The
`Vitamins
`
`Fundamental Aspects in Nutrition
`and Health
`
`SECOND EDITION
`
`Gerald F. Combs, Jr., Ph.D.
`Professor of Nutrition
`Division of Nutritional Sciences
`Cornell University
`Ithaca, New York
`
`HEALTH SCIENCES LIBRARY
`ST. FRANCIS HOSPITAL
`& MEDICAL CENTER
`
`ACADEMIC PRESS
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`Copyright© 1998, 1992 by ACADEMIC PRESS
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`16
`
`Folate
`
`Using Streptococcus lactis R as a test organism, we have obtained in a
`highly concentrated and probably nearly pure form an acid nutrilite with
`interesting physiological properties. Four tons of spinach have been
`extracted and carried through the first stages of concentration .... This
`acid, or one with similar chemical and physiological properties, occurs in
`a number of animal tissues of which liver and kidney are the best sources
`. . .. It is especially abundant in green leaves of many kinds, including
`grass. Because of this fact, we suggest the name "folic acid" [Latin,
`folium-leaf]. Many commercially canned greens are nearly lacking in the
`H. K. Mitchell, E. S. Snell, and R. J. Williams
`substance.
`
`I. Significance of Folate
`II. Sources of Folate
`III. Absorption of Folate
`IV. Transport of Folate
`V. Metabolism of Folate
`VI. Metabolic Functions of Folate
`VII. Folate Deficiency
`VIII. Pharmacologic Uses of Folate
`IX. Folate toxicity
`X. Case Study
`Case Questions
`Study Questions and Exercises
`Recommended Reading
`
`378
`379
`382
`383
`386
`388
`395
`397
`398
`398
`400
`400
`400
`
`Anchoring Concepts
`
`1. Folate is the generic descriptor for folic acid (pteroylmonoglutamic acid)
`and related compounds exhibiting qualitatively the biological activity of
`folic acid. The termfolates refers generally to the compounds in this group,
`including mono- and polyglutamates.
`2. Palates are active as coenzymes in single-carbon metabolism.
`3. Deficiencies of folate are manifested as anemia and dermatologic lesions.
`
`Learning Objectives
`
`1. To understand the chief natural sources of folates
`2. To understand the means of absorption and transport of the folates
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`378
`
`16. Folate
`
`Vocabulary
`
`3. To understand the biochemical functions of the folates as coenzymes in sin(cid:173)
`gle-carbon metabolism, and the relationship of that function to the physio(cid:173)
`logical activities of the vitamin
`4. To understand the metabolic interrelationship of folate and vitamin B 12 and
`its physiological implications
`
`5-Formimino-FH4
`Betaine
`5-Formyl-FH4
`Cervical paralysis
`10-Formyl-FH4
`7 ,8-Dihydrofolate
`10-Formyl-FH, synthe-
`reductase
`FH2 (dihydrofolic acid)
`tase
`FH4 (tetrahydrofolic acid) Homocysteine
`Leukopenia
`Folate
`Folate-binding proteins Macrocytic anemia
`(FBPs)
`Megaloblasts
`5,10-Methenyl-FH4
`Folate receptors
`Methionine synthetase
`Folic acid
`Folyl conjugase
`Methotrexate
`5,10-Methylene-FH4
`Folyl y-glutamyl hydro-
`5, 10-Methylene-FH,
`lase
`Folyl polyglutamates
`dehydrogenase
`5, 10-Methylene-FH4
`Folyl polyglutamate
`reductase
`synthetase
`
`5-Methyl-FH4
`Methyl-folate trap
`Pernicious anemia
`Pterin ring
`Pteroylglutamic acid
`Purines
`Serine hydroxymethyl-
`transferase
`Single-carbon metabo-
`!ism
`Sulfa drugs
`Thymidylate (dTMP)
`Vitamin B 12
`
`I. Significance of Folate
`
`FOLATE IS a vitamin that is often not appreciated, either for its importance in normal
`metabolism or its relevance to the etiologies of .chronic diseases and birth defects. Widely
`distributed among foods, particularly those of plant foliar origin, this abundant vitamin is
`underconsumed by people whose food habits do not emphasize plant foods. Intimately
`related in function with vitamins B12 and B6
`, its status at the level of subclinical deficien(cid:173)
`cy can be difficult to assess and the full extent of its interrelationships with these vitamins
`and with amino acids remains incompletely elucidated.
`Folate deficiency is an important problem in many parts of the world, particularly
`where there is poverty and malnutrition. It is an important cause of anemia, second only
`to nutritional iron deficiency in that regard. Evidence shows that marginal folate status can
`support apparently normal circulating folate levels while still limiting single-carbon
`metabolism. Thus, folate is emerging as having an important role in the etiology of homo(cid:173)
`cysteinemia, which has been identified as a risk factor for occlusive vascular disease,
`cancer, and birth defects. It has been estimated that an increase in mean folate intake of
`200 !J.g/day could reduce coronary artery disease deaths in the United States by
`13,500-50,000; and the finding of markedly reduced neural tube defects risk by pericon(cid:173)
`ceptional folate treatment has driven folate supplementation effotts in the United States.
`Still, there must be concern about the use of folate supplements, particularly high-level
`supplements, as folate is known to mask the macrocytic anemia of vitamin B12 deficien(cid:173)
`cy, which will lead to neuropathy if not corrected.
`For these several reasons, it is important to understand folate nutrition.
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`II. Sources of Folate
`
`11. Sources of Folate
`
`379
`
`Distribution in Foods
`Folates (folyl polyglutamates) occur in a wide variety of foods and feedstuffs of both plant
`and animal origin (Table 16-1). Liver, mushrooms, and green leafy vegetables are rich
`sources of folate in human diets; while oilseed meals (for example, soybean meal) and
`animal by-products are importaJ?.t sources of folate in animal feeds. The folates in foods
`and feedstuffs are almost exclusively in reduced form as polyglutarnyl derivatives of
`tetrahydrofolic acid (FH4). Very little free folate (folyl monoglutamate) is found in foods
`or feedstuffs.
`Analyses of foods have revealed a wide distribution of general types of folate deriva(cid:173)
`tives, the predominant forms being 5-methyl-FH4 and 10-formyl-FH4. The folates found
`in organ meats (for example, liver and kidney) are about 40% methyl derivatives, where(cid:173)
`as that in milk (and erythrocytes) is exclusively the methyl form. Some plant materials
`also contain mainly 5-methyl-FH4 (for example, lettuce, cabbage, orange juice), but
`others (for example, soybean) contain relatively little of that form ( -15% ), the rest occur(cid:173)
`ring as the 5- and 10-formyl derivatives. Most of the folates in cabbage are hexa(cid:173)
`and heptaglutamates, whereas half of those in soybean are monoglutamates. More than a
`third of the folates in orange juice are present as monoglutamates and nearly half are
`present as pentaglutamates. Liver and kidney contain mainly pentaglutamates, and -60%
`of the folates in milk are monoglutamates (with only 4-8% each of di- to heptagluta(cid:173)
`mates).
`
`Table 16-1. Folate Contents of Foods
`
`Food
`Dairy products
`
`Milk
`Cheese
`Meats
`Beef
`Liver
`
`Beef
`Chicken
`Tuna
`Cereals
`Barley
`Corn
`Rice
`Polished
`Unpolished
`Wheat, whole
`
`Wheat bran
`
`Folate
`(!J.g/100 g)
`
`5-12
`20
`
`5-18
`
`140-1070
`
`1810
`15
`
`15
`35
`
`15
`25
`30-55
`
`80
`
`Food
`Other
`Eggs
`Brewers' yeast
`
`Vegetables
`Asparagus
`
`Beans
`Broccoli
`Brussels sprouts
`Cabbage
`Cauliflower
`
`Peas
`Soybeans
`Spinach
`Tomatoes
`
`Fruits
`Apples
`Bananas
`
`Oranges
`
`Folate
`(!J.g/100 g)
`
`70
`1500
`
`70-175
`
`70
`180
`90-175
`15-45
`55-120
`
`90
`360
`50-190
`5-30
`
`5
`30
`25
`
`,
`.
`
`j.
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`Stability
`
`16. Folate
`
`Most folates in foods and feedstuffs (that is, folates other than folic acid 1 and S-formyl(cid:173)
`FH4) are easily oxidized and, therefore, are unstable to oxidation under aerobic conditions
`of storage and processing. Under such conditions (especially in the added presence of
`heat, light, and/or metal ions), FH4 derivatives can readily be oxidized to the correspond(cid:173)
`ing derivatives of dihydrofolic acid (FH2) (partially oxidized) or folic acid (fully oxi(cid:173)
`dized), some of which can react further to yield physiologically inactive compounds. For
`example, the two predominant folates in fresh foods, 5-methyl-FH4 and 10-formyl-FH
`,
`4
`are converted to 5-methyl-5,6-FH2 and 10-formylfolic acid, respectively. For this reason,
`5-methyl-5,6-FH2 has been found to account for about half of the folate in most prepared
`foods. Although it can be reduced to the FH4 form (e.g., by ascorbic acid), in the acidity
`of normal gastric juice it isomerizes to yield 5-methyl-5,8-FH2 which is completely inac(cid:173)
`tive. It is of interest to note that, owing to their gastric anacidosis, this isomerization does
`not occur in pernicious anemia patients, who are thus able to utilize the partially oxidized
`form by absorbing it and subsequently activating it to 5-methyl-FH4
`• Because some folate
`derivatives of the latter type can support the growth responses of test microorganisms used
`to measure folates,Z some information in the available literature may overestimate the
`biologically useful folate contents of foods and/or feedstuffs. Substantial losses in the
`folate contents of food can occur as the result of leaching in cooking water when boiling
`(losses of total folates of 22% for asparagus and 84% for cauliflower have been observed),
`as well as oxidation, as described above.
`
`Bioavailability
`
`The biological availability of folates is variable among foods (30-80%, in comparison
`with folic acid); it is generally less well utilized from plant-derived foods than from ani(cid:173)
`mal products (Table 16-2). Several factors affect the biologic availability of food folates:
`
`The character of the diet Folates can bind to the food matrices; many foods contain
`inhibitors of the intestinal brush border folate conjugase and/or folate transport
`
`Inherent characteristics of various folates Folate vi tamers vary in biopotency
`
`Nutritional status of the host Deficiencies of iron and vitamin C status are associated
`with impaired utilization of dietary folate3
`
`The interactions of these factors complicate the task of predicting the bioavailability of
`dietary folates. This problem is exacerbated by the methodological difficulties in evaluat(cid:173)
`ing folate utilization, which can be done through bioassays with animal models, 4 balance
`
`1 Throughout, the term folic acid is used as the specific trivial name for the compound pteroylglutamic acid.
`2 Lactobacillus casei, Streptococcus faecium (fmmerly, S.
`lactis R. and S. faecalis, respectively), and
`Pediococcus cerevisiae (formerly, Leuconostoc citrovonan) have been used. Of these, L. casei responds to the
`widest spectrum of folates.
`3 Some anemic patients respond optimally to oral folate therapy only when they are also given iron. Patients
`with scurvy often have megaloblastic anemia, apparently owing to impaired utilization of folate. In some scor(cid:173)
`butic patients, vitamin C has an antianemic effect; others require folate to cotTect the anemia.
`4 As with any application of information from studies with animal models, the validity of extrapolation is an
`issue important in assessing folate bioavailability. For example, the rat and many other species have little or
`no jejunal brush border conjugase activity, these species relying on the pancreatic conjugase for folate decon(cid:173)
`jugation. This contrasts with the pig and human, which deconjugate folates primarily by jejunal brush border
`activity, with activity of only secondary importance in the pancreatic juice.
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`II. Sources of Folate
`
`381
`
`Table 16-2. Biologic Availability to Humans
`of Folates in Foods
`
`Food/feedstuff
`Bananas
`
`Cabbage
`Eggs
`Lima beans
`Liver (goat)
`Orange juice
`
`Bioavailability•
`(range of reported values, %)
`
`0-148
`0--127
`35-137
`
`0-181
`9- 135
`
`29-40
`Spinach
`26-99
`24-7 1
`Tomatoes
`0- 64
`Wheat germ
`10- 100
`Brewers' yeast
`0-83
`Soybean meal
`Sources: Baker, H., Jaslow, S. P., and Frank, 0. (1978). J. Am.
`Geriatr. Soc. 26, 218-221; Baker, H., and Srikantia, S. G.
`(1976). Am. J. Clin. Nutr. 29, 376- 379; Tamura, T., and
`Stokstad, E. L. R. (1973). Br. J. Haematol. 25, 513-532.
`•Results expressed relative to folic acid.
`
`studies with humans, or isotopic methods to measure the appearance of folates in blood,
`excreta, and tissues.
`Under fasting conditions, folic acid, 5-methyl-FH4
`, and 5-formyl-FH4 are virtually
`completely absorbed and most polyglutamyl folates seem to be 60-80% absorbed,
`although slightly higher and lower efficiencies have been reported. The general finding
`of lower absorptive efficiency of polyglutamyl folates relative to folic acid suggests that
`the hydrolytic cleavage of the polyglutamyl side chains of food folates is important to
`their physiologic utilization. Folate absorption responds markedly to changes in pH:
`marginally acidic conditions in the intestinal lumen (such as in patients with pancreatic
`insufficiency) can increase folate utilization,5 whereas more strongly acidic conditions
`(such as in patients with atrophic gastritis) can inhibit folate utilization. 6 Acid condi(cid:173)
`tions inhibit the enzymatic cleavage of folyl poly glutamate side chains (by the so-called
`conjugase). Natural conjugase inhibitors are contained in certain foods: cabbage,
`oranges, yeast, beans (red kidney, pinto, lima, navy, soy), lentils, and black-eyed peas .7
`The presence of conjugase inhibitors reduces folate bioavailability; for example, this
`effect appears to be the basis for the low availability of the vitamin in orange juice.
`Folate absorption can also be reduced by certain drugs including cholestyrarnine (which
`binds folates), ethanol (which inhibits deconjugation), salicylazosulfapyridine, 8
`diphenylhydantoin,9 aspirin and other salicylates, and several nonsteroidal antiinflam(cid:173)
`matory drugs.
`
`' This occurs by the loss of bicarbonate, which results in a shift of lumenal pH closer to the optimum for folate
`absorption.
`6 Elderly plllicnts with alrop'hi gn. tritis I'CSpond lo simultaneous LroalJuent with folate and acid.
`7 The conjugasc inhibitors in beans and peas ·reside in the ~ecd coats nnd are heal labile.
`8 This dru g, al~o cal led A~ulficli ne and sul t'rtS(tlazi ne, U, used to treat in.flamrua tory bowel disorder.
`9 This drug, also called Dilanli.n, is an unticonvuls:tnL
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`382
`
`16. Folate
`
`III. Absorption of Folate
`
`Active Transport and Diffusion
`
`Dietary folates are absorbed as folic acid, 5-methyl-FH4, and 5-formyl-FH4 in most
`species, although some (e.g., dogs) appear also to absorb folyl polyglutamates. Folic acid
`is actively transported across the jejunum, and perhaps the duodenum, by an Na+-coupled,
`carrier-mediated process that is stimulated by glucose and shows a pH maximum at about
`pH 6. Folic acid can also be absorbed passively, presumably by diffusion. Whereas the sat(cid:173)
`urable transport process is maximal at lumenal folate concentrations of 10-20 11M, the
`nonsaturable process is linearly related to lumenal folate concentrations and can account
`for 20-30% of folate absorption at high folate intakes. The overall efficiency of folate
`absorption appears to be -50% (10-90% ). Malabsorption of the vitamin occurs in diseases
`affecting the intestinal mucosa.
`A microclimate hypothesis has been proposed for the enteric absorption of folates. It
`holds that folate absorption is dependent on the pH of the proximal jejunum, with an opti(cid:173)
`mum at pH 6.0-6.3. According to this hypothesis, the elevated absorption of folate in indi(cid:173)
`viduals with pancreatic exocrine insufficiency may be due to the low pancreatic excretion
`of bicarbonate in that condition. Loss of buffering capacity renders the intestinal lumenal
`milieu slightly more acidic, minimizing the charge on the folate molecule, thus facilitat(cid:173)
`ing its diffusion across the brush border membrane. Under more basic conditions (i.e., pH
`>6.0), folate absorption falls off rapidly. Two jejunal brush border folate-binding proteins
`(FBPs), thought to be involved in this process, have been isolated.
`
`Folyl Conjugase
`
`Because the majority of food folates occur as reduced polyglutamates, they must be
`cleaved to the mono- or diglutamate forms for absorption. This is accomplished by the
`action of an exocarboxypeptidase folyl 'Y·glutamyl hydrolase, more commonly called folyl
`conjugase. Conjugase activity is widely distributed in the mucosa of the proximal small
`intestine, both intracellularly and in association with the brush border. These appear to be
`different enzymes. 10 The intracellular enzyme is localized in the lysosomes and has an opti(cid:173)
`mum of pH 4.5-50, whereas the brush border enzyme has an optimum of pH 6.5-7.0.
`Although the latter enzyme is present in lower amounts, it appears to be important for the
`hydrolysis of dietary folyl polyglutamates. Folyl conjugase activities have also been found
`in bile, pancreatic juice, kidney, liver, placenta, bone marrow, leukocytes, and plasma,
`although the physiological importance of the activity in these tissues is uncertain. In the
`uterus, conjugase activity is induced by estrogen. Conjugase activity is reduced by nutii(cid:173)
`tional zinc deficiency or by exposure to naturally occurring inhibitors in foods (Table 16-3).
`Loss of conjugase activity by either means results in impaired folate absorption. Studies
`with several animal models have demonstrated that chronic ethanol feeding can decrease
`intestinal hydrolysis of folyl polyglutamates and can impair the absorption, transport, cel(cid:173)
`lular release, and metabolism of folates. Effects of this nature are thought to contribute to
`the folate deficiencies frequently observed among chronic alcoholics; however, there are
`other likely contiibuting factors, as enterocytes are known to be sensitive to ethanol toxic(cid:173)
`ity, and many chronic alcoholics can have insufficient dietary intakes of the vitamin.
`
`10 The human brush border enzyme has a molecular mass of 700 kDa, whereas the lysosomal enzyme has a mol(cid:173)
`ecular mass of only 75 kDa.
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`IV. Transport of Folate
`
`383
`
`Table 16-3.
`Inhibition of Jejunal Folate Conjugase
`Activities in Vitro by Components of Selected Foods
`
`Food
`
`Pig conjugase
`(% inhibition)
`
`Human conjugase
`(% inhibition)
`
`15.9
`33.2
`35.2
`19.3
`28.3
`0
`14.2
`46.0
`15.3
`13.9
`73.4
`5.3
`
`35.5
`Red kidney beans
`35.1
`Pinto beans
`35.6
`Lima beans
`Black -eyed peas
`25.9
`35.3
`Yellow cornmeal
`-2.0
`Wheat bran
`8.1
`Tomato
`45.9
`Banana
`25.2
`Cauliflower
`21.1
`Spinach
`80.0
`Orange juice
`11.5
`Egg
`13.7
`Milk
`12.1
`Cabbage
`Whole wheat flour
`0.3
`2.2
`Medium rye flour
`Source: Bhandari, S. D., and Gregory, J. F. (1990). Am. J. Clin. Nutr. 51,
`87-94.
`
`Limited Methylation
`
`, which can either be
`Folic acid taken up by the intestinal mucosal cell is reduced to FH4
`transferred without further metabolism to the portal circulation or alkylated (e.g., by
`methylation to 5-methyl-FH4) before being transferred.
`
`IV. Transport of Folate
`
`Free in Plasma
`
`In most species, folate is transported to the tissues mostly as monoglutamate derivatives
`in free solution in the plasma. 11 The predominant form in portal plasma is the reduced
`form, tetrahydrofolic acid (FH4). This is taken up by the liver, which releases it to the
`peripheral plasma after converting it primarily to 5-methyl-FH4
`, but also to 10-formyl(cid:173)
`FH4. The concentration of 10-formyl-FH4 is tightly regulated12 whereas that of 5-methyl(cid:173)
`FH4 is not; thus the latter varies in response to folate meals, etc. Thus, folates of dietary
`origin are absorbed and transported to the liver as FH4
`, which is converted to the methy(cid:173)
`lated form and transported to the peripheral tissues.
`Plasma and erythrocyte folates are reduced by cigarette smoking; habitual smokers
`show plasma folate levels that are more than 40% less than those of nonsmokers. 13 While
`
`11 The notable exception is the pig, in which FH4 is the predominant circulating form of the vitamin. The meta(cid:173)
`bolic basis for this anomaly is not clear.
`12 In humans, .the plasma level is held at about 80 ng/dl.
`13 These findings probably relate to the inactivation of cobalamins by factors (cyanides, hydrogen sulfide, nitrous
`oxide) in cigarette smoke (see Chapter 17).
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`384
`
`16. Folate
`
`serum folates have been found to be normal among middle-class drinkers of moderate
`amounts of ethanol, more than 80% of impoverished chronic alcoholics show abnormally
`low serum levels and some 40% show low erythrocyte levels. This corresponds to a simi(cid:173)
`lar incidence (34-42%) of megaloblastosis of the bone mauow in alcoholic patients. These
`effects probably relate to Lbe displacement of foods containing folates by alcoholic bever(cid:173)
`ages, which are virtually devoid of the vitamin, as well as to direct metabolic effects: inhi(cid:173)
`bition of intestinal folyl conjugase activity and decreased urinary recovery of the vitamin.
`
`Cellular Uptake
`
`Cellular uptake of fo lates occurs exclusively with monoglutamate derivatives found in the
`plasma, as the polyglulamales cannot cross biological membranes. The cellular uptake of
`folate involves two speci.fi proce. ses. One is mediated by a binding protein occurring as
`differenli ·oforms of the ~ late receptor; tltis carrier-mediated process requires energy and
`Na". A ec od process involves a reduced folate carrier anion-exchange system.
`, which is bound to intracellular
`Within cells, FH4 is methylated to yield 5-methyl-FH4
`macromol cules. As a Tesult of methylation in the mammary gland, 5-methyl-FH4 com(cid:173)
`prises three-quarters of the fo lates in breast milk. Folate is held in cells by conversion to
`folyl polyglutamaLes; poJyglutamation trap folates inside cells at concentrations greater
`(by one to tw orders of magnitude) than those of extracellular fluids.
`
`Folate-Binding Proteins
`
`Folate-binding proteins (FBPs, also called folate receptors) have been identified in
`plasma, milk, and several other tissues (e.g., erythrocytes, leuko yte , intestinal. mucosa,
`kidney, liver, placenta, choroid plexus, and urine 11). Each FBP binds folates ooncovalently
`with high affinity, such thatlhe complex does not dissociate under physiologici;tl condj(cid:173)
`cions.15 Low concent:rat.ions (e.g., binding <10 ng of folic acid per deciliter) of FBPs have
`been found in sera of healthy humans; greater concentrations of FBP · have been found in
`folate-deficient subjects, pregnant women, human milk, and leukemic leukocytes. It is
`hypothesized that the FBPs found in plasma and milk are derived from cellular mem"
`to be true of the FBPs in
`branes in which they serve transport functions. TI1is appear
`milk16
`; they have been shown to stimulate Lhe enteric absoqJtion of folate from tl1al food.
`Liver contain. two FBP ; one is the enzyme dlmelhylglycioe dehydrogenru e, the other l
`the enzyme sarc siJ1e dehydrogenase. Each binds reduced fo lates with much greater affin(cid:173)
`ity (about 100-fold) Lhan the nom·educed forms . Intestine contains three FBPs of cliffi rent
`molecular weights; these are associated with the brush border, and two appear to be aggre(cid:173)
`gates of the first.
`Isofonns of FBP cDNAs have been cloned: three from humans (FBP-a, -~, and -y) and
`two from murine Ll 2 10 cells. These have identical open reading frames and 3'-untrans(cid:173)
`lated regions; but their 5' -unlranslated regions d.i[fer in both sequence and length. Four
`buman FBP genes have been lo ated in chromosome llql3 . 2~ql3 . 5 within a 140-kilo(cid:173)
`base region 17; t heir promotor lacks TATA or CAAI elements but has sequences recognized
`
`14 Urinary FBP is presumed to be of plasma origin.
`15 Oth'!r proteins ('!.g., albumin) bind folat'!s non specifically, forming complexes that dissociate readily.
`16 There ar'! two FBPs in milk. Each is a glycoprotein; one may be a degradation product of the other.
`17 FBP-a. and FBP-~ genes are found less than 23 kb apart in sequence, with two additional FBP-relat'!d genes
`located upstream of the FBP-a. gene.
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`IV. Transport of Folate
`
`385
`
`Table 16-4. Differential Binding of Folate
`Metabolites by Folate-Binding Protein Isoforms
`
`FBP isoform binding affinity
`
`Folate/metabolite
`Folate
`5'-Methyl-FH4
`5'-Formyl-FH4
`Folate antagonist
`Methotrexate
`FBP-a > FBP-~ by 20-fold
`FBP-a > FBP-~ by 10-fold
`Dideaza-FH4
`Source: Antony, A. C. (1996). Annu. Rev. Nutr. 16, 501-521.
`
`FBP-a > FBP-~ by 50-fold
`FBP-a > FBP-~ by 100-fold
`
`by transcription factors (ets oncogene-encoded transcription factor and SPl) that are
`thought to regulate FBP expression. The genes appear to have a single promotor located
`within an intron and containing a triplet of clustered SPl-binding sites and an initiator
`region. The regulation of FBP expression is not well understood but it is clear that extra(cid:173)
`cellular folate concentration plays an important role in that process, serving as an inverse
`stimulus to FBP expression.
`The membrane FBPs appear to be anchored by glycosyl-phospholipids, probably via
`serinyl or asparaginyl residues. Because FBPs show different binding affinities for vari(cid:173)
`ous folates (and folate antagonists; Table 16-4), it is assumed that the transport of folates
`into cells depends on the relative expression of FBP isoforms. The best evidence for a
`transport role of the FBPs come from studies of the matemal-to-fetal transfer of folate.
`That process has been shown to involve the concentration of 5'-methyl-FH4 by placental
`FBPs on the maternally facing chorionic surface followed by the transfer of the vitamers
`to the fetal circulation down a descending concentration gradient. Folate uptake in liver
`presumably occurs similarly; studies show the process to be electroneutral, involving the
`cotransport of H+.
`
`Tissue Distribution
`
`In humans, the total body content of folate is 5-10 mg, about half of which resides in the
`liver in the form of tetra-, penta-, hexa- and heptaglutamates of 5-methyl-FH4 and 10-
`formyl-FH4.18 The relative amounts of these single-carbon derivatives vary among tissues,
`depending on the rate of cell division. In tissues with rapid cell division (e.g., intestinal
`mucosa, regenerating liver, carcinoma) relatively low concentrations of 5-methyl-FH4 are
`found, usually with concomitant elevations in 10-formyl-FH4. In contrast, in tissues with
`low rates of cell division (e.g., normal liver), 5-methyl-FH4 predominates. Brain folate
`(mostly 5-methyl-FH4) levels tend to be very low, with a subcellular distribution .(penta(cid:173)
`and hexaglutamates mostly in the cytosol and polyglutamates mostly in the mitochondria)
`the opposite of that found in liver. That folate-deficient animals show relatively low hepat(cid:173)
`ic concentrations of shorter chain-length folyl polyglutamates compared with longer chain(cid:173)
`length folates suggests that the longer chain-length metabolites are better retained within
`cells. In the rat, uteline concentrations of folates show cyclic variations according to the
`
`18 The hepatic reserve of folate should be sufficient to support normal plasma concentrations of the vitarnin (>400
`ng/dl) for at least 4 weeks. (Signs of megaloblastic anernia are usually not observed within 2-3 months of
`folate deprivation.) However, some evidence suggests that the release of folate from the liver is independent
`of nutritional folate status, resulting instead from the deaths of hepatocytes.
`
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`
`386
`
`16. Folate
`
`o· · ·;·~~0 OH ~DP . ·~" :·?<Y-~-<-Oii
`.. ·;-oJ-N~[1_0tl ~ ~DP
`):
`<.a
`,. ;If I T
`"-...-"'f ~ " { ; .
`'
`•
`.] I
`:
`'
`;
`0
`0
`H,ti:"N
`HH
`:
`H,H''N
`.'
`.
`.
`• . . ... o' HO~
`HO~ •..
`HO~
`•• . ... ••
`
`:
`
`)
`>1,
`
`folic acid
`
`7,8-dihydrofolic acid
`
`5,6, 7,8-tetrahydrofolic acid
`
`Figure 16-1. Two-step reduction of folate by 7,8-dihydrofolate reductase.
`
`menstrual cycle with maxima coincident with peak e Lrogenic activity just before vu.la(cid:173)
`tion. 19 It has been uggested that tissue FBPs, which bind poly glutamate forms of the vita(cid:173)
`min, may play important roles in stabilizing folates wilhin cells, thu, reducing their rates
`of metabolic turnover and increasing their intracellular: retention. Becau ·e mo
`ingle(cid:173)
`carbon (1-C) folate derivatives in cells ure bound to enzymes or FBPs the concentrati ns
`appear to be low i.e., in the oanomolru· range.
`of lhe free pools of 1-C folate coenzyrn
`
`V. Metabolism of Folate
`
`There are three aspects of folate metabolism:
`
`• Reduction of the pterin ring system
`• Reactions of the polyglutamyl side chain
`• Acquisition of single-carbon moieties at certain positions (i.e., N-5 or N-10) on the
`pterin ring
`
`Ring Reduction
`
`f the pterin ring from the two nonreduced state . folic acid and ilihydrofolic
`R ductio.n
`acid (F~), t
`the fully reduced fonn ten·ahydrofolic acid (FH4) that is capable of a cept(cid:173)
`ing a si.ngle-ca1bon unit i accomplished by the cyto olic enzyme 7,8-dillydJ·ofolate
`reducta e (Fig. 16-i).20 This activity ls found in higb amo unts in liver and kidney and in
`rapidly divicling cells (e.g., tumor). The reducta e is inhibited by several impmtanl drugs
`including the cancer chemotherapeutic drug methoh·e:xate,21 •22 which appears to exert its
`f ltunor ceUs .
`antitumor action by inhibiting th reductase activity
`
`19 On the basis of this type of observation, it has been suggested that estrogen enhancement of folate turnover in
`hormone-dependent tissues may be the basis of the effects of pregnancy and oral contraceptive steroids in
`potentiating low folate status.
`20 Also called tetrahydrofolate dehydrogenase, this 65-kDa NADPH-dependent enzyme can reduce folic acid to
`FH2 and, of greater importance, FH2 to FH4
`.
`2 14-Amino-10-methylfolic acid:
`
`22 Other inhibitors include the antimalarial drug pyrimethamine and the antibacterial drug trimethoprim.
`
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`
`V. M etabolism of Folate
`
`387
`
`5-methyl-
`
`5-formyl-
`
`1G-formyl-
`. ' '
`'•
`
`5, 10-melhylene-
`....
`·''
`
`5, 10-melhenyl-
`
`5-formlmlno-
`·····-..
`
`HN=HC HN .
`~
`~
`'
`'
`' ~ I
`.. ......
`.
`.
`
`Figure 16-2. Single-carbon units carried by folate.
`
`Side-Chain Reactions
`
`The folyl monoglutamates that are taken up by cells are trapped therein as polyglutamate
`derivatives that cannot cross cell membranes. This is accomplished by the action of the
`ATP-dependent folyl polyglutamate synthetase, which links glutamyl residues to the vit(cid:173)
`amin by peptide bonds involving the y-carboxyl groups.23 The enzyme requires prior
`reduction of folate to FH4 or demethylation of the circulating 5-methyl-FH4 (by vitamin
`B12-dependent methionine synthetase). It is widely distributed at low concentrations in
`many tissues . That folyl polyglutamate synthetase is critical in converting the monoglu(cid:173)
`tamyl transport forms of the vitamin to the metabolically active polyglutamyl forms was
`demonstrated by the discovery of a mutational loss of the synthetase activity, which pro(cid:173)
`duced lethal folate deficiency. Folyl polyglutamates are converted to derivatives of short(cid:173)
`er chain length by cellular conjugases, some of which appear to be zinc-metalloenzymes.
`
`Acquisition of Single-Carbon Units
`
`Folate is metabolically active as a variety of derivatives with single-carbon units at the oxi(cid:173)
`dation levels of formate, formaldehyde, or methanol 24 substituted at the N-5 and/or N-10
`positions of the pterine ring system (Fig. 16-2). The main source of singl