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Biotin content of human milk during early lactational stages
NUTRIIION RESEARCH, Vol. 2, pp. 579-583, 1982 0271-5317/82/050579-05503.00/0 Printed in the USA. Copyright (c) 1982 Pergamon Press Ltd. All rights reserved.
BIOTIN CONTENT OF HUMAN MILK DURING EARLY LACTATIONAL STAGES I Sara J. Goldsmith, M.S., Ronald R. Eitenmiller, Ph.D., Ruth M. Feeley, Ph.D., Harold M. Barnhart, Ph.D., and Frances C. Maddox Department of Food Science, University of Georgia, Athens, Georgia
ABSTRACT
In order to expand and update the nutritional information of human milk, the biotin content was determined in early transitional (three to eight days postpartum), transitional (I0 to 14 days postpartum), and mature (30 to 47 days postpartum) human milk from 84 donors. The women ranged in age from 16 to 38 years. The biotin level increased significantly (P < .01) as time postpartum increased from 0.07 ~g/100 g at the early transitional stage to 0.30 and 0.47 ~g/100 g at the transitional and mature stages, respectively. Early morning and late evening samples were collected at the transitional and mature stages; however, no significant diurnal variations were observed. Key terms:
human milk, biotin, lactation.
INTRODUCTION
The infants requirement for biotin is not known; however, in 1976 the Committee on Nutrition of the American Academy of Pediatrics recommended that infant formulas contain a minimum of 1.5 ~g of biotin per i00 kcal. This level is similar to the biotin content of commonly used milk-based formulas and represents an intake that is adequate to avoid any signs of biotin deficiency (i). While breast-fed infants generally exhibit lower incidence of gastrointestinal infections than bottle-fed infants (2, 3), it has also been reported that sudden infant death syndrome (SIDS) is less common among breast-fed infants (4). A recent report that diets marginally deficient in biotin, coupled with stress, may be linked to SlDS (5), suggests that the biotin content of an infant's diet may have more importance than previously recognized. The recentconcern over biotin in the infant's diet was reflected in the Infant Formula Act of 1980 (6) which established 1,5 ~g/100 kcal (1.05 ~g/lO0 g) as the minimum biotin content in non-milk based formulas. Previous standards, such as the 1971 FDA regulations for infant formulas (7) did not set minimum biotin standards.
This research was supported by the National Institute of Child Health and Human Development Grant No. 5 R01 HD14880-02.
579
580
S.J. Goldsmith et al.
The major objective of this study was to obtain current data on the biotin content of human milk during early stages of lactation. Further, diurnal variation in the biotin content was assessed. This information is needed for a better understanding of the biotin requirements of infants.
MATERIALS AND METHODS
Description of Donors: The donors for this study were all residents of the Athens, Georgia area. They were contacted initially one day postpartum at either of the two major hospitals in Athens. Over a 2.5 year period, a total of 87 women agreed to participate and signed an informed consent. The women were of middle socio-economic status and ranged in age from 16 to 38 years. Of the women who participated, 79 were Caucasian, 3 were black, and 2 were of Oriental descent. All of the women were in good health, encountered no unusual difficulties during delivery, and delivered healthy full-term infants. Collection of Milk Samples: Donors collected the milk samples by manual expression or by using a Lopuco breast pump (Lopuco Ltd., Woodbine, MD). The milk was collected at the following three stages of lactation: early transitional (three to eight days postpartum), transitional (10-14 days postpartum), and mature (30-47 days postpartum). At the early transitional stage, each donor collected one composite sample from both the late evening and early morning feedings. Two samples were collected at the transitional and mature stages. For these samples, donors were instructed to collect approximately equal volumes of milk from the beginning, middle, and end of one late evening and one early morning feeding. The samples were refrigerated immediately after collection, picked up after the morning collection, and transported on ice to the laboratory. Biotin Assay: Biotin content was determined by a microbiological assay adapted from the procedure of Skeggs (8) using Lactobacillus plantarum ATCC 8014 as the test organism. Milk samples were diluted with an equal volume of 6N H~SO. and autoclaved at 20 psi, 120~ for 60 min. After hydrolysis, the extractlon medium was cooled, adjusted to pH 4.5 with 1.0 N NaOH and clarified by filtration. Final dilution of the extract was completed with distilled water. The assay was run on two or more sample aliquot volumes using Difco biotin assay medium (Difco Laboratories, Detroit, MI). After incubation at 37~ for 16-20 h, absorbance of the growth medium was determined at 620 nm in a Bausch and Lomb Spectronic 20 Spectrophotometer (Bausch and Lomb, Inc., Atlanta, GA). Growth response was proportional to the volume of extract assayed. Recovery of added biotin averaged 110%. Statistical analysis was performed using the general linear models procedure and Duncan's multiple range test in the Statistical Analysis System (9) package.
RESULTS AND DISCUSSION
Data showing the mean biotin content of early transitional, transitional and mature milk are presented in Table i. No significant (P < .05) diurnal variations were noted at either the transitional or mature stages. For all additional statistical comparisons the morning and evening results were combined. The biotin content was the lowest in early transitional milk (0.07 ~g/lO0 g) and increased significantly (P < 0.01) to 0.30 ~g/100 ml at the transitional stage and 0.47 ~g/100 ml at the mature stage. Macy (i0) reported similar biotin levels in early lactational stage milk. She reported a biotin content of 0.06 ~g/100 ml in colostrum (one to five days postpartum) and 0.35 ~g/100 ml in transitional milk (six to 12 days postpartum); however a higher level of 0.81 ~g/100 ml was
Biotin in Human Milk
581
reported for mature milk collected 15 days to 15 months postpartum. Since a study by Hood and Johnson (ii) indicated that the biotin content of human milk continues to increase as lactation time increases, the long range of lactation time represented by Macy's (i0) compilation of data may account for the higher biotin value at the mature stage. Hood and Johnson (ii) found that the biotin content increased rapidly between day one and day seven from 0.30 to 0.68 ~g/100 ml followed by an increase to 1.25 and 1.27 ~g/100 ml at days 49 and 70, respectively. The fact that these values are much higher than the results reported by Macy (i0) and in our study could be due to geographical location and/or differences in diet. Another study (12) on samples collected in five separate locations in Great Britain, but all transported to one laboratory for analysis, reported large geographical differences with mean values ranging from 0.52 ~g/100 ml in one area to 1.13 ~g/100 ml in another area. A mean of 0.72 ~g/100 ml was reported for samples from each of the other three locations, resulting in an overall average of 0.76 ug/100 ml. These mean values were determined on pooled samples of mature milk collected 14-63 days postpartum. TABLE i Mean Biotin Content of Early Transitional, Transitional and Mature Human Milk
*
Lactational Stage*
Mean • SD** (Range) pg/100 g
Early transitional n = 84
0.07 • 0.09 a (0.00 - 0.72)
Transitional n = 67
0.30 • 0.22 b (0.01 - 1.18)
Mature n ffi 64
0.47 • 0.22 b (0.05 - 1.41)
n denotes the number of donors
** Means followed by different postscripts are significantly different at P <.01 as determined by Duncan's multiple range test. The frequency distribution of the biotin values are shown in Fig. I. As time postpartum increased, the range of biotin values also increased. At the early transitional stage, 95% of the samples were in the range 0.0-0.2 Mg/100 g, with 3% in the range 0.2-0.4 ~g/100 g and 2% in the range of 0.6-0.8 ~g/100 g. The range at the transitional stage was from 0.01-1.2 Mg/100 g with 95% of the samples ranging from 0.01-0.7 Mg/100 g. The remaining 5% were in the range from 0.7-1.2 ~g/100 g. At the mature stage, 95% of the samples ranged from 0.05-1.0 Bg/100 g while the remaining 5% ranged from 1.0-1.4 Mg/100 g. Although a Recommended Dietary Allowance (RDA) for biotin has not been established, the Committee on Dietary Allowances estimated the safe and adequate intake of biotin for infants up to six months of age to be 35 ~g/day (13). Based on an estimated average daily consumption of 600 ml of milk (14) for infants one month old, the infants in the present study consumed an average of 2.8 ~g biotin/day. This level is considerably lower than the 6.3 ~g/day provided by infant formulas containing a mlnimum of 1.5 ~g/100 kcal and more than ten-fold lower than the estimated safe and adequate level recommended by the National Research Council (NRC) (13). Therefore, these findings indicate that there is a large difference between the estimated safe and adequate level suggested for biotin by the NRC and the actual intakes of beast-fed infants during the neonatal period.
582
(/') hi ,,--I O,.
S.J. Goldsmith et al.
6O
(/) i. o
5o
t~ LsJ a~
qo
'~
30
z 20
10
0.1
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0.3
0,5
0.7
: -
o.g
-
I.I
4
1.3
0.1
I
0.3
-
-
0.5
0,7
0.9
1.1
a
1.3
I
0,1
I
0.3
0.5
0.7
s
0.9
1.1
1.3
I
8Io'fZN MIDPOINT
5TRGE
UG/IO0 GRAMS
FIG. i Biotin frequency distribution for early transitional, transitional, and mature milk (stages i, 2, and 3, respectively)
With respect to the report by Johnson et al. (5) that SIDS may be linked to biotin deficiency, the evidence here seems to contradict their theory. As mentioned earlier, SIDS is less prevalent among breast-fed infants (4), yet our study shows that the biotin concentration of human milk is one-half of that required in infant formula. If in fact biotin deficiency is linked to SlDS, breast-fed infants with a lower biotin intake would be more susceptible than formula-fed infants.
ACKNOWLEDGEMENTS
We wish to express our appreciation to the staff members of St. Mary's and Athens General hospitals in Athens, Georgia for their assistance and support of this project.
REFERENCES
i.
American Academy of Pediatrics, Committee on Nutrition. Commentary on breast-feeding and infant formulas, including proposed standards for formulas. Pediatrics, 57:278-285, 1976.
Biotin in Human Milk
583
2.
PLANK, S. J. and MILANESl, M. L. Infant feeding and infant mortality in rural Chile. Bull WHO 48:203-210, 1973.
3.
LARSEN, S.A. and HOMER, D. R. Relation of breast versus bottle feeding to hospitalization for gastroenteritis in a middle-class U.S. population. ~. Pediatr. 92:417-418, 1978.
4.
GUNTHER, M. The neonate's immunity gap, breast feeding, and cot death. Lancet, 1:441-442, 1975.
5.
JOHNSON, A. R., HOOD, R. L. and EMERY, J. L. death syndrome. Nature, 285:159-160, 1980.
6,
Infant Formula Act of 1980.
7.
Food and Drug Administration Rules and Regulations (pt 125). Label statements concerning dietary properties of food purporting to be or represented for specific dietary uses. Federal Register, 36:23553, 1971.
8.
SKEGGS, H. R. Biotin. In: Analytical Microbiology. Academic Press, Inc., New York, 1963, pp. 421-430.
9.
HELWIG, J. T. and COUNCIL, K. A., eds. Institute Inc., Raleigh, 1979.
Biotin and the sudden infant
Public Law 96-359.
F. Kavanagh (ed).
SAS User's Guide.
1979 ed.
SAS
i0.
MACY, I. G. Composition of human colostrum and milk. 78:589-603, 1949.
ii.
HOOD, R. L. and JOHNSON, A. R. Supplementation of infant formulations with biotin. Nutr. Reports International, 21:727-731, 1980.
12.
Department of Health and Social Security. The composition of mature human milk. Her Majesty's Stationery Office, London, 1977.
13.
National Research Council. Reco~ended Dietary Allowances. tional Academy of Sciences, Washington, 1980, p. 178.
14.
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.
Am. J. Dis. Child.
9th ed.
Na-
BIOTIN CONTENT OF HUMAN MILK DURING EARLY LACTATIONAL STAGES I Sara J. Goldsmith, M.S., Ronald R. Eitenmiller, Ph.D., Ruth M. Feeley, Ph.D., Harold M. Barnhart, Ph.D., and Frances C. Maddox Department of Food Science, University of Georgia, Athens, Georgia
ABSTRACT
In order to expand and update the nutritional information of human milk, the biotin content was determined in early transitional (three to eight days postpartum), transitional (I0 to 14 days postpartum), and mature (30 to 47 days postpartum) human milk from 84 donors. The women ranged in age from 16 to 38 years. The biotin level increased significantly (P < .01) as time postpartum increased from 0.07 ~g/100 g at the early transitional stage to 0.30 and 0.47 ~g/100 g at the transitional and mature stages, respectively. Early morning and late evening samples were collected at the transitional and mature stages; however, no significant diurnal variations were observed. Key terms:
human milk, biotin, lactation.
INTRODUCTION
The infants requirement for biotin is not known; however, in 1976 the Committee on Nutrition of the American Academy of Pediatrics recommended that infant formulas contain a minimum of 1.5 ~g of biotin per i00 kcal. This level is similar to the biotin content of commonly used milk-based formulas and represents an intake that is adequate to avoid any signs of biotin deficiency (i). While breast-fed infants generally exhibit lower incidence of gastrointestinal infections than bottle-fed infants (2, 3), it has also been reported that sudden infant death syndrome (SIDS) is less common among breast-fed infants (4). A recent report that diets marginally deficient in biotin, coupled with stress, may be linked to SlDS (5), suggests that the biotin content of an infant's diet may have more importance than previously recognized. The recentconcern over biotin in the infant's diet was reflected in the Infant Formula Act of 1980 (6) which established 1,5 ~g/100 kcal (1.05 ~g/lO0 g) as the minimum biotin content in non-milk based formulas. Previous standards, such as the 1971 FDA regulations for infant formulas (7) did not set minimum biotin standards.
This research was supported by the National Institute of Child Health and Human Development Grant No. 5 R01 HD14880-02.
579
580
S.J. Goldsmith et al.
The major objective of this study was to obtain current data on the biotin content of human milk during early stages of lactation. Further, diurnal variation in the biotin content was assessed. This information is needed for a better understanding of the biotin requirements of infants.
MATERIALS AND METHODS
Description of Donors: The donors for this study were all residents of the Athens, Georgia area. They were contacted initially one day postpartum at either of the two major hospitals in Athens. Over a 2.5 year period, a total of 87 women agreed to participate and signed an informed consent. The women were of middle socio-economic status and ranged in age from 16 to 38 years. Of the women who participated, 79 were Caucasian, 3 were black, and 2 were of Oriental descent. All of the women were in good health, encountered no unusual difficulties during delivery, and delivered healthy full-term infants. Collection of Milk Samples: Donors collected the milk samples by manual expression or by using a Lopuco breast pump (Lopuco Ltd., Woodbine, MD). The milk was collected at the following three stages of lactation: early transitional (three to eight days postpartum), transitional (10-14 days postpartum), and mature (30-47 days postpartum). At the early transitional stage, each donor collected one composite sample from both the late evening and early morning feedings. Two samples were collected at the transitional and mature stages. For these samples, donors were instructed to collect approximately equal volumes of milk from the beginning, middle, and end of one late evening and one early morning feeding. The samples were refrigerated immediately after collection, picked up after the morning collection, and transported on ice to the laboratory. Biotin Assay: Biotin content was determined by a microbiological assay adapted from the procedure of Skeggs (8) using Lactobacillus plantarum ATCC 8014 as the test organism. Milk samples were diluted with an equal volume of 6N H~SO. and autoclaved at 20 psi, 120~ for 60 min. After hydrolysis, the extractlon medium was cooled, adjusted to pH 4.5 with 1.0 N NaOH and clarified by filtration. Final dilution of the extract was completed with distilled water. The assay was run on two or more sample aliquot volumes using Difco biotin assay medium (Difco Laboratories, Detroit, MI). After incubation at 37~ for 16-20 h, absorbance of the growth medium was determined at 620 nm in a Bausch and Lomb Spectronic 20 Spectrophotometer (Bausch and Lomb, Inc., Atlanta, GA). Growth response was proportional to the volume of extract assayed. Recovery of added biotin averaged 110%. Statistical analysis was performed using the general linear models procedure and Duncan's multiple range test in the Statistical Analysis System (9) package.
RESULTS AND DISCUSSION
Data showing the mean biotin content of early transitional, transitional and mature milk are presented in Table i. No significant (P < .05) diurnal variations were noted at either the transitional or mature stages. For all additional statistical comparisons the morning and evening results were combined. The biotin content was the lowest in early transitional milk (0.07 ~g/lO0 g) and increased significantly (P < 0.01) to 0.30 ~g/100 ml at the transitional stage and 0.47 ~g/100 ml at the mature stage. Macy (i0) reported similar biotin levels in early lactational stage milk. She reported a biotin content of 0.06 ~g/100 ml in colostrum (one to five days postpartum) and 0.35 ~g/100 ml in transitional milk (six to 12 days postpartum); however a higher level of 0.81 ~g/100 ml was
Biotin in Human Milk
581
reported for mature milk collected 15 days to 15 months postpartum. Since a study by Hood and Johnson (ii) indicated that the biotin content of human milk continues to increase as lactation time increases, the long range of lactation time represented by Macy's (i0) compilation of data may account for the higher biotin value at the mature stage. Hood and Johnson (ii) found that the biotin content increased rapidly between day one and day seven from 0.30 to 0.68 ~g/100 ml followed by an increase to 1.25 and 1.27 ~g/100 ml at days 49 and 70, respectively. The fact that these values are much higher than the results reported by Macy (i0) and in our study could be due to geographical location and/or differences in diet. Another study (12) on samples collected in five separate locations in Great Britain, but all transported to one laboratory for analysis, reported large geographical differences with mean values ranging from 0.52 ~g/100 ml in one area to 1.13 ~g/100 ml in another area. A mean of 0.72 ~g/100 ml was reported for samples from each of the other three locations, resulting in an overall average of 0.76 ug/100 ml. These mean values were determined on pooled samples of mature milk collected 14-63 days postpartum. TABLE i Mean Biotin Content of Early Transitional, Transitional and Mature Human Milk
*
Lactational Stage*
Mean • SD** (Range) pg/100 g
Early transitional n = 84
0.07 • 0.09 a (0.00 - 0.72)
Transitional n = 67
0.30 • 0.22 b (0.01 - 1.18)
Mature n ffi 64
0.47 • 0.22 b (0.05 - 1.41)
n denotes the number of donors
** Means followed by different postscripts are significantly different at P <.01 as determined by Duncan's multiple range test. The frequency distribution of the biotin values are shown in Fig. I. As time postpartum increased, the range of biotin values also increased. At the early transitional stage, 95% of the samples were in the range 0.0-0.2 Mg/100 g, with 3% in the range 0.2-0.4 ~g/100 g and 2% in the range of 0.6-0.8 ~g/100 g. The range at the transitional stage was from 0.01-1.2 Mg/100 g with 95% of the samples ranging from 0.01-0.7 Mg/100 g. The remaining 5% were in the range from 0.7-1.2 ~g/100 g. At the mature stage, 95% of the samples ranged from 0.05-1.0 Bg/100 g while the remaining 5% ranged from 1.0-1.4 Mg/100 g. Although a Recommended Dietary Allowance (RDA) for biotin has not been established, the Committee on Dietary Allowances estimated the safe and adequate intake of biotin for infants up to six months of age to be 35 ~g/day (13). Based on an estimated average daily consumption of 600 ml of milk (14) for infants one month old, the infants in the present study consumed an average of 2.8 ~g biotin/day. This level is considerably lower than the 6.3 ~g/day provided by infant formulas containing a mlnimum of 1.5 ~g/100 kcal and more than ten-fold lower than the estimated safe and adequate level recommended by the National Research Council (NRC) (13). Therefore, these findings indicate that there is a large difference between the estimated safe and adequate level suggested for biotin by the NRC and the actual intakes of beast-fed infants during the neonatal period.
582
(/') hi ,,--I O,.
S.J. Goldsmith et al.
6O
(/) i. o
5o
t~ LsJ a~
qo
'~
30
z 20
10
0.1
I
0.3
0,5
0.7
: -
o.g
-
I.I
4
1.3
0.1
I
0.3
-
-
0.5
0,7
0.9
1.1
a
1.3
I
0,1
I
0.3
0.5
0.7
s
0.9
1.1
1.3
I
8Io'fZN MIDPOINT
5TRGE
UG/IO0 GRAMS
FIG. i Biotin frequency distribution for early transitional, transitional, and mature milk (stages i, 2, and 3, respectively)
With respect to the report by Johnson et al. (5) that SIDS may be linked to biotin deficiency, the evidence here seems to contradict their theory. As mentioned earlier, SIDS is less prevalent among breast-fed infants (4), yet our study shows that the biotin concentration of human milk is one-half of that required in infant formula. If in fact biotin deficiency is linked to SlDS, breast-fed infants with a lower biotin intake would be more susceptible than formula-fed infants.
ACKNOWLEDGEMENTS
We wish to express our appreciation to the staff members of St. Mary's and Athens General hospitals in Athens, Georgia for their assistance and support of this project.
REFERENCES
i.
American Academy of Pediatrics, Committee on Nutrition. Commentary on breast-feeding and infant formulas, including proposed standards for formulas. Pediatrics, 57:278-285, 1976.
Biotin in Human Milk
583
2.
PLANK, S. J. and MILANESl, M. L. Infant feeding and infant mortality in rural Chile. Bull WHO 48:203-210, 1973.
3.
LARSEN, S.A. and HOMER, D. R. Relation of breast versus bottle feeding to hospitalization for gastroenteritis in a middle-class U.S. population. ~. Pediatr. 92:417-418, 1978.
4.
GUNTHER, M. The neonate's immunity gap, breast feeding, and cot death. Lancet, 1:441-442, 1975.
5.
JOHNSON, A. R., HOOD, R. L. and EMERY, J. L. death syndrome. Nature, 285:159-160, 1980.
6,
Infant Formula Act of 1980.
7.
Food and Drug Administration Rules and Regulations (pt 125). Label statements concerning dietary properties of food purporting to be or represented for specific dietary uses. Federal Register, 36:23553, 1971.
8.
SKEGGS, H. R. Biotin. In: Analytical Microbiology. Academic Press, Inc., New York, 1963, pp. 421-430.
9.
HELWIG, J. T. and COUNCIL, K. A., eds. Institute Inc., Raleigh, 1979.
Biotin and the sudden infant
Public Law 96-359.
F. Kavanagh (ed).
SAS User's Guide.
1979 ed.
SAS
i0.
MACY, I. G. Composition of human colostrum and milk. 78:589-603, 1949.
ii.
HOOD, R. L. and JOHNSON, A. R. Supplementation of infant formulations with biotin. Nutr. Reports International, 21:727-731, 1980.
12.
Department of Health and Social Security. The composition of mature human milk. Her Majesty's Stationery Office, London, 1977.
13.
National Research Council. Reco~ended Dietary Allowances. tional Academy of Sciences, Washington, 1980, p. 178.
14.
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.
Am. J. Dis. Child.
9th ed.
Na-