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Folate content of human milk during early lactational stages
NUTRITION RESEARCH, Vol. 4, pp. 391-397, 1984 0271-5317/84 $3.00 + .00 Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved.
FOLATE CONTENTOF HUMANMILK DURING EARLYLACTATIONAL STAGESl Ronald R. Eitenmiller, Ph.D. Wonda D. Bryan, Indra K. Khals~, Ph.D., Ruth M. Feeley, Ph D. and Harold M. Barnhart, Ph.D. Department of Food Science, University of Georgia, Athens, Georgia
ABSTRACT Folate content of human milk was determined in early transitional milk (4-7 days postpartum), transitional milk (lO-14 days postpartum) and mature milk (36-45 days postpartum). Mature milk contained a mean total folate level of 4.9 ug/lO0 g. No diurnal variations were observed. At each stage, total folate was significantly higher (p< .05) than free folate. As lactation progressed to the mature milk stage, both free and total folate increased significantly (p<.05). The percentage of folate in the free form decreased as length of lactation increased. Based on folate levels determined in this study, a one month old breast-fed infant would have a folate intake of 6.7 ug/kg/day which is somewhat higher than the recommended dietary allowance for infants of 5 ~g/kg/day. INTRODUCTION Increased interest in breast feeding has led to increased knowledge of the nutrient composition of human milk. Such information is important for the understanding of dietary requirements of the infant and establishment of dietary allowances and standards for infant formulas. Wide variability exists in the literature regarding folate content of human milk (l-13). Values range from 0.07 ~g/lO0 ml (2) to 14.1 ~g/lO0 ml (IO). Since early investigators when assaying for human milk folate content used extraction procedures that did not adequately protect labile folates, the values reported were low (14). However, even among recent studies, large variability exists in values reported for mature human milk.
1
This research was supported by the National Institute of Child Health and 2 Human Development Grant No. 5 ROl HD14880-02. Author Feeley is affiliated with the Division of Nutrition, Emory University, Atlanta, Georgia. 391
392
R.R. EITENMILLER et al.
Karlin (5) analyzed the milk of 12 women d a i l y from 2 to 8 days postpartum. There was an increase in mean f o l a t e concentrations from 0.5 ug/lO0 ml to I . I ~g/lO0 ml during the 6 day period. Karlin (5) u t i l i z e d milk samples from 230 women at various stages of l a c t a t i o n and reported that f o l a t e levels remained constant between 4 and 9 months postpartum. The mean t o t a l f o l a t e level a f t e r 1 month postpartum was between 5 and 6 ug/lO0 ml. In 1977, the results of a large B r i t i s h study (7) showed that pooled mature human milk contained 5.2 ug/lO0 ml. S i m i l a r i l y , Thomas et al (9) reported a f o l a t e value of 5.5 and 5.0 ug/lO0 ml f o r 6 well-nourished mothers with f o l a t e supplementation, and without supplementation, r e s p e c t i v e l y , at 6 months postpartum. The researchers concluded that supplementation had l i t t l e e f f e c t on the f o l a t e content of the milk of well nourished mothers. Sneed et al ( I I ) in a s i m i l a r study of 16 low socio-economic women showed a s t a t i s t i c a l l y s i g n i f i c a n t difference between the f o l a t e values of a unsupplemented group compared to a f o l a t e supplemented group. The unsupplemented group had a mean f o l a t e level of 4.2 ~g/lO0 ml at 5-7 days postpartum and 4.3 ug/lO0 ml at 43-45 days postpartum. The supplemented group had the same mean value of 4.9 ~g/lO0 ml at each of the t e s t times. Studies by Yamada (8) and Tamura et al (I0) reported higher milk f o l a t e levels than the previously noted studies. Yamada (8), using an extraction procedure incorporating the use of the p r o t e o l y t i c enzyme Pronase , reported the mean t o t a l f o l a t ~ content of 21 samples of human milk to be 7.75 ~g/lO0 ml. The Pronase treatment of some milk samples more than doubled the measurable f o l a t e levels. Tamura et al (I0) in a study of the r e l a t i o n s h i p between infant serum and plasma f o l a t e levels and the f o l a t e concentration in maternal milk found that the mean f o l a t e content of 39 milk samples was 14.1 ~g/lO0 ml. This represents the highest human milk f o l a t e value reported to date. In contrast to the f o l a t e values reported by Tamura et al ( I 0 ) , Cooperman et al (12) recently reported that mature human milk contains 2.6 ~g/lO0 ml. These authors did not show a difference between free (unconjugated) and t o t a l f o l a t e values in the milk which d i f f e r s from the observations of Yamada (8). The present i n v e s t i g a t i o n was undertaken to determine the free and t o t a l f o l a t e content of human milk at early stages of l a c t a t i o n during which f o l a t e concentration rapidly changes. Also, diurnal v a r i a t i o n in the f o l a t e content was assessed. A modification of the e x t r a c t i o n procedure of Yamada (8) was used to ensure complete l i b e r a t i o n of bound folacin. In view of the many inconsistencies in the l i t e r a t u r e , i t was hoped that t h i s study would help to improve the understanding of the f o l a t e content of human milk and thus, the understanding of the f o l a t e intake of breast-fed infants. Materials and Methods Description of Donors: All donors were residents of the Athens, Georgia area. They were contacted i n i t i a l l y one day postpartum at e i t h e r of the two major hospitals in Athens. Over a three year period 57 women provided milk f o r the study. The mothers were in good health, encountered no unusual d i f f i c u l t i e s during d e l i v e r y , and delivered healthy f u l l - t e r m infants.
FOLATE IN HUMANMILK
Collection of Milk Samples: Milk samples" were collected at the following three stages of lactation: early t r a n s i t i o n a l (4-7 days postpartum), t r a n s i t i o n a l (10-14 days postpartum), and mature (30-45 days postpartum). Samples were collected by either a Lopuco breast pump (Lopuco Ltd, Woodbine, MD) or by manual expression. Hand breast pumps and polyethylene wide-mouth v i a l s were provided. The mothers were instructed to collect approximately equal volumes of milk from the beginning, middle, and end of a feeding. I t was hoped that the donor's judgement would be s u f f i c i e n t to provide milk representative of the entire feeding. However, Lucas et al (15) determined that during a I0 min feeding 80-90% of the total milk was delivered to the infant in the f i r s t 4 min. Therefore, milk used in this study might not be e n t i r e l y representative of the entire feeding i f the middle sample was taken a f t e r 4 min from the i n i t i a t i o n of nursing. The samples of early t r a n s i t i o n a l milk contained a combined sample of milk from both a late evening (PM) feeding and an early morning (AM) feeding. For the t r a n s i t i o n a l and mature milk stages, two samples were collected to represent a PM feeding and an AM feeding. The samples were refrigerated immediately a f t e r c o l l e c t i o n , picked up a f t e r the morning c o l l e c t i o n , and transported on ice to the laboratory. Folate Assay: Free and total folate were assayed using Lactobacillus casei 7469 by the procedure o f Yamada (8) as modified by Khalsa (13). folate was determined by adding 3.0 ml of 0.I M sodium phosphate buffer, pH 6.8, containing I% ascorbic acid to 1.0 g of milk. For each g of milk, 1 ml of chicken pancreas extract (conjugase) was added. The conjugase extract was prepared by grinding dry acetone washed chicken pancreas powder (Difco Laboratories, D e t r o i t , MI 48232) to which a few drops of glycerol was added with a mortar and pestle, adding one ml of the phosphate-ascorbic acid buffer per 4 mg of pancreas powder and grinding u n t i l the powder was in suspension. The suspension was f i l t e r e d through glass wool. After addition of the conjugase preparatisn, the mixture was overlayered with tolueneRand incubated overnight at 37 C. The following morning, 3 ml of Pronase (Calbiochem-Beh~ing Corp., LaJolla, CA 92037) solution prepared by mixing 0.2 g Pronase powder with I00 ml of the phosphate-ascsrbic acid buffer were added. The digest was then incubated for 4 h at 37 C. After digestion, the samples were steBmed at I00 C for 5 min to remove the toluene and to inactivate the Pronase"o Enzyme controls consisting of 1.0 ml ~f HpO, 3 ml of phosphate-ascorbic acid buffer and the conjugase and Pronase preparations were prepared for each assay. Standard curves were prepared using pteroylglutamic acid. Recovery of added pteroylglutamic acid based on the total f o l i c acid extraction procedure was 97.8 • 2.9% (8 t r i a l s ) . Free folate was determined on milk samples that were not digested with conjugase or Pronase . S t a t i s t i c a l analysis was performed using the general linear models procedure and Duncan's multiple range test in the S t a t i s t i c a l Analysis System (16) package. RESULTS AND DISCUSSION No significant (p <.05) differences were found in transitional and mature milk samples collected at the late evening (PM) or early morning (AM) feeding when the influence of diurnal variation was tested. For all additional statistical comparisons, the morning and evening results were combined.
393
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Data on the free and t o t a l f o l a t e content of the milk is given in Table I. The value f o r t o t a l f o l a t e of 4.9 ~g/lO0 g f o r mature milk is similar to the values reported in Great B r i t a i n (7), by Thomas et al (9) and by Sneed et al ( I I ) . This value d i f f e r s considerably from the low value of 2.6 ~g/lO0 ml reported by Cooperman et al (12) and the higher value of 14.1 ug/lO0 ml reported by Tamura ( I 0 ) . At each stage, the free f o l a t e value was s i g n i f i c a n t l y d i f f e r e n t ( p < . 0 5 ) from the t o t a l f o l a t e value. As l a c t a t i o n progressed, both free and t o t a l f o l a t e increased. In the one week period as l a c t a t i o n progressed from the early t r a n s i t i o n a l (4-7 days postpartum) stage to the t r a n s i t i o n a l (10-14 days postpartum) stage, the t o t a l f o l a t e content increased by 1.4 ug/lO0 g from 2.1 to 3.5 ug/lO0 g. Likewise, as the milk passed from the t r a n s i t i o n a l stage to the mature stage (30-45 days postpartum), there was another increase of 1.4 ug/lO0 g from 3.5 ug/lO0 g to 4.9 ~g/lO0 g f o r t o t a l f o l a t e . TABLE 1
Mean Folate Content of Early Transitional, Transitional, and Mature Human Milk Mean • SD* (Range) Lactational Stage**
pg/lOOg Free Folate
Total Folate
Early Transitional
1.2 -+ O.7a ( 0 . I I - 2.86) N = 36
2.2 +_ 1.2a (.32-5.96) N = 36
Transitional
1.8 • O.7b (0.12 - 3.51) N : 70
3.5 .+ 1.6b (0.76 - 9.31) N = 65
Mature
2.2 -+ 1.2c (0.26 - 7.46) N : 72
4.9.+ 1.9c ( I . 0 - 9.38) N = 65
* Means in each column with d i f f e r e n t postscripts are s i g n i f i c a n t l y d i f f e r e n t at p < . 0 5 as determined by Duncan's multiple range t e s t . Free f o l a t e was s i g n i f i c a n t l y lower ( p < . 0 5 ) than t o t a l f o l a t e at each stage. ** N = number of samples As the total f o l a t e concentration increased with length of l a c t a t i o n , the percentage of f o l a t e in the free form decreased. Decreases from 54.5% free f o l a t e in the early t r a n s i t i o n a l milk to 51.4% free f o l a t e in the t r a n s i t i o n a l milk to 44.9% free f o l a t e in mature milk were noted. The increase in conjugated and/or bound forms may correspond to physiological processes that regulate f o l a t e concentration in the milk.
FOLATE IN HUMANMILK
395
More complex forms of folate may be used to transport or sequester folate in milk at the higher concentrations found in mature milk. The Recommended Dietary Allowance (RDA) for infants has been set at 5 ~g/kg/day (17). In order to compare intake to the RDA, estimates of infant folate intake at each stage of lactation were calculated using milk volumes of 400, 500 and 600 ml (18, 19, 20) and average infant weights of 3.5, 4.0 and 4.5 kg at the early t r a n s i t i o n a l , t r a n s i t i o n a l and mature (30 days postpartum) stages of l a c t a t i o n , respectively. Folate values determined in this study were converted to ug/lO0 ml by multiplying by the factor 1.03 (7). This gave values of 2.27 ug/lO0 ml at the early t r a n s i t i o n a l stage, 3.61 ug/lO0 ml at the t r a n s i t i o n a l stage and 5.05 ug/lO0 ml at the mature stage. Using these folate levels, calculated mean intakes were 2.5 ug/kg/day at the early t r a n s i t i o n a l stage, 4.5 ug/kg/day at the t r a n s i t i o n a l stage and 6.7 ug/kg/day 30 days postpartum. These values show that folate intake rapidly approaches recommended levels and that by 1 month postpartum mature milk provides recommended levels of folate to the infant. Further, because of the low folate levels in early milk during the f i r s t 2 weeks of l a c t a t i o n , the proper folate n u t r i t i o n a l status of the mother during pregnancy is emphasized to ensure adequate neonatal folate status during the time required for the milk to reach an adequate folate level. ACKNOWLEDGEMENTS We wish to express our appreciation to the s t a f f members of St. Mary's and Athens General Hospitals in Athens, Georgia for t h e i r assistance and support of this project. Also, the authors thank Dr. Mohan Rao and Ms. Sara J. Goldsmith of the Department of Food Science, University of Georgia f o r s t a t i s t i c a l analysis of the data. REFERENCES I.
MACY, I. G. Composition of human colostrum and milk. Child. 78:589-603, 1949.
Am. J. Dis.
2.
COLLINS, R. A., HARPER, A. E., SCHREIBER, M. and ELVEHJEM, C. A. The f o l i c acid and vitamin B1e content of the milk of various species. J. Nutr. 43:313-327, 1951.
3.
MATOTH, Y., PINKAS, A. and SROKA, C. Studies on f o l i c acid in infancy. I I I . Folate in breast fed infants and t h e i r mothers. Am. j . Clin. Nutr. 16:356-359, 1965.
4.
RAMASASTRI, B. Folate a c t i v i t y in human milk. 19:581-586, 1965.
5.
KARLIN, R. Etude sur les taux d'acide folique de l a i t humain et du l a i t bovin. Int. J. Vitamin Res. 27:334-341, 1967.
Br. J. Nutr.
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6.
JATHAR, V. S., KAMATH, S. A., PARIKH, M. N., REGE, D. V. and SATOSKAR, R. S. Maternal milk and serum vitamin Bl~, folic acid and protein levels in Indian subjects. Arch. Dis.'~hild. 45:236-241, 1970.
7.
Department of Health and Social Security. The composition of human milk. Report on Health and Social Subjects, No. 12. Her Majesty's Stationery Office, London, 1977.
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YAMADA, M. 1979.
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THOMAS, M. R., SNEED, S. M., WEI, C., NAIL, P. A., WILSON, M. AND SPRINKLE I l l , E. E. The effects of vitamin C, vitamin BA, vitamin BI~, f o l i c acid, riboflavin, and thiamin on the breast milk and m~ernal status of well-nourished women at 6 months postpartum. Am. J. Clin. Nutr. 33:2151-2156, 1980.
lO.
TAMURA, T., YOSHIMURA, Y. AND ARAKAWA, T. Humanmilk and folate status in lactating mothers and their infants. Am. J. Clin. Nutr. 33:193-197, 1980.
II.
SNEED, S. M., ZANE, C. AND THOMAS, M. R. The effects of ascorbic acid, vitamin B6, vitamin B]2 , and folic acid supplementation on the breast milk and maternal nutritional status of low socioeconomic lactating women. Am. J. Clin. Nutr. 34:1338-1346, 1981.
12.
COOPERMAN, J. M., DWECK, H. S., NEWMAN, L. J., GARBARINO, C. and LOPEZ, R. The folate in human milk. Am. J. Clin. Nutr. 36:576-580, 1982.
13.
KHALSA, I. K. Folate and folate binding capacity of human milk. Ph.D. Dissertation, University of Georgia, 1982.
14.
PICCIANO, M. F. The volume and composition of human milk. In: Infant and Child Feedin9. J. T. Bond, L. J. Filer, G. A. Leveille, A. M. Thomson and W. B. Weil, Jr. (eds.). Academic Press, Inc., New York, 1981, p. 47.
15.
LUCAS, A., LUCAS, P. J. and BAUM, J. D. The Nipple-Shield sampling system: a device for measuring the dietary intake of breast fed infants. Early Hum. Dev. 4:365-372.
16.
HELWIG, J. T. AND COUNCIL, K. A., eds. SAS User's Guide. 1979 ed. SAS Institute Inc., Raleigh, N. C., 1979.
17.
National Research Council. Recommended Dietary Allowances. 9th ed. National Academy of Sciences, Washington, 1980, p. I09.
Folate contents in milk.
Vitamins (Japan), 53:221-227,
FOLATE IN HUMAN MILK
18.
LONNERDAL, B., FORSUM, E. and HAMBRAEUS, L. A longitudinal study of the protein, nitrogen and lactose contents of human milk from Swedish well-nourished mothers. Am. J. Clin. Nutr. 29:1127-1133, 1976.
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19.
PAO, E. M., HIMES, J. M. and ROCHE, A. F. Milk intakes and feeding patterns of breast-fed infants. J. Am. Diet. Assoc. 77:540-545, 1980.
20.
PlCClANO, M. F., CALKINS, E. J., GARRICK, J. R. and DEERING, R. A. Milk and mineral intakes of breast-fed infants. Acta Paediatr. Scand. 70:189-194, 1981.
Accepted for publication March 4, 1984.
397
FOLATE CONTENTOF HUMANMILK DURING EARLYLACTATIONAL STAGESl Ronald R. Eitenmiller, Ph.D. Wonda D. Bryan, Indra K. Khals~, Ph.D., Ruth M. Feeley, Ph D. and Harold M. Barnhart, Ph.D. Department of Food Science, University of Georgia, Athens, Georgia
ABSTRACT Folate content of human milk was determined in early transitional milk (4-7 days postpartum), transitional milk (lO-14 days postpartum) and mature milk (36-45 days postpartum). Mature milk contained a mean total folate level of 4.9 ug/lO0 g. No diurnal variations were observed. At each stage, total folate was significantly higher (p< .05) than free folate. As lactation progressed to the mature milk stage, both free and total folate increased significantly (p<.05). The percentage of folate in the free form decreased as length of lactation increased. Based on folate levels determined in this study, a one month old breast-fed infant would have a folate intake of 6.7 ug/kg/day which is somewhat higher than the recommended dietary allowance for infants of 5 ~g/kg/day. INTRODUCTION Increased interest in breast feeding has led to increased knowledge of the nutrient composition of human milk. Such information is important for the understanding of dietary requirements of the infant and establishment of dietary allowances and standards for infant formulas. Wide variability exists in the literature regarding folate content of human milk (l-13). Values range from 0.07 ~g/lO0 ml (2) to 14.1 ~g/lO0 ml (IO). Since early investigators when assaying for human milk folate content used extraction procedures that did not adequately protect labile folates, the values reported were low (14). However, even among recent studies, large variability exists in values reported for mature human milk.
1
This research was supported by the National Institute of Child Health and 2 Human Development Grant No. 5 ROl HD14880-02. Author Feeley is affiliated with the Division of Nutrition, Emory University, Atlanta, Georgia. 391
392
R.R. EITENMILLER et al.
Karlin (5) analyzed the milk of 12 women d a i l y from 2 to 8 days postpartum. There was an increase in mean f o l a t e concentrations from 0.5 ug/lO0 ml to I . I ~g/lO0 ml during the 6 day period. Karlin (5) u t i l i z e d milk samples from 230 women at various stages of l a c t a t i o n and reported that f o l a t e levels remained constant between 4 and 9 months postpartum. The mean t o t a l f o l a t e level a f t e r 1 month postpartum was between 5 and 6 ug/lO0 ml. In 1977, the results of a large B r i t i s h study (7) showed that pooled mature human milk contained 5.2 ug/lO0 ml. S i m i l a r i l y , Thomas et al (9) reported a f o l a t e value of 5.5 and 5.0 ug/lO0 ml f o r 6 well-nourished mothers with f o l a t e supplementation, and without supplementation, r e s p e c t i v e l y , at 6 months postpartum. The researchers concluded that supplementation had l i t t l e e f f e c t on the f o l a t e content of the milk of well nourished mothers. Sneed et al ( I I ) in a s i m i l a r study of 16 low socio-economic women showed a s t a t i s t i c a l l y s i g n i f i c a n t difference between the f o l a t e values of a unsupplemented group compared to a f o l a t e supplemented group. The unsupplemented group had a mean f o l a t e level of 4.2 ~g/lO0 ml at 5-7 days postpartum and 4.3 ug/lO0 ml at 43-45 days postpartum. The supplemented group had the same mean value of 4.9 ~g/lO0 ml at each of the t e s t times. Studies by Yamada (8) and Tamura et al (I0) reported higher milk f o l a t e levels than the previously noted studies. Yamada (8), using an extraction procedure incorporating the use of the p r o t e o l y t i c enzyme Pronase , reported the mean t o t a l f o l a t ~ content of 21 samples of human milk to be 7.75 ~g/lO0 ml. The Pronase treatment of some milk samples more than doubled the measurable f o l a t e levels. Tamura et al (I0) in a study of the r e l a t i o n s h i p between infant serum and plasma f o l a t e levels and the f o l a t e concentration in maternal milk found that the mean f o l a t e content of 39 milk samples was 14.1 ~g/lO0 ml. This represents the highest human milk f o l a t e value reported to date. In contrast to the f o l a t e values reported by Tamura et al ( I 0 ) , Cooperman et al (12) recently reported that mature human milk contains 2.6 ~g/lO0 ml. These authors did not show a difference between free (unconjugated) and t o t a l f o l a t e values in the milk which d i f f e r s from the observations of Yamada (8). The present i n v e s t i g a t i o n was undertaken to determine the free and t o t a l f o l a t e content of human milk at early stages of l a c t a t i o n during which f o l a t e concentration rapidly changes. Also, diurnal v a r i a t i o n in the f o l a t e content was assessed. A modification of the e x t r a c t i o n procedure of Yamada (8) was used to ensure complete l i b e r a t i o n of bound folacin. In view of the many inconsistencies in the l i t e r a t u r e , i t was hoped that t h i s study would help to improve the understanding of the f o l a t e content of human milk and thus, the understanding of the f o l a t e intake of breast-fed infants. Materials and Methods Description of Donors: All donors were residents of the Athens, Georgia area. They were contacted i n i t i a l l y one day postpartum at e i t h e r of the two major hospitals in Athens. Over a three year period 57 women provided milk f o r the study. The mothers were in good health, encountered no unusual d i f f i c u l t i e s during d e l i v e r y , and delivered healthy f u l l - t e r m infants.
FOLATE IN HUMANMILK
Collection of Milk Samples: Milk samples" were collected at the following three stages of lactation: early t r a n s i t i o n a l (4-7 days postpartum), t r a n s i t i o n a l (10-14 days postpartum), and mature (30-45 days postpartum). Samples were collected by either a Lopuco breast pump (Lopuco Ltd, Woodbine, MD) or by manual expression. Hand breast pumps and polyethylene wide-mouth v i a l s were provided. The mothers were instructed to collect approximately equal volumes of milk from the beginning, middle, and end of a feeding. I t was hoped that the donor's judgement would be s u f f i c i e n t to provide milk representative of the entire feeding. However, Lucas et al (15) determined that during a I0 min feeding 80-90% of the total milk was delivered to the infant in the f i r s t 4 min. Therefore, milk used in this study might not be e n t i r e l y representative of the entire feeding i f the middle sample was taken a f t e r 4 min from the i n i t i a t i o n of nursing. The samples of early t r a n s i t i o n a l milk contained a combined sample of milk from both a late evening (PM) feeding and an early morning (AM) feeding. For the t r a n s i t i o n a l and mature milk stages, two samples were collected to represent a PM feeding and an AM feeding. The samples were refrigerated immediately a f t e r c o l l e c t i o n , picked up a f t e r the morning c o l l e c t i o n , and transported on ice to the laboratory. Folate Assay: Free and total folate were assayed using Lactobacillus casei 7469 by the procedure o f Yamada (8) as modified by Khalsa (13). folate was determined by adding 3.0 ml of 0.I M sodium phosphate buffer, pH 6.8, containing I% ascorbic acid to 1.0 g of milk. For each g of milk, 1 ml of chicken pancreas extract (conjugase) was added. The conjugase extract was prepared by grinding dry acetone washed chicken pancreas powder (Difco Laboratories, D e t r o i t , MI 48232) to which a few drops of glycerol was added with a mortar and pestle, adding one ml of the phosphate-ascorbic acid buffer per 4 mg of pancreas powder and grinding u n t i l the powder was in suspension. The suspension was f i l t e r e d through glass wool. After addition of the conjugase preparatisn, the mixture was overlayered with tolueneRand incubated overnight at 37 C. The following morning, 3 ml of Pronase (Calbiochem-Beh~ing Corp., LaJolla, CA 92037) solution prepared by mixing 0.2 g Pronase powder with I00 ml of the phosphate-ascsrbic acid buffer were added. The digest was then incubated for 4 h at 37 C. After digestion, the samples were steBmed at I00 C for 5 min to remove the toluene and to inactivate the Pronase"o Enzyme controls consisting of 1.0 ml ~f HpO, 3 ml of phosphate-ascorbic acid buffer and the conjugase and Pronase preparations were prepared for each assay. Standard curves were prepared using pteroylglutamic acid. Recovery of added pteroylglutamic acid based on the total f o l i c acid extraction procedure was 97.8 • 2.9% (8 t r i a l s ) . Free folate was determined on milk samples that were not digested with conjugase or Pronase . S t a t i s t i c a l analysis was performed using the general linear models procedure and Duncan's multiple range test in the S t a t i s t i c a l Analysis System (16) package. RESULTS AND DISCUSSION No significant (p <.05) differences were found in transitional and mature milk samples collected at the late evening (PM) or early morning (AM) feeding when the influence of diurnal variation was tested. For all additional statistical comparisons, the morning and evening results were combined.
393
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R.R. EITENMILLER et al.
Data on the free and t o t a l f o l a t e content of the milk is given in Table I. The value f o r t o t a l f o l a t e of 4.9 ~g/lO0 g f o r mature milk is similar to the values reported in Great B r i t a i n (7), by Thomas et al (9) and by Sneed et al ( I I ) . This value d i f f e r s considerably from the low value of 2.6 ~g/lO0 ml reported by Cooperman et al (12) and the higher value of 14.1 ug/lO0 ml reported by Tamura ( I 0 ) . At each stage, the free f o l a t e value was s i g n i f i c a n t l y d i f f e r e n t ( p < . 0 5 ) from the t o t a l f o l a t e value. As l a c t a t i o n progressed, both free and t o t a l f o l a t e increased. In the one week period as l a c t a t i o n progressed from the early t r a n s i t i o n a l (4-7 days postpartum) stage to the t r a n s i t i o n a l (10-14 days postpartum) stage, the t o t a l f o l a t e content increased by 1.4 ug/lO0 g from 2.1 to 3.5 ug/lO0 g. Likewise, as the milk passed from the t r a n s i t i o n a l stage to the mature stage (30-45 days postpartum), there was another increase of 1.4 ug/lO0 g from 3.5 ug/lO0 g to 4.9 ~g/lO0 g f o r t o t a l f o l a t e . TABLE 1
Mean Folate Content of Early Transitional, Transitional, and Mature Human Milk Mean • SD* (Range) Lactational Stage**
pg/lOOg Free Folate
Total Folate
Early Transitional
1.2 -+ O.7a ( 0 . I I - 2.86) N = 36
2.2 +_ 1.2a (.32-5.96) N = 36
Transitional
1.8 • O.7b (0.12 - 3.51) N : 70
3.5 .+ 1.6b (0.76 - 9.31) N = 65
Mature
2.2 -+ 1.2c (0.26 - 7.46) N : 72
4.9.+ 1.9c ( I . 0 - 9.38) N = 65
* Means in each column with d i f f e r e n t postscripts are s i g n i f i c a n t l y d i f f e r e n t at p < . 0 5 as determined by Duncan's multiple range t e s t . Free f o l a t e was s i g n i f i c a n t l y lower ( p < . 0 5 ) than t o t a l f o l a t e at each stage. ** N = number of samples As the total f o l a t e concentration increased with length of l a c t a t i o n , the percentage of f o l a t e in the free form decreased. Decreases from 54.5% free f o l a t e in the early t r a n s i t i o n a l milk to 51.4% free f o l a t e in the t r a n s i t i o n a l milk to 44.9% free f o l a t e in mature milk were noted. The increase in conjugated and/or bound forms may correspond to physiological processes that regulate f o l a t e concentration in the milk.
FOLATE IN HUMANMILK
395
More complex forms of folate may be used to transport or sequester folate in milk at the higher concentrations found in mature milk. The Recommended Dietary Allowance (RDA) for infants has been set at 5 ~g/kg/day (17). In order to compare intake to the RDA, estimates of infant folate intake at each stage of lactation were calculated using milk volumes of 400, 500 and 600 ml (18, 19, 20) and average infant weights of 3.5, 4.0 and 4.5 kg at the early t r a n s i t i o n a l , t r a n s i t i o n a l and mature (30 days postpartum) stages of l a c t a t i o n , respectively. Folate values determined in this study were converted to ug/lO0 ml by multiplying by the factor 1.03 (7). This gave values of 2.27 ug/lO0 ml at the early t r a n s i t i o n a l stage, 3.61 ug/lO0 ml at the t r a n s i t i o n a l stage and 5.05 ug/lO0 ml at the mature stage. Using these folate levels, calculated mean intakes were 2.5 ug/kg/day at the early t r a n s i t i o n a l stage, 4.5 ug/kg/day at the t r a n s i t i o n a l stage and 6.7 ug/kg/day 30 days postpartum. These values show that folate intake rapidly approaches recommended levels and that by 1 month postpartum mature milk provides recommended levels of folate to the infant. Further, because of the low folate levels in early milk during the f i r s t 2 weeks of l a c t a t i o n , the proper folate n u t r i t i o n a l status of the mother during pregnancy is emphasized to ensure adequate neonatal folate status during the time required for the milk to reach an adequate folate level. ACKNOWLEDGEMENTS We wish to express our appreciation to the s t a f f members of St. Mary's and Athens General Hospitals in Athens, Georgia for t h e i r assistance and support of this project. Also, the authors thank Dr. Mohan Rao and Ms. Sara J. Goldsmith of the Department of Food Science, University of Georgia f o r s t a t i s t i c a l analysis of the data. REFERENCES I.
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COOPERMAN, J. M., DWECK, H. S., NEWMAN, L. J., GARBARINO, C. and LOPEZ, R. The folate in human milk. Am. J. Clin. Nutr. 36:576-580, 1982.
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KHALSA, I. K. Folate and folate binding capacity of human milk. Ph.D. Dissertation, University of Georgia, 1982.
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LONNERDAL, B., FORSUM, E. and HAMBRAEUS, L. A longitudinal study of the protein, nitrogen and lactose contents of human milk from Swedish well-nourished mothers. Am. J. Clin. Nutr. 29:1127-1133, 1976.
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PAO, E. M., HIMES, J. M. and ROCHE, A. F. Milk intakes and feeding patterns of breast-fed infants. J. Am. Diet. Assoc. 77:540-545, 1980.
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PlCClANO, M. F., CALKINS, E. J., GARRICK, J. R. and DEERING, R. A. Milk and mineral intakes of breast-fed infants. Acta Paediatr. Scand. 70:189-194, 1981.
Accepted for publication March 4, 1984.
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