THE INHIBITION OF CELLULAR UPTAKE OF FOLATE BY FOLIC ACID-BINDING PROTEIN Studies o f the uptake o f labeled folates by HeLa cells in tissue culture show that folic acid-binding proteins are not transport proteins and make folates less available for cellular uptake. Key Words: folic acid-binding proteins, pteroylglutarnic acid, tetrahydrofolic acid, HeLa cells, cellular uptake of folates
Specific binding proteins for folic acid (FABP) have been demonstrated in a number of materials. These appear to be present in both bovine and human milk' and in the sera from folic acid deficient patients. Folic acid-binding materials have also been demonstrated in the sera from patients suffering from a number of pathological states and in women taking oral contraceptives.* The identity of the binding agents in these various sources has not been established but they share some common characteristics. These are a rapid association rate and a slow dissociation rate for the binding of pteroyl glutamic acid and preferential binding of oxidized folyl mono- and polyglutamates compared with reduced folates. There appear t o be two proteins involved in folate binding, one a beta-globulin with a molecular weight of around 50,000 and another much larger protein. The amounts of FABP in milk are much higher than in serum. It has been suggested that FABP may be a cellular, perhaps membrane-derived protein with the role of regulating cellular uptake, distribution, and storage of the various folate coenzymes. The true physiological significance of FABP is yet t o be established. Waxman and Schreiber3 recently described some studies on the role of these proteins in the cellular uptake of folates. The uptake of labeled pteroyl glutamic acid (" H PGA) and methyl-tetrahydrofoiic acid ( 3 H methyl-THFA) was studied using HeLa cells in monolayer cultures. Some
HeLa cells were adapted t o folate deficiency by passage through a folic acid free medium. The fetal calf serum which is usually added to tissue culture media was dialyzed and as used contained less than 1 ng of folate per milliliter. The uptake of the labeled folates was measured a t 37" and a t 4°C; H teropterin (a folyl polyglutamate) was also studied under the same conditions. The cells cultured in a normal medium showed an increased uptake of both forms for some three hours with slight evidence of a plateau after one hour. The uptakeof H methyl-THFA was greater than for H PGA a t 37°C whereas the uptake of the polyglutamate was virtually zero. Uptake a t 4°C was very low and Dilantin (0.1 mg per milliliter) and ethanol ( 1 percent) had no effect on the uptake. The folate-depleted cells showed a reduced growth rate. It was only half that of the normal cells after one week and cell death was increased in the folate-deficient cells after three weeks. There was also evidence of deranged DNA synthesis, as judged by the suppression of the incorporation of 3 H thymidine by deoxyuridine. DNA synthesis was restored t o normal in these cells by the addition of pteroyl glutamic acid. Uptake of the labeled folates by these depleted cells was much greater, by a factor of five for 3 H PGA and three for 3 H methyl-THFA compared with the normal cells. There was no difference in the uptake of the two forms. When the cells were incubated with various substances (normal sera, folatedeficient and uremic sera, cow's milk, NUTRITION REVIEWSIVOL. 33, NO. 6 I JUNE 1975
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beta-lactoglobulin, and human milk) containing varying amounts of FABP, there was a considerable reduction in the uptake of the two forms of labeled folates compared with cells incubated with normal human albumin. The inhibition of uptake was related t o the FABP content of the various test substances as measured by their capacity to bind H PGA.4 HeLa cell cultures are rapidly doubling lines so that a large proportion of thecells are actively synthesizing DNA a t any one time. One would therefore expect them t o have a high requirement for folate. The temperature dependence of uptake together with the absence of an effect of Dilantin or ethanol suggest that an energydependent active transport mechanism is involved in both PGA and methyl-THFA uptake. The FABP in the various substances and sera tested appeared t o make the labeled folates less available for uptake into the HeLa cells. In the sera tested the inhibition of 3 H PGA uptake was directly proportional t o their FABP contents. It therefore appears that FABP in the serum will retard the delivery of folates into the cell and that FABP is not a serum-delivery protein for folate. It is possible that under certain conditions serum FABP could produce i n t race I I u Ia r folate deficiency despite adequate serum folate levels. This situation has been suggested as the cause of megaloblastic marrow maturation in some uremic patients whose sera contain FABP. Milk FABP has been shown to depress the bacterial uptake of folate. FABP in the mammary gland may act as a mechanism for accumulating folate from the blood plasma into the milk, and in the gut facilit a t e absorption by preventing folate uptake by the intestinal microorganism^.^ The milk FABP may influence the availability of folate t o the neonate as well as influencing the ecology of the gut microflora.
184 NUTRITION REVIEWSIVOL. 33, NO. 6 I JUNE 1975
FABP could not be detected in portions of the culture media from the HeLa cells cultured in the absence of folate although it is released into the media by cultures of c h r o n ic m y e logenous leukemia cells6 FABP has been demonstrated in the brush border cells of r a t small intestine and human lymphocytes. These suggest that FABPs are cellular-derived proteins which may be involved in the intracellular accumulation of folate. FABP in the membrane may be the rate limiting factor in folate uptake because it could act as a folate carrier during transport across the membrane, i t s availability then being determined by the folate state of the cell. Much remains t o be done before the true physiological role of these folic acidbinding proteins is established and the place of these proteins in folic acid metabolism i s understood. Folic acid nutrition represents many puzzling features a t present and the elucidation of the role of these binding proteins may resolve some of these problems. 0 ~
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1. J. Ghitis: The Folate Binding in Milk. Am. J. Clin. Nutrition 20: 1-4,1967 2. Folate Binder in Leukocytes and Serum. Nutrition Reviews 33:9-10, 1975 3. S.Waxman and C. Shreiber: The Role of Folic Acid Binding Proteins (FABP) in the Cellular Uptake of Folates. Proc. SOC.Exp. Biol. Med.
147:760-764,1974 4. S. Waxman and C. Schreiber: Measurement of Serum Folate Levels and Serum Folic Acidb i n d i n g Protein by 3H-PGA Radioassay. Blood 42: 281-290.1973 5. J. E. Ford: Some Observations on the Possible Nutritional Significance of Vitamin BIZFolate-Binding Proteins in Milk. Brit. J. Nutrition 31:243-257,1974 6. M. da Costa and S. P. Rothenberg: Studies of the Folate Binding Factor in Cultures and Subcellular Fractions of Chronic Myelogenous Leukemia (CML) Cells. Clin. Res. 22: 486A,
1974
Specific binding proteins for folic acid (FABP) have been demonstrated in a number of materials. These appear to be present in both bovine and human milk' and in the sera from folic acid deficient patients. Folic acid-binding materials have also been demonstrated in the sera from patients suffering from a number of pathological states and in women taking oral contraceptives.* The identity of the binding agents in these various sources has not been established but they share some common characteristics. These are a rapid association rate and a slow dissociation rate for the binding of pteroyl glutamic acid and preferential binding of oxidized folyl mono- and polyglutamates compared with reduced folates. There appear t o be two proteins involved in folate binding, one a beta-globulin with a molecular weight of around 50,000 and another much larger protein. The amounts of FABP in milk are much higher than in serum. It has been suggested that FABP may be a cellular, perhaps membrane-derived protein with the role of regulating cellular uptake, distribution, and storage of the various folate coenzymes. The true physiological significance of FABP is yet t o be established. Waxman and Schreiber3 recently described some studies on the role of these proteins in the cellular uptake of folates. The uptake of labeled pteroyl glutamic acid (" H PGA) and methyl-tetrahydrofoiic acid ( 3 H methyl-THFA) was studied using HeLa cells in monolayer cultures. Some
HeLa cells were adapted t o folate deficiency by passage through a folic acid free medium. The fetal calf serum which is usually added to tissue culture media was dialyzed and as used contained less than 1 ng of folate per milliliter. The uptake of the labeled folates was measured a t 37" and a t 4°C; H teropterin (a folyl polyglutamate) was also studied under the same conditions. The cells cultured in a normal medium showed an increased uptake of both forms for some three hours with slight evidence of a plateau after one hour. The uptakeof H methyl-THFA was greater than for H PGA a t 37°C whereas the uptake of the polyglutamate was virtually zero. Uptake a t 4°C was very low and Dilantin (0.1 mg per milliliter) and ethanol ( 1 percent) had no effect on the uptake. The folate-depleted cells showed a reduced growth rate. It was only half that of the normal cells after one week and cell death was increased in the folate-deficient cells after three weeks. There was also evidence of deranged DNA synthesis, as judged by the suppression of the incorporation of 3 H thymidine by deoxyuridine. DNA synthesis was restored t o normal in these cells by the addition of pteroyl glutamic acid. Uptake of the labeled folates by these depleted cells was much greater, by a factor of five for 3 H PGA and three for 3 H methyl-THFA compared with the normal cells. There was no difference in the uptake of the two forms. When the cells were incubated with various substances (normal sera, folatedeficient and uremic sera, cow's milk, NUTRITION REVIEWSIVOL. 33, NO. 6 I JUNE 1975
103
beta-lactoglobulin, and human milk) containing varying amounts of FABP, there was a considerable reduction in the uptake of the two forms of labeled folates compared with cells incubated with normal human albumin. The inhibition of uptake was related t o the FABP content of the various test substances as measured by their capacity to bind H PGA.4 HeLa cell cultures are rapidly doubling lines so that a large proportion of thecells are actively synthesizing DNA a t any one time. One would therefore expect them t o have a high requirement for folate. The temperature dependence of uptake together with the absence of an effect of Dilantin or ethanol suggest that an energydependent active transport mechanism is involved in both PGA and methyl-THFA uptake. The FABP in the various substances and sera tested appeared t o make the labeled folates less available for uptake into the HeLa cells. In the sera tested the inhibition of 3 H PGA uptake was directly proportional t o their FABP contents. It therefore appears that FABP in the serum will retard the delivery of folates into the cell and that FABP is not a serum-delivery protein for folate. It is possible that under certain conditions serum FABP could produce i n t race I I u Ia r folate deficiency despite adequate serum folate levels. This situation has been suggested as the cause of megaloblastic marrow maturation in some uremic patients whose sera contain FABP. Milk FABP has been shown to depress the bacterial uptake of folate. FABP in the mammary gland may act as a mechanism for accumulating folate from the blood plasma into the milk, and in the gut facilit a t e absorption by preventing folate uptake by the intestinal microorganism^.^ The milk FABP may influence the availability of folate t o the neonate as well as influencing the ecology of the gut microflora.
184 NUTRITION REVIEWSIVOL. 33, NO. 6 I JUNE 1975
FABP could not be detected in portions of the culture media from the HeLa cells cultured in the absence of folate although it is released into the media by cultures of c h r o n ic m y e logenous leukemia cells6 FABP has been demonstrated in the brush border cells of r a t small intestine and human lymphocytes. These suggest that FABPs are cellular-derived proteins which may be involved in the intracellular accumulation of folate. FABP in the membrane may be the rate limiting factor in folate uptake because it could act as a folate carrier during transport across the membrane, i t s availability then being determined by the folate state of the cell. Much remains t o be done before the true physiological role of these folic acidbinding proteins is established and the place of these proteins in folic acid metabolism i s understood. Folic acid nutrition represents many puzzling features a t present and the elucidation of the role of these binding proteins may resolve some of these problems. 0 ~
~~~~
~~~~
~
~~~
1. J. Ghitis: The Folate Binding in Milk. Am. J. Clin. Nutrition 20: 1-4,1967 2. Folate Binder in Leukocytes and Serum. Nutrition Reviews 33:9-10, 1975 3. S.Waxman and C. Shreiber: The Role of Folic Acid Binding Proteins (FABP) in the Cellular Uptake of Folates. Proc. SOC.Exp. Biol. Med.
147:760-764,1974 4. S. Waxman and C. Schreiber: Measurement of Serum Folate Levels and Serum Folic Acidb i n d i n g Protein by 3H-PGA Radioassay. Blood 42: 281-290.1973 5. J. E. Ford: Some Observations on the Possible Nutritional Significance of Vitamin BIZFolate-Binding Proteins in Milk. Brit. J. Nutrition 31:243-257,1974 6. M. da Costa and S. P. Rothenberg: Studies of the Folate Binding Factor in Cultures and Subcellular Fractions of Chronic Myelogenous Leukemia (CML) Cells. Clin. Res. 22: 486A,
1974
