Milk folate binding protein (FBP): A secretory protein for folate?

NUTRITION RESEARCH,Vol. 4, pp. 181-187, 1984 0271-5317/84 $3.00 + .00 Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved.

MILK FOLATE BINDING PROTEIN (FBP): A SECRETORY PROTEIN FOR FOLATE? Jacob Selhub, Ph.D.1, Ralph Arnold, B.S.1, Anne M. Smith, M.S.2 and Mary F. Picciano, Ph.D.2 1The Department of Medicine, Section of Gastroenterology, University of Chicago, Chicago, Illinois 60637 2The Department of Foods and Nutrition, Nutritional Sciences Program, University of Illinois, Urbana, Illinois 61801 ABSTRACT Analyses of human milk samples reveal that although folate binding protein (FBP) concentration varies considerably among individuals, a positive correlation with milk folate concentrations exists. The equation expressing this relationship is Y = 29.2 + 0.78 X, where Y and X represent FBP and folate concentrations, respectively. Since folylpolyglutamates comprise at least half of total milk folate and are known not to cross biological membranes, these data suggest that folate is likely to be secreted from the lactating cell as s complex with FBP. This complex could exit from the lactating cell by a route which is common to other milk proteins. KEY WORDS:

Folate-binding protein, human milk, lactation, milk folate INTRODUCTION

In 1967 Ghitis reported the presence in bovine milk of a heat labile macromolecular factor that protected milk folate against adsorption to activated charcoal (1). This observation led to the conclusion that bovine milk contains high affinity folate binding proteins (FBP's). SubseQuent to this landmark finding, intensified studies were directed principally toward purification and determination of binding properties as well as identification of similar proteins in other tissues (2-5). A limited number of studies also were concerned with the possible roles of these proteins in mediation of folate transport across cellular membranes or in regulation of cellular folate metabolism (6-9). The o r i g i n a l observation of Ghitis that milk f o l a t e s are Q u a n t i t a t i v e l y bound to these proteins has been l a r g e l y neglected. This feature i s unique, since in other tissues the f o l a t e binding capacity comprises a small f r a c t i o n of the t o t a l f o l a t e content. While studying maternal f o l a t e status, human milk f o l a t e content and t h e i r r e l a t i o n s h i p to i n f a n t f o l a t e status, i t was noted that f o l a t e concentration in human milk varies considerably both within the same period of lactation in individuals and among individuals (lO). The present study was undertaken to examine the relationship between folate binding capacity and folate content of human milk samples.

Address reprint requests to: M. F. Picciano, Department of Foods and Nutrition, University of Illinois, 905 South Goodwin, Urbana, IL 61801. 181

182

J. SELHUB et al. MATERIALS AND METHODS

Subjects. This study is a part of a broad project which investigated folate nutriture of infants fed either human milk or proprietary formula. The use of human subjects in these investigations was annually reviewed and approved by the Institutional Review Board of the University of Illinois. Mothers of 21 to 38 years of age were studied at six weeks and three months post partum. All but three mothers received multivitamin and mineral supplement with 0.8 to 1.O mg folate. All infants were apparently healthy and received human milk as the sole nutritional source from birth to three months of age. Milk collection. At six weeks and three months of the infant age, three-day assessments of milk FBP and milk folate were performed. In absence of direct light, samples of fore- and hind-milk were collected by the mothers manually or with a polypropylene breast pump (Davol, Inc., Providence, RI), and then immediately frozen at -20oc. A total of twelve human milk samples were collected from each mother. At six weeks and three months post partum, fore- and hind-milk samples were collected on three consecutive days. At each collection period, day one samples were obtained at the morning feeding, day two samples at the midday feeding, and day three samples at the evening feeding. Mothers were provided with opaaue vials containing approximately 50 mg of sodium ascorbate to protect labile folate from oxidative destruction during collection. Determination of FBP. FBP was determined by a modification of the procedure which utilized cellulose-nitrate filter to selectively trap bound folate (ll,12). The milk sample was first acidified by dilution with 19 volumes (v/v) O.1 M acetic acid to dissociate bound endogenous folate. A 0.05 ml aliquot was then incubated for 50 min at room temperature with 8 ng (G-3H) folic acid, (28.4 mCi/pmol; Amersham, Arlington Heights, IL) in lO0 mM potassium phosphate buffer at a final pH of 7.2. The final volume was 1 ml. The mixture was then passed under a household vacuum through a cellulose-nitrate filter (0.45 , Schleicher and Schull, Dassel, Germany). The filter was washed twice, each time with 15 ml O . 1 M potassium phosphate buffer, pH 7.0, to remove remaining unbound radioactivity, then dried and counted in a scintillation counter as described previously (ll,12). The conditions of this assay were designed to obviate the need to remove endogenous folate, as hasbeen the practice with other methods that measured total folate binding capacity (13,14). These endogenous folates, which were dissociated from FBP by acid, amount to about 2% (range of 0.8 to 9%) of the radioactive folate added to the incubation mixture. Interference by these endogenous folates may actually be even smaller than their concentration in the incubated samples, since they consist primarily of methylated derivatives which compete poorly for binding sites at neutral pH against the test folic acid (2). Values obtained by this newly devised assay were consistently higher (lO to 20%) than those obtained by the twostep procedure in which the protein sample was first freed of its folate by acidification and charcoal treatment, then incubated with radioactive folate at neutral pH followea by a second treatment with coated charcoal to remove unbound radioactive folate. Determination of folate concentration. Milk folate was determined with Lactobacillus casei (ATCC 7469) after treatment with partially purified chicken pancreas conjugase as described by Cooperman and Shimizu (15) with the following modifications. Because of the smaller proportions of casein to whey in human milk (40:60) as compared to cow's milk (82:18), the

MILK FOLATE BINDING PROTEIN

183

precipitation step of casein with rennin was omitted as sample turbidity was not apparent. Statistical analysis. Analysis of variance, student's "t" test for paired data and correlation statistics were used in tests of significance. RESULTS Variation in milk FBP concentrations. Data presented in Table i show that FBP concentration varies considerably among the various milk samples. Most noticeable is the variation among individuals ranging from 21+6 (subject code T) to 224+--95 (subject code O) ng folate binding/ml mTlk. A second, less pronounced, yet statistically significant (P < 0.05) variation is found when the FBP concentrations of fore- and hind-milk were compared. Mean F ~ concentrations of fore-milk at six weeks and three months are 82.1+36.2 and 82.9+--56.7, respectively, compared to 103+45.5 and 119.6+63.5 for The hind-milk at the same two periods. Difference--in concentratio--ns was also seen when samples from morning, midday and evening were compared. The extent of these variations is indicated from the often large standard deviation of some of the individual means given in Table 1. TABLE 1 FBP Concentration in Fore- and Hind-Human Milk at 6 Weeks and 3 Months Post Partum

Subject Code

B C H L M 0 P Q R S T U AA CC Means

FBP Concentration (ng Folate Binding/ml Milk)l 6 Weeks 3 Months Fore Hind Fore Hind

56+18 I18T78 80+4 91~38 37~10 83+--62 84+--34 70~32 97+--23 87+--13 47-~13 i15~28 I05~54 82+--35

71+4 169~99 97+32 98"$13 73+--34 174"$'37 69-~17 I08T9 122~25 101"$-4 3~12 121+--26 142+--20 84+--6

50+22 54"$'31 88+67 82~29 41T7 186~107 66+--42 108"$-73 91+--42 63~32 30+--12 120+--65 97+--24 75u

129+10 91"$'25 84+43 187T21 50~13 224"$'95 i05~ii 135~76 14~43 I09T61 21"$-6 175"~65 13~35 70~14

82.1+36.2

103.0+45.5

82.9+56.7

i19.6+63.5

lEach value represents the average + S.D. of FBP concentration of three milk samples (morning, midday and evening) collected before infants were nursed (foremilk) and after infants nursed (hindmilk) from a single breast.

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J. SELHUB et al.

Milk folate concentrations. Data presented in Table 2 show that milk folate concentration is different among various individuals and within the same lactation period; hind-milk contained more folate than fore-milk. TABLE 2 Folate Concentration in Fore- and Hind-Milk at 6 Weeks and 3 Months Post Partum

Subject Code

B

Folate Concentration (ng/ml Milk) 1 6 Weeks 3 Months Fore Hind Fore Hind

45+28

L M 0

36+14 77+30 76+33 6~15 26+6 70~87

66+25 111+48 112+61 87+--27 46+18 85+--68

43+30 85+96 61+--54 32+10 102+--71

108+16 76+27 81+74 14~46 83+54 146~74

P Q

8~18 43+--29

82+--32

100~24

151~63

76+-20 i0~ii 80~6 49+11 82+-2 89+-22 74+-15

146+-71 i07~32 64~29 49+10 67~17 61+-ll 82+-57

19~71 160~90 90+-'50 34+4 130~67 83+-50 117T78

77+51

114+61

C

H

R S T U AA CC

Means

105"/38 7~5 48+4 71+-22 76~23

79+-24 67+32

(37_~21)2

83+28

(44+~21)

(41~30)

(59~_351

lEach value represents the average + S.D. as determined by L. casei assay following incubation with partiall~purified chicken pancreas conjugase. 2Values in parentheses represent the mean folate activity of samples prior to incubation with partially purified chicken pancreas conjugase.

The data in Table 2 were obtained with L. casei after treating the milk samples with chicken pancreas conjugase. At the bottom of the table, the mean folate concentration based on L. casei responses before conjugase treatment is given. The values are-abou-~alf of those obtained after conjugase treatment, indicating that about half of the folate in these milk samples consist of polyglutamyl derivatives with at least 4 glutamic acid residues. These results are in agreement with recent analyses of human milk samples (16,17) which also showed that considerable portions of milk folates consist of polyglutamate derivatives. Interrelation between milk FBP and milk folate concentrations. The plot of folate concentrations ~etermined in over 200 milk samples vs. FBP concentrations in the same samples (Fig. l) shows a highly significant (P < O.OO1 for paired T test) positive correlation (r = 0.71). The line in Fig. 1 corresponds to Y = 0.79 X + 29.2 where Y and X represent FBP and folate concentrations, respectively.

MILK FOLATE BINDING PROTEIN

~0

0

iX

250

A

200

O (J

A

iX iXZX

%

iX%iXA

]50 -

185

iX/iX

iX ix

X


100 -

A iX

50

I

1

I

I

I

I

50

loo

15o

200

250

30o

Folate concentration (ng/ml) FIG. 1 Correlation between human milk folate concentrations and FBP concentrations. FBP concentrations are expressed in ng folic acid binding capacity per ml.

DISCUSSION This observed close correlation between folate concentration and folate binding capacity in human milk suggests that the secretion of these two from mammary glands is coordinated. The significance of these findings depends to a large degree on the identification of the stage at which the complex between FBP and folate takes place. Such complex may take place (1) immediately following the entry of folate from plasma into the lactating cell, (2) before secretion, or (3) after secretion. Presence of folylpolyglutamate in milk implies that in the process of its transfer into milk, plasma folate, mostly 5-methyl-tetrahydropteroylglutamate, undergoes extensive metabolism and polyglutamation. The occurrence of these metabolic products of folate strongly argues against the first possibility that complex of folate with FBP takes place immediately following the entry of folate into the lactating cell. The complex with FBP is too stable (Kd = lO-9-10-11M) to allow any significant enzymatic changes of the folate substrate (18). It has been shown that binding to FBP inhibits the reduction of folate by dihydrofolate reductase (4,19). Moreover, the observed low synthesis of dTMP from deoxyuridine by certain leukocyte populations was thought to be due to the high FBP concentration in these cells which, by forming a complex with folate, restricts folate coenzyme activities (20).

Presence of folylpolyglutamates also argues against the third possibility, namely, that complex with FSP occurs after secretion from the

186

J. SELHUB et al.

lactating cell. The consensus among investigators is that these forms of folates are incapable of crossing biological membranes (21). Crossing of these membranes is possible at the monoglutamate or at the most extreme triglutamate level as in the case with L. casei cells. Cells which lost the ability to synthesize folylpolyglutamat~ a ~ o w n to have also lost the ability to retain folate (21). Presence of folylpolyglutamate in milk can best be explained by the second possibility. That is, milk folylpolyglutamates are secreted from the lactating cell as a complex with FBP, perhaps through a similar route which allows the secretion of milk proteins. Folate polyglutamates do bind to FBP with the same avidity as do other monoglutamyl folates (22). ACKNOWLEDGMENT

This work is supported by grant AM-15351 from the National Institutes of Health, by USDA/SEA grant 5901-0410-9-0506-0 and the Illinois Agricultural Experiment Station. We thank Ann Carrillo for preparation of the manuscript. REFERENCES i.

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The folate binding in milk.

2.

FORD, 3. E., SALTER, D. N. and SCOTT, K. 3. The folate binding proteins in milk. O. Dairy Res. 36: 455-442, 1969.

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COLMAN, N., HETTIARACHCHY, N. and HERBERT, V. Detection of a milk factor that facilitates folate uptake by intestinal cells. Science 211: 1427-1428, 1981.

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WAXMAN, S. and SCHREIBER, C. The role of folic acid binding protein (FABR) in the cellular uptake of folates. Proc. Soc. Exptl. Biol. Med. 14.__Z7:760-764, 1974.

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RUBINOFF, M., ABRAMSON, R., SCHREIBER, C. and WAXMAN, S. Effect of a folate-binding protein on the plasma transport and tissue distribution of folic acid. Acta Haematol. 65: 145-152, 1981.

10.

SMITH, A. M., PICCIANO, M. F. and DEERING, R. Folate supplementation during lactation: Maternal folate status, human milk folate content, and their relationship to infant folate status. O. Ped. Gastro. Nutr. (in press).

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SELHUB, O. and ROSENBERG, I. H. Demonstration of high-affinity folate binding activity associated with the brush border membranes of rat kidney. Proc. Natl. Acad. Sciences 75: 3090-3093, 1978.

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SELHUB, J., GAY, A. C. and ROSENBERG, I. H. Folate binding activity in epithelial brush border membranes. A system for investigating membrane associated folate binding proteins. In: The Chemistry and Biology of Pterins. R. Kisliuk and G. M. Brown (eds). Elsevier, North Holland

Press, Amsterdam, 1979, pp. 393-396. 13.

KAMEN, B. A. and CASTON, D. J. Identification of a folate binder in hog kidney. O. Biol. Chem. 250: 2203-2205, 1975.

14.

COLMAN, N. and HERBERT, V. Total folate binding capacity of normal human plasma and variations in uremia, cirrhosis, and pregnancy. Blood 48: 911-912, 1976.

15.

COOPERMAN, O. M. and SHIMIZU, N. An improved method to assay folates in milk by a turbidimetric microbiological assay. Anal. Letters 12: 1443-1449, 1979.

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AREEKFUL, S., VONGYUTHITHAM, M., YAMARAT, P. and SUNTHORNTHOM, S. Folic acia and folic acid binding proteins in milk. O. Med. Assoc. Thailand 61: 81-86, 1978.

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TAMURA, T., YOSHIMURA, Y. and ARAKAWA, T. Human milk folate and folate status in lactating mothers and their infants. Am. O. Clin. Nutr. 33: 193-197, 1980.

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HANSEN, S. I., HOLM, J. and LYNGBYE, J. Cooperative binding of folate to a protein isolated from cow's whey. Bioch. Biophys. Acta. 535: 309-318, 1978.

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ROTHENBERG, S. P. A macromolecular factor in some leukemic cells which binds folate acid. Proc. Soc. Exptl. Biol. Med. 133: 428-438, 1970.

20.

DA COSTA, M., ROTHENBERG, S. P. and KAMEN, B. DNA synthesis in chronic myelogenous leukemia ceils: Comparison of results in cells containing folate binding factor to replicating cells without binders. Blood 39: 621-627, 1972.

21.

MCGUIRE, O. O. and BERTINO, O. R. Enzymatic synthesis and function of folylpolyglutamates. Mol. Cell. Biochem. 38: 19-48, 1981.

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Accepted for publication December 15, 1984.