels (Munch Peterson, 1950). Several studies have found no relation between maternal dietary copper intakes and breast milk copper levels (Kirksey et al., 1979; Vaughan et al., 1979; Vuori et al., 1980; Feeley et al., 1983; Finley et al., 1985; Mbofung and Atinmo, 1986; Moser et al., 1988). Lower maternal serum copper levels due to long-term oral contraceptive use did not result in low breast milk copper levels (Kirksey et al., 1979). Although relatively low breast milk copper levels were found in the milk from Finnish (Salmenpera et al., 1986) and Bangladeshi (Simmer et al., 1990) mothers, the reasons for this are not known. Despite the fact that zinc or iron can affect copper absorption, there was no evidence that zinc or iron supplements given to lactating women influenced their breast milk copper levels (Picciano and Guthrie, 1976; Fischer et al., 1980). Apart from being the result of a genetic abnormality (Wilson's disease), copper toxicity in humans appears relatively rare. 7.7. CHROMIUM Marginal chromium deficiency can result in disturbances in glucose or lipid metabolism. Breast milk contains trace amounts of chromium (Kumpulainen et al., 1980; Casey et al., 1985a; Cocha et al., 1992; Anderson et al., 1993), but relatively little is known about its biological and nutritional significance. Neither Kumpulainen et al. (1980) nor Anderson et al. (1993) could detect any relation between maternal chromium intake and breast milk chromium levels; the latter also failed to find evidence of any relation with serum or urinary chromium. It is not known yet whether any specific subgroups of infants are at increased risk of chromium deficiency, nor how this might relate to breast-feeding practices. 7.8. MANGANESE Manganese is an essential component of several enzymes, and a deficiency in animals has resulted in growth retardation, impaired glucose tolerance and impaired reproduction. Manganese deficiency in humans, however, is virtually unknown, apart from a tentative association with certain convulsive disorders (Tanaka, 1982). Manganese is found in the whey fraction and lipid fraction of human milk (L/Snnerdal et aL, 1985). The changes recorded during the course of lactation include an initial decrease during the first week and then a rise during the latter stage of lactation, possibly associated with weaning (Vuori, 1979; Vaughan et al., 1979; Stastny et al., 1984; Casey et al., 1985a, 1989; Saner and Garibagaoglu, 1988). Parr et al. (1991) found considerable differences in breast milk manganese levels between populations. Little is known about the effects of maternal deficiency or supplementation on breast milk manganese levels, but two reports (Vaughan et al., 1979; Vuori et al., 1980) described a positive correlation between maternal intakes and breast milk levels. Clearly this needs to be investigated further, e.g. by controlled supplementation studies. 7.9. MOLYBDENUMAND NICKEL Studies on these two ultra-trace elements are in their infancy, but recent studies (Dang et al., 1984; Casey and Neville, 1987; Bermejo-Barrera et al., 1990) have described ng/mL amounts as being present. Neither their biological significance, nor the possible effects of variations of maternal intakes, have yet been investigated. 7.10. SELENIUM Selenium is an essential component of certain enzymes, of which the best known is glutathione peroxidase, which helps to protect lipids, including those in cell membranes, from oxidative damage. It is also involved in thyroid hormone production. Both deficiency and toxic oversupply have been described in different parts of China, where extremes of soil selenium content occur (Levander, 1989). In other parts of the world, such variations are less extreme, but marginal

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deficiencies have, for instance, been identified in Finland and in New Zealand. There is considerable current interest in the possible protective effects of selenium against various degenerative diseases. However, there have not been any recorded cases of clinical selenium deficiency in breast-fed infants (term or preterm). Selenium in breast milk is correlated with the protein content and specifically with the glutathione peroxidase activity (Picciano and Mannan, 1986; Hojo, 1986; Debski et aL, 1987; Milner et al., 1987). Fractionation of milk by gel chromatography revealed 8-12 different seleno-protein fractions (Debski et al., 1987), but very little of the selenium is present in the lipid fraction. Selenium concentrations are highest in colostrum and lowest in mature milk (Kumpulainen et al., 1984; Robberecht et al., 1985; Walivaara et al., 1986; Levander et al., 1987; Ellis et al., 1990). In Finland, Kumpulainen et al. (1984) observed a progressive fall in breast milk selenium throughout lactation, possibly due to progressive maternal depletion. In West African (Gambian) women, both parity and the fluctuations of nutritional status with season had significant effect on breast milk selenium levels (Funk et al., 1990). Several studies have shown that variations in maternal selenium intakes do influence breast milk selenium levels considerably (Picciano, 1985; Kumpulainen et al., 1984, 1985; Mannan and Picciano, 1987; Schramel et al., 1988; Levander, 1989; Litov and Combs, 1991). Finnish mothers who had a naturally low selenium intake exhibited a significant increase in breast milk selenium levels after the importation of selenium-rich grain (Kumpulainen et al., 1984), or after the provision of extra selenium in a yeast base (Kumpulainen et al., 1985). Yeast-selenium (100pg/day) was judged to be both safe and effective as a supplement, and seleno-methionine was found to be more effective than inorganic selenite as a supplement (Mangels et al., 1990). The huge differences in selenium intakes between different regions of China are associated with parallel differences in breast milk selenium levels (Levander, 1989). Because of the potential toxicity of excessive selenium intakes, and because dietary selenium clearly can affect breast milk levels quite considerably, lactating mothers should be cautioned against self-megadosage. In those regions where soil selenium levels are suboptimal, and especially if most of the available foods are grown locally, controlled selenium supplements are likely to be beneficial. 7.11. IODINE Dietary iodine deficiency is a common nutritional problem throughout the world, especially in some developing countries, leading to goitre in adults and cretinism in young children because of the iodine requirement for thyroid hormone production. As an avoidable problem with potentially devastating consequences, it ought not to be neglected. The minimum daily requirement for adults is around 70 p g (0.6/~ mol) (Department of Health, 1991) and the current RNI values in the United Kingdom are 140 pg/day for adults and 50-60/~g/day for formula-fed children. There is some risk of thyrotoxicosis with chronic intakes (in adults) above 5000/~g/day, and this may vary with previous exposure. Whereas in Europe there remain regions of borderline iodine deficiency (Delange, 1985), in the United States iodine toxicity is an emerging problem (Kidd et aL, 1974). Ionic iodide comprised more than 80% of total breast milk iodine (Gushurst et al., 1984), and there was no relation between iodine concentration and duration of lactation in this study, although an earlier study (Turner, 1934) recorded a rise followed by a fall after ca. 4 months' lactation. Etling et al. (1986) recorded a progressive rise from colostrum to mature milk, and Johnson et al. (1990) found that milk samples collected more than 60 days after parturition contained less iodine than earlier samples. Thus, the effect of stage of lactation is unclear. Breast milk iodine concentrations clearly are influenced by the maternal dietary intake (Gushurst et aL, 1984; Johnson et al., 1990), although the mammary gland can also compensate by concentrating iodine, if dietary intakes are low (Vermiglio et al., 1992). Parr et al. (1991) observed much lower breast milk iodine levels in women from Zaire (a deficient population) than from other countries. The wide range of breast milk iodine levels observed in American women was correlated with the use of iodized table salt (Gushurst et al., 1984). Very high intakes, associated with consumption of algae in Japan, are related to high breast milk iodine levels (Murumatsu et aL,

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1983). Even topical iodine preparations can be absorbed and can affect breast milk iodine levels (Postellon and Aronow, 1982; Danziger et al., 1987). This could pose a health problem, since transient hypothyroidism in the infant was observed in one study (Danziger et al., 1987). Clearly, as with selenium, lactating mothers should be aware of the dangers of both iodine deficiency and iodine toxicity, and should regulate their intakes and exposure accordingly. 7.12. FLUORINE The fluoride ion is deposited in bones and teeth in proportion to dietary intake, whereas blood fluoride levels are strictly controlled. Moderate intakes of fluoride are thought to protect against dental caries and possibly also against periodontal disease and osteoporosis (Department of Health, 1991). However, excessive intakes cause discoloration of the teeth and very high intakes are associated with more severe toxicity (fluorosis), affecting the bones. Ionic fluoride is typically present in breast milk at 4-11/~g/L (Esala et al., 1982), but another (bound) fraction may constitute 20-92% of the total fluoride content. There is no firm evidence for differences with stage of lactation (Spak et al., 1983), and the milk levels are 150-200 times lower than those found in fluoridated tapwater (Ekstrand, 1989). Although the level of fluoride in breast milk is affected by maternal dietary intake, this effect is small in comparison with variations in tapwater fluoride levels (Dirks et al., 1974; Ekstrand et al., 1981, 1984; Esala et al., 1982; Spak et al., 1983; Machari, 1984; Dabeka et al., 1986; Opinya et al., 1991). Even when a large amount of fluoride was given to a lactating mother as treatment for osteoporosis, only 0.2% of the maternal dose was secreted into the milk. However, there have been occasional early reports of dental fluorosis in the suckling infants of occupationally exposed mothers (Brinch and Roholm, 1934). Parr et al. (1991) observed very big differences in breast milk fluoride levels between populations.

8. SYNOPSIS AND CONCLUSIONS There are three important aspects of the subject of variability of vitamin, mineral and trace element concentrations in human breast milk. The first is the existence of deficiency or toxicity syndromes in young infants, which may be influenced by the choice of breast vs formula feeds. The second is the question of responsiveness of breast milk to variations in maternal nutrient intakes. The third is the existence of other factors that determine breast milk concentrations, such as stage of lactation, and inherent variations between subjects. Clearly, each nutrient must be assessed separately, because there is a very wide range of characteristics within such a diverse group of nutrients. Nevertheless, some generalizations can be made. Among the fat-soluble vitamins, overt deficiencies have been identified in infants and young children; these deficiencies are associated with inadequate supplies of vitamins A, D or K. Apparently, vitamin E is not responsible for any characteristic deficiency disease in 'normal' subjects, but its protective role is, nevertheless, of particular importance to vulnerable (especially preterm) infants. Vitamins A and D are also associated with toxicity syndromes at excessive intakes, and vitamin D is the only vitamin for which maternal megadosage has been reported to cause toxicity in the suckling infant. Vitamin A and E exhibit a characteristic decline in levels with the progress of lactation, and for all four fat-soluble vitamins, there is some evidence of a response to changes in maternal intake; however, quantitation of this is lacking. For the water-soluble vitamins, there is little evidence for overt deficiencies because breast milk takes precedence over the maternal tissues for access to the available vitamin supplies. Nevertheless, there are significant variations in breast milk concentrations associated with variations in maternal intakes, especially for riboflavin, vitamin B6, vitamin B~2and vitamin C. For the other five water-soluble vitamins, the evidence is less compelling. Changes during the course of lactation are variable but, in contrast to the fat-soluble vitamins, the overall trend is upward. Toxicity is not a problem for breast-fed infants, and problems of deficiency have only been encountered in formula-fed infants, e.g. where the formula was inappropriate or damaged during pretreatment.

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For the cationic electrolytes, including magnesium, breast milk levels are extremely wellregulated and do not vary either with maternal diet or with stage of lactation. Variability of breast milk calcium (and phosphorus) levels is currently under investigation, and the differences in protein (especially the casein) contents between breast milk and formula feeds can result in major differences in the bioavailability of calcium and of other divalent metal ions. The amounts of calcium, iron, zinc and copper that are secreted into breast milk apparently are not influenced by short-term variations in maternal intakes, provided the mother is well-nourished. Some changes in zinc concentrations may occur after long-term, high-level supplementation, but these changes are relatively small in comparison with the natural variations that are seen during the course of lactation. Inherent maternal variability in secretion seems to be a major consideration for zinc, and there are instances of breast-fed infants who received insufficient zinc and needed zinc supplements. Several divalent metal ions in breast milk decline in concentration during the course of lactation, this change being especially pronounced for zinc. For the other (minor) trace element cations (chromium, manganese, molybdenum, nickel, etc.), the available information is generally inadequate, but of these, only manganese is thought to show some variation with maternal intake. The trace anions (selenium, iodine and fluorine) all exhibit significant responsiveness to variations in maternal dietary intake, so that mothers living in regions where the soil is deficient may secrete inadequate amounts into their breast milk. For selenium or iodine this could be a public health problem in some regions, (Parr et al., 1991), though probably not for fluorine, because drinking water usually contains much more fluorine than breast milk does. Maternal supplementation with selenium, or with iodine, might achieve important benefits for the suckling infant in regions of endemic deficiency, and prolonged high-level intakes or exposures might result in toxicity. Several micronutrients are absorbed more efficiently from breast milk than from cows' milk formulae. In some cases, this effect may be due to protection by specific binding proteins (e.g. folic acid), whilst for others, such as the divalent metal ions, the ratio of casein to why proteins seems to be especially important. Some nutrients, including metal ions, are partly associated with the lipid fraction (e.g. the fat globule membranes) in breast milk, but the resulting effects on availability have not been extensively studied yet. Clearly, there are many unresolved questions: first, about the requirements of young (and especially preterm) infants for specific micronutrients; second, about the relative merits of breast milk and formula feeds in being able to deliver them effectively; and third, about maternal characteristics, including maternal nutritional status and nutrient intake, on the composition and, hence, the nutritional adequacy, of breast milk.

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(1989) Studies in human lactation: secretion of zinc, copper and manganese in human milk. Am. J. clin. Nutr. 49: 773-785. CELADA,A., BUSSET,R., GUTIERREX,J. and HERRERA,V. (1982) No correlation between iron concentration in breast milk and maternal iron stores. Helv. Paediatr. Acta. 37: il-16. CHANG, S. J. and KIRKSEY,A. (1990) Pyridoxine supplementation of lactating mothers: relation to maternal nutrition status and vitamin B6 concentrations in milk. Am. J. clin. Nutr. 51: 826-831.

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COCHA,J. A., CERVILLA,J. R., REY-GOLDAR,M. L., FDEz-LORENZO,J. R. and FRAGA,J. M. (1992) Chromium content in human milk, cows milk and infant formulas. Biol. Trace Elem. Res. 32: 105-107. COMMITTEE 1/5 OF THE INTERNATIONALUNION OF NUTRITIONALSCIENCES(1983) Recommended dietary intakes around the world. Nutr. Abstr. Rev. 53: 939-1015. COOPERMAN, J. M, and LOpEZ, R. (1982) Pteroylglutamates in human milk. Am. J. din. Nutr. 54: 760-761. COOPERMAN, J. M., DWECK, H. S., NEWMAN, L. J., GARaARINO,C. and LOPEZ, R. (1982) The folate in human milk. Am. J. clin. Nutr. 36: 576-580. CORYELL, M. N., HARRIS, M. E., MILLER, S. E., WILLIAMS,H. H. and MACY, I. G. (1945) Human milk studies XXII. Nicotinic acid, pantothenic acid and biotin contents of colostrum and mature human milk. Am. J. Dis. Child. 70: 150-161. CRAFT, I. L., MATTHEWS,D. M. and LINNELL,J. C. (1971) Cobalamins in human pregnancy and lactation. J. clin. Pathol. 24: 449-455. DABEKA, R. W., KARPINSKI,K. F., MC~ENZIE, A. D. and BAJOIK,C. D. (1986) Survey of lead, cadmium and fluoride in human milk and correlation of levels with environmental and food factors. Food Chem. Toxic. 24: 913-921. DALLMAN, P. R. (1986) Iron deficiency in the weanling: a nutritional problem on the way to resolution. Acta Pediatr. Scand. 323 (Suppl.): 59-67. DANG,H. S., JAISWAL,D. D., SOMASUNDARAM,S., DESHPANDE,A. and DACOSTA,H. (1984) Concentrations of four essential trace elements in breast milk of mothers from two socioeconomic groups: preliminary observations. Sci. Total Environ. 35: 85-89. DANZIGER,Y., PERTZELAN,A. and MIMOUNI, M. (1987) Transient congenital hypothyroidism after topical iodine in pregnancy and lactation. Arch. Dis. Child. 62: 295-296. DEBSKI, B., PICCIANO,M. F. and MILNER, J. A. (1987) Selenium content and distribution of human, cow and goat milk. J. Nutr. 117: 1091-1097. DEBUSE, P. J. (1992) Shoshin beriberi in an infant of a thiamine-deficient mother. Acta Paediatr. 81: 723-724. DE FILIPPI, J. P., KAANDERS,H. and HOFMAN, A. 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J. olin. Nutr. 38: 929- 935. EKSTRAND,J. (1989) Fluoride intake in early pregnancy. J. Nutr. 119: 1856-1860. EKSTRAND,J., BOREUS,L. O. and DECHATEAU,P. (1981) NO evidence of transfer of fluoride from plasma to breast milk. Br. reed. J. 283: 761-762. EKSTRAND, J., SPAK, C-J., FALCH, J., AFSETH,J. and ULVESTAD,H. (1984) Distribution of fluoride to human breast milk following intake of high doses of fluoride. Caries Res. 18: 93-94. ELLIS, L., PICCIANO, M. F., SMITH, A. M., HAMOSH, M. and MEHTA, N. R. (1990) The impact of gestational length on human milk selenium concentration and glutathione-peroxidase activity. Pediatr. Res. 27: 32-35. EREMAN, R. R., LONNERDAL,B. and DEWEY, K. G. (1987) Maternal sodium intake does not affect postprandial sodium concentration in human milk. J. Nutr. 117: 1154-1157. ESALA,S., VUORI,E. and HELLE,A. (1982) Effect of maternal fluorine intake on breast milk fluorine content. Br. J. Nutr. 48: 201-204. ETLING, N., PADOVANI,E., FOUQUE, F. and TATO, L. 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FAO (1988) Requirements of vitamin A, iron, folate and vitamin BiT Report of a joint FAO/WHO Expert Consultation. FAO Food and Nutrition Series No. 23, Rome. FAWZl, W. W., CHALMERS,T. C., HERRERA,G. and MOSTELLER,F. (1993) Vitamin A supplementation and child mortality. A meta-analysis. J. Am. IVied. Ass. 269: 898-903. FEELEY,R. M., EITENMILLER,R. R., JONES,J. B. and BARNHART,H. (1983) Calcium, phosphorus and magnesium contents of human milk during early lactation. J. Pediatr. Gastroenterol. Nutr. 2: 262-267. FIGGE, H. L., FIGGE,J., SOUNEY,P. F., MUTNICK,A. H. and SACKS,F. (1988) Nicotonic acid: a review of its clinical use in the treatment of lipid disorders. Pharmacotherapy 8: 287-294. FINLEY,D. A., LONNERDAL,B., DEWEY,K. G. and GRIVETTI,L. E. (1985) Inorganic constituents of breast milk from vegetarian and non-vegetarian women: relationships with each other and with organic constituents. J. Nutr. 115: 772-781. FISCHER,P. W. F., GIROUX,A. and ABBE,M. R. L. 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