PROGRESS IN GASTROENTEROLOGY

Transcription

1 GASTROENTEROLOGY Copyright 1971 by The Williams & Wilkins Co. Vol. 60, No.3 Printed in U. S. A. PROGRESS IN GASTROENTEROLOGY THE DIGESTION AND ABSORPTION OF DIETARY FOLATE IRWIN H, RoSENBERG, M,D" AND HERMAN A, GODWIN, M,D. Thorndike Memorial Laboratory, Harvard Medical Service, Boston City Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts Twenty-five years ago folic acid, or pteroylglutamic acid, was synthesized chemically and in the same year conjugated folate was isolated from yeast and crystallized and its structure was identified, It was appreciated at this time that much natural folate is conjugated, and several studies employing the newly isolated conjugated material demonstrated that this form of folate could be absorbed. However, investigations of absorption and metabolism of folate since 1948 have largely utilized synthetic pteroylmonoglutamic acid (PGA), probably because of the lack of adequate amounts of chemically defined conjugate. (Also in 1948, a strong impetus for research into the nature of conjugated folate was lost when the isolation of vitamin Bl2 excluded conjugated folate as a contender for Castle's extrinsic factor.) The recent renewal of interest in the absorption of conjugated and reduced folates has led to the successful synthesis of pteroylheptaglutamate (Pte Glu 7 ) and other conjugates as well as preparation of reduced forms of folate. With the availability of adequate amounts of such materials for study, many questions of long standing are now being reconsidered. It is, therefore, an Received September 1, 1970, Address requests for reprints to: Dr, Irwin H. Rosenberg, Department of Medicine, University of Chicago Medical School, Chicago, lllinois Supported in part by United States Public Health Service Grants AM-795, AM-539I, AM-54I3, HEI AM-7652, AM/9115, and FR-76 from the National Institutes of Health, Bethesda, Maryland. Dr. Rosenberg is the recipient of Career Development Award I-K4-AM-235I6-0I from the National Institutes of Health. 445 appropriate time to review current information concerning the digestion and absorption of conjugated and free folate. Isolation of Folic Acid and Its Conjugate The synthesis of pteroylglutamic acid by Angier and his associates l at Lederle and American Cyanamid in 1943 and confirmation of its structural identity with "folic acid" or "Lactobacillus casei factor" represented the culmination of more than a decade of investigation into the nature of a variety of seemingly unrelated growth principles or antianemia factors (table 1). The first of these was a "new hemopoietic factor" in yeasp or liver 4 which cured tropical macrocytic anemia in India as well as an experimental anemia in monkeys induced by similar dietary deficiency.27 This unknown substance was later named "Wills' factor" by Watson and Castle 28 when they confirmed that the "new hemopoietic" principle, or Wills' factor, was found in a different fraction of liver extract from that which was curative for pernicious anemia. Additional factors were described during the same period which were active against nutritional pancytopenia in monkeys6, 7 or were required as growth 8, 9 or antianemia factorslo,l1 in chicks, Because these substances were impure and their identity with Wills' factor could neither be deduced nor tested, they received a variety of individual designations ranging from vitamin M (monkey)7 to vitamin Be (chick).l1 The report of vitamin Be by Hogan and Parrott ll also provided investigators with a useful tool, the chick anemia assay, for

2 446 PROGRESS IN GASTROENTEROLOGY Vol. 60, No.3 TABLE 1. Milestones in the isolation and synthesis of free and conjugated folates a Factor Description Date Folate form Referenee(s) "Wills' factor" Vitamin M Factor U Factors Rand S Vitamin Be Norite eluate factor "Folic acid" Pure L. casei factor Crystalline vitamin Be Vitamins B10 and B" Vitamin Be conjugate Crystalline vitamin Be conjugate Pteroylglutamic acid synthetic Pteroylheptaglutamic acid synthetic Yeast extract effective against tropical macrocytic anemia in man and monkeys Yeast and liver extract effective against nutritional cytopenia in monkeys Growth factor for chicks found in yeast extract Yeast factors required for chick nutrition Chick antianemia factor obtained from liver Growth factor for Lactobacillus casei present in yeast and liver Concentrate from spinach leaves, active for Streptococcus faecalis From yeast and liver, microbiologically active From liver, active in chick and microbiological assays From liver, active in chicks, inactive for bacteria From yeast, active in chicks, but active for bacteria only after digestion From yeast, active in chicks, but active for bacteria only after digestion Active for bacteria, chicks, monkeys, and man Active for bacteria after deconjugation, active in man 1931 Unknown 3,4, Unknown 6, Unknown Unknown Free 10, Free 12, Free 14, Free Free Conjugated? 18, Conjugated 20, Pte Glu 7 22, PGA 1968 Pte Glu 7 24, 25, 26 a Modified from Jukes and Stokstad. 2 evaluating unidentified hematopoietic substances. Another critical step leading toward the isolation and identification of these factors was the discovery at the University of Wisconsin that a factor in a charcoal eluate of yeast served as a growth factor for L. casei.12 Later, as L. casei factor and vitamin Be were also found to have antianemia potency, a striking inconsistency appeared. Some yeast extracts which were curative in the macrocytic anemia of chicks and monkeys would not support the growth of L. casei.20 It thus appeared that the chick anemia assay and the L. casei assay were not measuring the same principle. This apparent paradox was clarified by Bird and his co-workers 29 who showed that the antianemia principle of yeast would support the growth of L. casei if the yeast extract were first digested with crude tissue homogenates obtained from animal sources. They gave the name "vitamin Be conjugate" to the complex form of the antianemia principle already known as vitamin Be, and they named the enzymes which release vitamin Be from the complex "conjugases." Exploiting the microbiological and chick assays, Stokstad 16 and Pfiffner and his co-workers l7 independently isolated the free vitamin from yeast and liver. Chemical identification of the isolated vitamin was followed quickly by its synthesis as pteroylglutamic acid. I Pfiffner et al. 22

3 March 1971 PROGRESS IN GASTROENTEROLOGY 447 then utilized conjugases as an adjunct to microbiological methods and accomplished the isolation and identification of the conjugated vitamin from yeast in Only in retrospect was it possible to weave the disparate threads into a pattern which indicated that all of the variously named factors were variants of PGA or its conjugate. Nutritional Evaluation of Free and Conjugated Folates Early studies, most of them performed prior to the isolation of PGA or its heptaglutamate conjugate, indicated that the folate forms in yeast were well utilized by chickens,30 turkeys,31 monkeys,6 and patients with macrocytic anemia. 5 Although the greatest portion of yeast folate is conjugated,32 any response to the feeding of yeast extracts could reflect absorption and utilization of free folate derived from either the conjugate or residual free dietary folate. The ready availability of crystalline PGA permitted studies which quickly demonstrated that this vitamin form is effectively utilized by man when administered either orally or parenterally.33, 34 In fact, daily oral doses of 1 mg or less were found to be adequate in the treatment of severe nutritional macrocytic anemia. 35 However, the question of efficiency with which conjugated folate is utilized by man remains controversial. The first use in man of the relatively pure heptaglutamic folate conjugate isolated by Pfiffner and his colleagues 22 was reported by Bethell and co-workers.36 Three of 9 patients with pernicious anemia in relapse and 1 of 2 patients with macrocytic anemia following subtotal gastrectomy exhibited little or no evidence of erythrocyte regeneration when given oral doses of crude yeast concentrate. Urinary excretion of free vitamin in these patients was low. However, when a purified preparation of heptaglutamic folate was administered, or when the conjugate was digested in vitro with conjugase in order to release free folate prior to administration, the hematological responses and urinary excretion patterns were similar to those seen after administration of equimolar amounts of crystalline PGA. Interpretation of these studies is complicated by variable states of nutrition and gastrointestinal function in the patients studied. The first studies attempting to compare the efficiency of absorption of synthetic PGA with conjugated folate from yeast in normal subjects were made by Swendseid et al. 37 Comparison depended upon assay of microbiologically active folates excreted in the urine following large (4 mg) doses of PGA or equimolar amounts of conjugate. When the source of conjugate was a crude yeast extract, or even a partially purified norite eluate from yeast, the quantity of folate excretion was small when compared with the excretion after molar equivalents of PGA. However, the apparent absorptive advantage for the free vitamin diminished markedly when a highly purified heptaglutam ate from yeast was compared with crystalline PGA. These workers suggested that differences in absorption of crude yeast conjugates and the purified heptaglutamate from yeast could be explained by the presence of natural inhibitors of conjugase present in the crude extracts. This concept was supported by in vitro experiments from the same laboratory38 showing that decreasing amounts of hog kidney conjugase were required to release free folate from yeast conjugate as the yeast extract increased in purity. Later studies by Spray and Witts 39, 40 demonstrated that ingested yeast failed to produce an elevation of serum folate, while equal amounts of PGA produced a large rise. In our laboratory, we confirmed the findings of Spray and Witts with crude yeast extract, but we observed enhanced serum folate levels if the conjugated folate from yeast is purified by ion exchange chromatography before feeding. 41 The study of Jandl and Lear 42 demonstrated that the prior deconjugation of yeast extract in vitro would enhance urinary excretion of folate in normal subjects 2-fold but still to reach only 60% of the excretion of an equimolar dose of PGA. This discrepancy in utilization ofconju 'gated and free folate was again reported recently by Perry and Chanarin 43 who, employing 1500-J,Lg doses of conjugated folate (obtained as a charcoal eluant from

4 448 PROGRESS IN GASTROENTEROLOGY Vol. 60, No. 3 yeast) and equivalent amounts of PGA, estimated that conjugated folate is utilized only one-third as well as is free folate. Before attempting to resolve the question of relative availability of free and conjugated folate in terms of mechanism of digestion and absorption, it may be helpful to review basic information concerning folate chemistry and metabolism, assay employing microbiological organisms, and distribution in foods. Functional Chemistry of Folate Structure. The structures of PGA and its principal conjugate are shown in figure 1. PGA consists of a 2-amino, 4-hydroxypteridine joined with p-aminobenzoic acid and a single L-glutamic acid molecule. The majority of naturally occurring folate compounds are conjugates containing additional L-glutamic acids in "( peptide linkage. 23 Pteroylheptaglutamic acid is the principal molecular form in yeast,20 and, by inference, similar conjugates predominate in other plant and animal tissues. 32 Pteroyltriglutamic acid appears to be the major folate form in bacteria The pk values of the a- and,,(-carboxyl groups of glutamic acid are 2.19 and 4.28, respectively, with an isoelectric ph value of Therefore, at physiological ph, both carboxyl groups of PGA and all eight carboxyl groups of pteroylheptaglutamate are negatively charged. Assuming that the secondary amine in the NIO position has a net positive charge, PGA has one net negative charge and heptaglutamic folate has seven. The negatively charged peptide chain accounts for the increased water solubility of the heptaglutamate and poses special problems in membrane transport. The polyglutamic structure of conjugated folate is almost unique in nature. 48 Certain bacteria produce pure polyglutamates,49 but only Bacillus subtilis synthesizes a "( linked polypeptide of L-glutamic acid. 50 It has been assumed, and generally accepted, that all the glutamates in conjugated folate are linked by a "( rather than by the more common a peptide bond. The evidence for this is indirect. A shorter conjugate, pteroyitriglutamate, or "fermentation factor," which was isolated from a Corynebacterium species,44 was found to Pteroic Acid 1 ( L -Glutamic Acid H N N N 2 YO:'.} 3' 2' COOH N 4 I,,,: CH - N H C O - N H -. C: H x bc O H - C H N OH ' ' - 6 J '--v----! nooh '-- -y-, Pteridine P- Aminobenzoic NH - CH - CH 2 - CH 2-CO Acid COOH I NH- CH - CH 2 -CH 2 -CO NH-CH - CH 2 -CH 2 -CO NH - CH - CH 2- CHz- CO NH -CH - CH2-CH2- CO NH - CH - CH 2 - CH 2 - COOH PTEI?OYL GLfJTAMIC ACIO AI. W 441 PTEI?OYLHEPTAGLfJTAMIC ACID AI. W 1215 FIG. 1. Structure of pteroylglutamic acid (PGA) and its principal conjugate, pteroylheptaglutamic acid (Pte Glu 7 ). Note the 'Y-linked glutamyl peptide of the conjugate.

5 March 1971 PROGRESS IN GASTROENTEROLOGY 449 contain a ')'-linked peptide chain. Biosynthesis of the conjugate by cell-free fractions from Escherichia coli substantiated this linkage by demonstrating that chain elongation occurred when reduced pteroylglutamyl-,),-glutamate was used while pteroylglutamyl-a-glutamate was inert. 51 Synthetic pteroylglutamyl-,),-diglutamate possessed all the characteristics of natural fermentation factor, thus confirming the structure of this conjugate. The early assumption that the longer conjugate contains exclusively,), linkages is an extrapolation from this observation. Again, the accuracy of this extrapolation has been confirmed by the identical spectral, microbiological, and chromatographic behaviors of natural yeast conjugate and of pteroylheptaglutamic acid produced by different synthetic methods in two laboratories It should be noted that only the heptaglutamate from yeast has been isolated and identified. The evidence that conjugates in other plant and animal tissues are polyglutamates is indirect and derives from the microbiological behavior of these conjugates before and after deconjugation, and, in the case of liver 52 and erythrocytes, 53 from similar characteristics to known polyglutamates on ion exchange chromatography. The possibility exists, however, that seven is the average, rather than the exclusive, chain length of conjugated folate in yeast as well as in other tissues. In fact, other aminoacyl conjugates have been reported in bacteria 54 and may be present in the diet in small amounts. The preponderance of conjugated folate in animal as well as plant cells suggests that it is the conjugated form which the cell employs for storage, while the free or monoglutamic form is used as a co-factor. It therefore follows, and experimentally it has been established,55-57 that tissues which store folate contain enzymes which are capable of releasing free folate from the conjugate, probably either for distribution to other organs or for local intracellular utilization. Metabolically active forms. Double bonds at positions 5, 6 and 7, 8 in the pteridine ring are readily reduced, either chemically or enzymatically. Reduction in vivo is accomplished in the presence of reduced pyridine nucleotides by an enzyme known as dihydrofolate reductase which produces either dihydro- or tetrahydrocompounds. 58 Reduction to tetrahydrofolate is a basic requirement for coenzyme function. 32 Tetrahydrofolic acid is the precursor of coenzymes which act in the transfer of 1- carbon units (table 2). These units, at various levels of oxidation, are carried either at the N5 or N10 positions on the molecule, or they form a bridge between the two positions. Enzymatic reactions in which folate coenzymes are known to participate include: (1) certain aspects of purine and pyrimidine synthesis, (2) formation of methionine from homocysteine, (3) metabolism of other amino acids including the catabolism of histidine and interconversions of serine and glycine, (4) oxidationreduction reactions of some folic acid derivatives, and (5) formylation reactions. The details of these reactions have been recently reviewed. 32 Microbiological assay for folates. Chemical methods for assay of the small amounts of folate present in plants and animals are too insensitive to be useful in biological studies. The role of microbiological organisms with differential growth requirements for various forms of folate in the identification and quantitative determination of this group of compounds cannot be overemphasized. A brief summary of the microbiological growth activity for some folic acid compounds is presented in table 3. Folate distribution in foods. Folate is found in a wide variety of foodstuffs. The richest sources are yeast and liver, containing, respectively, 2 mg and 300 j.tg per 100 g of moist weight. 61 Significant quantities are present in nuts and vegetables-especially asparagus, lettuce, spinach, and dried beans Approximately 75 and 80% of natural folates exist as polyglutamyl conjugates; that is, they are detectable by microbiological assay only after enzymatic digestion. 64, 65 About 90% of folate in vegetables is present as 5- or 10-formyl deriva-

6 450 PROGRESS IN GASTROENTEROLOGY Vol. 60, No.3 TABLE 2. M etabolically active forms o f f ol ic acid a Fann I-Carbon unit Oxidation state NIO-formyltetrahydrofolic acid (lo-cho-h,pte Glu) N'-formyltetrahydrofolic acid (5-CHO-H,Pte Glu) ("folinic acid") -CHO - CHO Formate Formate N', Nlo-methenyltetrahydrofolic acid (5, 1G--CH Pte Glu) -CH Formate N' -formiminotetrahydrofolic acid (5-CHNH-H,Pte Glu) N', N,o-methylenetetrahydrofolic acid (5, 1G--CH 2 H,Pte Glu) N'-methyltetrahydrofolic acid (5-CH,-H,Pte Glu) -CH NH > CH 2 -CH, Formate Formaldehyde Methanol a Adapted from Stokstad and Koch 32 and Herbert.'9 Symbols in parentheses are those adopted by the Commission on Biochemical Nomenclature (IUPAC-IUB Commission, 1966). TABLE 3. Microbiological growth activity for some folic acid compounds Lacta Strepta Pediobacillus coccus coccus casei laecalis cerevisiae Pteroylglutamic acid (folic acid) Reduced pteroylmonoglutamates (except N'-methyl) N ' -methyltetrahydrofolic acid Pteroyldiglutamic acid Pteroyltriglutamic acid Pteroylheptaglutamic acid o Adapted from Johns and Bertino. 50 tives, with the remaining 10% or less existing as unreduced pteroylglutamate. Methylfolate compounds are found in yeast, liver, beans, and black-eyed peas, and the major portion of unprocessed liver folate exists as 5-methyltetrahydrofolate. 52, 64, 66, 67 Folates are sensitive to light, aerobic conditions, extremes of ph, and heat, especially boiling of foodstuffs,62 all of which result in losses of folate activity. A normal human daily folate intake has been estimated to vary from about 700 J.!g64, 65 to 1000 to 1500 J.!g4 2 per day. Digestion and Absorption of Folate As noted above, food folate exists as a mixture of free and conjugated forms with the latter predominating. We review the evidence that the polyglutamic conjugates are digested to free or monoglutamic forms before intestinal transport of the latter occurs. The data on transport mechanism of monoglutamic folate, whether derived directly from food or following deconjugation of the polyglutamates, are then considered. Digestion of conjugated folate. Pteroylheptaglutamate is a large molecule both in molecular weight (1215) and in molecular radius. It is strongly electronegative and highly water-soluble with its eight carboxyl groups all charged at physiological ph. By established concepts of cell membrane transport, this molecule would be expected to be excluded by the cells of most animal species. Even E. coli will not incorporate pure glutamyl peptides of chain lengths greater than three. 68 Folate in human blood circulates in the free or unconjugated form,69 presumably to facilitate entry into body cells. With but few exceptions,70, 71 it is the monoglutamic form of folate which is preferentially or exclusively utilized as cofactor for cellular enzymes. 32 Evidence that deconjugation does, in fact, occur in vivo was obtained in previously cited experiments 36, 37 in which purified yeast conjugates were fed to normal subjects and the resulting urinary excretion product was microbiologically active. If, as theory predicts, conjugated folate from food is converted to free folate before passage across the intestinal cell membrane into the circulation, we should expect to witness the appearance in plasma of only free folate after the ingestion of the conjugate. A representative experiment from our laboratory supporting this concept is shown

7 March 1971 PROGRESS IN GASTROENTEROLOGY 451 in figure 2. Yeast folate, purified by column chromatography,41, 72 was 95 to 98% conjugated. When normal volunteers took a physiological dose of 0.5 Jlmole by mouth, an elevation of serum folate active for both L. casei and Streptococcus faecalis was observed. No conjugated folate, as evidenced by the failure of deconjugating enzymes to release additional microbiologically active folate in serum following in vitro incubation, was detectable in the serum of any subjects. Other laboratories have reported similar observations employing either yeast folate or synthetic crystalline pteroylheptaglutamates as the test dose.43, Butterworth, Baugh, and Krumdieck,74 using 14C-Iabeled polyglutamates, provided additional support for the in vivo deconjugation of ingested pteroylpolyglutamates by demonstrating the appearance in serum of 14C-Iabeled pteroylmonoglutamate after oral ingestion of 14C-pteroylheptaglutamate labeled either in the pteroate moiety or the first glutamic acid. When the label was placed in the second glutamic acid of the peptide chain, plasma folate was not radioactive. Label appeared as 14C02 exhaled in the breath, suggesting that the terminal glutamates had been cleaved off and rapidly metabolized. Although there have been two reports suggesting the absorption of intact heptaglutamic folate,76, 77 the con- :::: '" --J " 1;: "J 10 L S. foecoi/s} Free Folate L. casel Conjugated Folate I HOURS FIG, 2, Change in serum folate concentration after oral administration of 0.5,.,mole of conjugated folate to a human subject. Note the similar rises in folate activity for Streptococcus faecalis and Lactobacillus casei. Conjugated folate, absorbed intact, could not be demonstrated. sensus of other experiments is sufficient, we believe, to establish that conjugated folate is quantitatively deconjugated before release into the circulation. Weare left, therefore, with a familiar situation in gastrointestinal physiology, namely that of a polymer in food whose units enter the circulation as monomers or oligomers. This is the case with starch, proteins, triglycerides, peptides, and polynucleotides. It is then logical to consider the usual sources of hydrolytic or digestive enzymes in the alimentary tract-saliva, gastric juice, and pancreatic juice-as potential sources of enzymes to deconjugate folate. However, none of the known enzymes in these fluids has a substrate spectrum which includes the unique l'-glutamyl bond 78 ; and, experimentally, the gastric and pancreatic proteases pepsin, trypsin, chymotrypsin, and carboxypeptidases A and B are all inactive against the l'-glutamyl peptide chain of yeast folate 79 or synthetic heptaglutamate (1. H. Rosenberg, J. Ribaya, and H. A. Godwin, unpublished observations). Attempts by Santini and coworkers 63 and Klipstein 80 to demonstrate deconjugating enzymes in fluid taken from the small intestinal lumen of man have given variable results. Among the gastrointestinal secretions collected in pure form, only bile has been reported to contain enzymatic activity against yeast conjugate. 81 However, this activity is demonstrable only at ph 4 to 5, which is much below the normal ph of bile. Deconjugation by intestinal mucosa. Several lines of evidence point to the intestinal mucosa as the source of enzymes which deconjugate food folate. The intestinal mucosa is known to perform several critical digestive functions in addition to its more widely studied role in absorption. Oligosaccharides are hydrolyzed to monosaccharides by enzymes of the intestinal brush border, 82 and peptidases of the intestinal mucosal cells complete the hydrolysis of oligopeptide products of gastric and pancreatic proteolysis. 83 Intestinal peptidases may be intracellular or brush border enzymes,84, 85 and they are responsible for the fact that amino acids enter the portal

8 452 PROGRESS IN GASTROENTEROLOGY Vol. 60, No.3 circulation as the major products of protein digestion. Clinical observations likewise focus upon the intestine as the critical organ in the assimilation of folate from food. Folate deficiency is rarely found in nonalcoholic liver disease or in pancreatic exocrine insufficiency, but folate deficiency complicates most cases of primary disease of the proximal intestinal mucosa. 86, 87 Celiac and tropical sprue are two of the most florid examples These clinical observations appear to exclude the other organs of the alimentary tract as indispensable participants in the digestion and absorption of dietary folate. Experimentally, it has been demonstrated that the isolated intestine is capable of releasing free folate from its conjugate. This was clearly shown by experiments with everted sacs of isolated rat jejunum as illustrated by figure When purified conjugated yeast folate was added to the fluid bathing the intestinal mucosa, microbiologically active folate appeared on the serosal as well as the mucosal side of the intestinal wall. All the folate which traversed the intestinal wall into the serosal fluid was free or unconjugated folate. Conjugated folate was converted almost quantitatively to free folate within 60 min. In vivo experiments by Baugh and coworkers 89 and Bernstein et al. 75 provide further physiological evidence for the role "- '" <> e: Conjugated 0.5 Folate "- Added '" "- 0.3 "'" "- '" SAMPLING TIME (min) FIG. 3. Absorption of folate from its conjugate by everted sacs of rat jejunum. Conjugated folate (0.75 JLffiole per ml) was added to the mucosal fluid, and the rise in free folate was measured by Lactobacillus casei assay. (Reproduced with permission of the New England Journal of Medicine.) of the intestine in the digestion of conjugated folate. Both groups perfused intact isolated loops of dog intestine with conjugated folates, and both identified deconjugated folate in the portal circulation. The liver could thus be excluded as a possible source of the postprandial elevations in systemic blood monoglutamic folate. Baugh and his co-workers 89 collected samples into tubes containing CuCl 2 in order to inhibit plasma-deconjugating enzymes, thereby minimizing the possibility that deconjugation was occurring after passage of folate into portal blood. In vitro, both plasma and liver have been found to contain enzymes which deconjugate folate, but the plasma enzyme is active only at ph values well below the ph of blood. It is likely that deconjugating enzymes of low ph optima in blood and in bile are released from tissues, and, like other acid hydrolases such as acid phosphatases, they probably have no function after release from cellular organelles. Both theoretical and experimental evidence thus supports the hypothesis that free folate is released from conjugated dietary folate by the action of enzymes present in the intestinal mucosa. Early surveys looking for sites of tissue conjugases demonstrated the intestinal mucosa as one of many active tissues. 55 However, in order to be considered as the physiological source of enzymes which deconjugate food folate, the intestinal mucosa must be able to produce from conjugated folate the monoglutamic folate product which appears in the circulation after ingestion of food folate. Support for this concept is derived from evidence showing that digestion of conjugated folate by intestinal homogenates releases the same product as that appearing in the blood after conjugated folate ingestion. This result has been obtained in several laboratories using microbiological techniques of e.nzyme assayy' 73, 75 Because L. casei and S'. faecalis assays cannot distinguish with certainty between monoglutamate and diglutamate products, we employed synthetic 3H -pteroylheptaglutamate as substrate for digestion by intestinal mucosal fractions. 90 The radioactive

9 March 1971 PROGRESS IN GASTROENTEROLOGY 453 product of this digestion was separated chromatographically and directly identified as 3H-pteroylmonoglutamic acid (fig. 4). Baugh and Krumdieck 91 used a different radioisotope technique to demonstrate conversion of 14C-pteroyltriglutamate to pteroylmonoglutamate by enzymes from intestinal mucosa. Site of hydrolysis of conjugated folate within the intestine. Alternate hypotheses for the site of conjugated folate hydrolysis in vivo are presented in figure 5. It seems likely that such hydrolytic enzymes would be located on the mucosal surface of the intestinal cell, much as are disaccharidases and certain peptidases. If this were true, the large folate molecule could be hydrolyzed before passage across the mucosal surface of the intestinal cell. Data from two laboratories 57 (I. H. Rosenberg, et al.) bearing on this anatomical point are summarized in table 4. Although the techniques for isolation of subcellular fractions in the guinea pig and rat are not strictly comparable, they are sufficiently similar to demonstrate that the largest percentages of,),-glutamyl peptidase activity are found in the mitochondrial-lysosomal fraction and in the supernatant, or soluble, fraction. There is an increase in specific activity over that for the whole homogenate only in the mitochondrial-lysosomal fraction. Contrary to theoretical expectation, in all studies the brush border fraction contains PTEROYL (GM n FIG. 4. Composite radioisotope strip count recordings of three electrophoretograms demonstrating the conversion by intestinal 'Y-glutamyl carboxypeptidase of 'H-pteroylheptaglutamate to 3H-pteroylmonoglutamate. Identification of products was made by comparison of product migration with that of synthetic marker compounds (shown along the bottom of the figure). Intestinal Lumen Intestinal Cell Mesenteric Circulation ( S : : : : : : : : ' ; Plet' I; Pte Glu - -t!=ilt:==========:::::±::li---+ BRUSH BOROER INTRACELLULAR Pte GlUT FIG. 5. Schematic demonstration of alternate hypotheses for the site of deconjugation of conjugated folate.

10 454 PROGRESS IN GASTROENTEROLOGY Vol. 60, No.3 TABLE 4.,,(-Glutamyl carboxypeptidase (conjugase) activity of subcellular fractions of intestinal mucosa Percentage of total activity Guinea piga Rat' Specific activity (cell fraction/ whole ho m o t e ) n a Guinea pig" mg/ml Rat' Whole homogenate Brush borders (pure) < 1.0 < Cell debris Mitochondria-lysosomes Microsomes Supernatant fluid a From Hoffbrand and Peters. 57 b Rosenberg, Ribaya, and Godwin, unpublished observations. no more than a very small portion of the total activity. Barring the unlikely possibility that this enzyme is eluted into the supernatant during the repeated washing required for purification, these cell fractionation data are not consistent with the concept that conjugated folate is hydrolyzed by intrinsic brush border enzymes. The localization of highest specific activity to the mitochondrial-lysosomal fraction suggests another hypothesis. Hoffbrand and Peters, 57 by further fractionation techniques, have located most of this activity in the lysosomes. The low ph optimum of the intestinal enzyme suggests the acidic lysosomal vacuole as a possible site for hydrolysis of the conjugate. Such a hypothesis could include passage of the poly glutamate into the lysosomal vacuole by a process which would not necessitate transport across the cell membrane. Pinocytosis would satisfy this requirement. However, the capability of mature mammalian intestine to incorporate large molecules by pinocytosis is by no means established. Still a third scheme supposes that the known continuous sloughing of intestinal villous cells releases enzyme into the lumen. Assuming that the mucosa "turns over" every 48 hr in the rat 92 and every 5 to 6 days in man 93 and using data on enzyme concentrations in intestinal mucosas 7 (I. H. Rosenberg, et al.), it is possible to calculate that the amount of enzyme sloughed from the intestinal mucosa into the succus entericus would greatly exceed the requirements for the hydrolysis of dietary folate. Again there is variable evidence concerning this hypothesis. Samplings of intestinal fluid (succus entericus) have not reproducibly demonstrated deconjugating enzymes. 63, 80 Such intraluminal hydrolysis should occur at the optimal ph for the enzyme (PH 4.6 to 5.6), and the ph of the jejunum (5.0 to 6.0) is well within its range of activity.9. Clearly, this problem must be reevaluated employing controlled assay conditions, including careful attention to ph and efforts to minimize autodigestion of succus enzymes by activated pancreatic proteases. The data upon which to select the correct alternative(s) from among those presented in the figure are not yet available. Absorption of Monoglutamic Folate After digestion or hydrolysis of conjugated folate, the next event in absorption involves the transport of monoglutamic folate either present as such in foods or released from conjugates by intestinal enzymes. Our concepts of the mechanism of transport come from studies in which monoglutamic folates are fed directly. It is theoretically possible that monoglutamic folate derived from conjugates is transported by a different mechanism than is monoglutamic folate ingested as such. Certain amino acids appear to be transported by different pathways when presented to the intestine as the free amino acid or as the dipeptide. 95 However, there are no experimental data suggesting separate transport pathways for mono-, di-, or the larger pteroylglutamates. We may, therefore, assume that concepts derived from the study of monoglutamic folate absorption are applicable to our understanding of the absorption of conjugated folate as well. Perhaps the best evidence against separate pathways of assimilation comes from reports of an isolated and specific gastrointestinal defect for monoglutamic folate absorption by Luhby et al. 96 and Lanzkowsky and co-workers.97 The severe defi-

11 March 1971 PROGRESS IN GASTROENTEROLOGY 455 ciency of folate which results suggests that the major portion of folate derived from the conjugate in food has no separate pathway by which to traverse the intestine and so to prevent the deficiency. Site of absorption of folate. Before considering the mechanism of transport of folate, it will be helpful to review the evidence that absorption occurs in the proximal intestine. A jejunal location is strongly suggested clinically by the high incidence of folate deficiency in diseases involving the proximal small bowel. 86, 87 Abnormal folic acid absorption is characteristic of disease of proximal jejunum or following its resection. 87, 98, 99 Folate malabsorption is particularly well documented in celiac disease, or gluten-sensitive enteropathy,loo which is a disease primarily involving proximal small intestine. On the other hand, folate deficiency does not develop in patients with ileal disease or following ileal resection. 87 Nearly all the available experimental evidence bearing on the question of absorptive site has been obtained using crystalline PGA. Herbert and Shapiro, 101 studying folate absorption with everted sacs of rat intestine, suggested preferential uptake in the jejunum. Hepner,102 employing in vivo perfusion of rat intestine, also found that the proximal small intestine is the major site of folate absorption. Burgen and Goldberg,103 however, using similar perfusion techniques, were unable to find significant differences between jejunal and ileal absorption. In the only study performed in man, absorption of perfused crystalline folic acid was shown by Hepner et al.104 to occur in the proximal small intestine with negligible absorption taking place in the ileum. Although there may be species variations, the evidence from clinical and experimental sources favors preferential absorption of folate in the proximal jejunum. Mechanism of absorption of monoglutamic folate. The mechanism by which pteroylmonoglutamic acid and its derivatives are transported across the intestinal mucosa continues to be a subject of uncertainty. PGA itself is a large (mol wt 441), moderately water-soluble molecule. As such, it could cross the intestinal cell by relatively slow passive diffusion, driven in part by the ph differential inside and outside the intestinal cell. Alternatively, its passage could take place by a more efficient structure-specific mechanism which may be termed active transport when driven by energy-requiring cellular processes. Much effort has been directed toward studies to establish the presence of an active transport system for folic acid in the intestine. Evidence for "active transport" has come from three types of experiments: (1) perfusion of intestine with increasing amounts of folate until a maximum rate of transport is achieved, thus suggesting a saturable, and therefore structure-specific system 102, 103; (2) the demonstration of net intestinal absorption in the face of high serum folate concentrations following a parenteral loading dose 1 0 3, 104; and (3) incubation of everted intestinal sacs in folatecontaining medium with demonstration of slightly higher concentrations of folic acid on the serosal as compared with the mucosal side of the sacs. 101, 105 Valid objections can be raised to the interpretation of these experiments. Other investigators, employing similar techniques, have been unable to confirm these observations None of the experiments claiming to show active transport has conclusively established a requirement for cellular energy. When metabolic inhibitors have been used experimentally, methyl folate absorption has not been affected (H. J. Binder, W. B. Strum, P. F. Nixon, and J. R. Bertino, personal communication). Even the evidence for a specific, saturable transport system has been challenged l1 It is important to note that all studies which have been interpreted as showing a saturable, concentrative transport system for folate have employed synthetic crystalline PGA. In nature, the majority of pteroylglutamates exist as reduced dihydro- or tetrahydro- derivatives of PGA. It is, therefore, important to know whether unreduced PGA behaves like other natural monoglutamic folates and can thus appropriately

12 456 PROGRESS IN GASTROENTEROLOGY Vol. 60, No. 3 be used as a prototype for study of the whole family of compounds. Evidence for methylation of folate by intestine. Data are accumulating which demonstrate that PGA is handled by the intestine quantitatively, if not qualitatively, in different manner than are dihydro- and tetrahydro-pga. Whitehead and Cooper,112 who studied patients undergoing umbilical vein catheterization for suspected liver disease, have shown that PGA in 1- mg doses crosses the intestine intact without undergoing metabolic conversion. Perry and Chanarin 113 have demonstrated that dihydro- and tetrahydro-pga given orally appears in the blood exclusively as methyltetrahydrofolate, while PGA is only partially methylated during transport. These investigators could not exclude participation of the liver in the observed metabolic alterations, but their conclusions have been confirmed recently by Whitehead and co-workers (V. M. Whitehead, R. Pratt, A. Viallet, and B. A. Cooper, personal communication) in studies of absorption of 5-formyltetrahydrofolic (folinic) acid in patients with umbilical vein catheters. Even at 2-mg oral doses, the folinic acid is converted almost quantitatively to methylfolate during passage from the intestinal lumen to the prehepatic mesenteric circulation (V. M. Whitehead, et ai.). The observed ease with which methylation of reduced folates occurs as compared with unreduced PGA is understandable in view of the apparent requirement for fully reduced folates by enzymes which add 1- carbon units to the pteroyl moiety. Thus, enzymatic reduction of PGA would need to precede methylation, and again the enzyme dihydrofolate reductase, as the name implies, exhibits a marked preference for partially reduced folates which are then further reduced to tetrahydrofolates Cohen,105 utilizing microbiological techniques, and Strum et ai., 108 analyzing folates in serosal fluid from everted sacs of rat jejunum by ion exchange chromatography, have found that PGA in low concentration can be partially converted to methylfolate during passage across the intestinal wall in vitro. However, when PGA is administered in large doses as described,112 it would be expected to cross the intestine largely intact because of the low affinity of the enzymes for unreduced folate. This interpretation may explain the observation of Hepner l02 that methotrexate, a potent competitive inhibitor of dihydrofolate reductase, impaired PGA uptake by rat intestine whether the drug was administered orally or parenterally. Observations on the absorption of yeast folate are in partial disagreement with the concept that reduced, natural folates are quantitatively methylated during intestinal transfer Yeast folates are assumed to be a mixture of reduced heptaglutamic folates. When yeast conjugate is taken orally, the folates appearing in the blood are active for both S. faecalis and L. casei, indicating, as noted previously, that these folates are mono- or diglutamates. Furthermore, these findings indicate that methyl folates, which would not support S. faecalis growth, represent only a portion of the absorbed folate. While these data, contrasted with the studies on the absorption of crystalline-reduced monoglutamic folates,"s (V. M. Whitehead, et al.) may suggest different handling of conjugated and of free reduced folates by the intestine; further clarification must await studies with fully defined conjugates of reduced folates. Thus, a third function of the intestinal mucosa during folate absorption appears to be the partial conversion of absorbed folates to methyl folate, the latter being the major form circulating in blood. 69 The possible implications of this conversion in the transport mechanism for monoglutamic folate by the intestine are farreaching. Analysis of experimental data must take into account the possibility that the molecular species of folate are not the same on both sides of the intestinal cell membrane and rules governing the distribution of a single species across a membrane may not be applicable. Future experiments attempting to clarify the still unresolved mechanism by which folate passes into and through the intestinal epithelium

13 March 1971 PROGRESS IN GASTROENTEROLOGY 457 must be designed to isolate the various components of the absorptive process schematically represented in figure 6. In this regard, the previously discussed case reports of an isolated defect in folate absorption 96,97 offer an important opportunity for study of factors which control specificity in man. With such studies, it may be possible to determine whether there is a specific uptake process for folate by intestine and, if so, how that process is influenced by, or even dependent upon, structural alterations of the folate molecule which occur in the mucosal cell. Quantitative aspects of folate absorption. The evidence thus far presented indicates that a variety of folate compounds are absorbed-free and conjugated, reduced and unreduced, formylated, and methylated. However, the quantitative efficiencies of absorption for the various folate forms are not presently well defined. Pte Glu H4 Pte Glu 5 CHOH 4 Pte Glu 5- CH,H 4 Pte Glu '"" } FIG. 6. Schematic representation of intracellular events occurring during absorption of various monoglutamic folates. The major pathways are indicated by the heavy arrow. Abbreviations used: Pte Glu, unreduced pteroylglutamic acid; H, Pte Glu, reduced (tetrahydro) folate; 5-CHOH, Pte Glu, 5-formyltetrahydrofolate ("folinic" acid); 5-CH.jf, Pte Glu, 5-methyltetrahydrofolate. Folate absorption in man has been assessed by a variety of techniques: (1) assay of rises in blood folate levels following an oral dose of either free or conjugated folate,39-u, , 113, (2) measurement of urinary excretion of microbiologically active folate compounds,37, and (3) determination of plasma rises of radioactivity or excretion of radioisotope in the urine and feces after oral radiolabeled folate , , 119, 120 When unlabeled PGA is given by mouth, a rise in microbiologically determined serum folate concentration is detected within minutes, followed by a peak of activity occurring usually at 1 to 2 hr.40, 116, 117 The amount of serum rise and the rapidity of return toward base line values is dependent to a great extent upon the folate dose administered and the degree of saturation of body folate stores. 117 Orally administered conjugated folate likewise produces a rise in serum folate concentration, but the peak of activity generally occurs somewhat later at 2 to 3 hr. 41 Urinary excretion studies have also been performed. Normally, only small amounts (2 to 4 /-Lg) of microbiologically active folate are excreted on unsupplemented diets. 37 When oral PGA is given, excretion varies from 2% of a 500-/-Lg dose 117 to almost 50% of a large 5-mg dose.1i8 As noted previously, it has been demonstrated that administration of crystalline-conjugated folate from yeast without conjugase inhibitor results in urinary excretion levels similar to those obtained with free vitamin, but the levels are markedly decreased when inhibitor is present. 37 There are significant problems of interpretation with both serum rises and renal excretion following oral folate doses. Dependence upon microbiological assay presents difficulties of specificity and accuracy. The degree of tissue saturation significantly affects results. Even the use of "saturating" doses of folate some days prior to study has not abolished the influen,ce of the variability in folate stores. Renal excretion of folate is influenced by the distribution of bound and circulating folate

14 458 PROGRESS IN GASTROENTEROLOGY Vol. 60, No.3 and by renal function. Equally as important is the fact that neither serum rises nor renal excretion allows for direct and accurate quantitation of the amount of folate absorbed. Earlier we discussed some of the studies in man attempting to quantitate absorption and to compare the efficiency of conjugated folate absorption with that for PG A. 36, 37, The introduction of tritium-labeled PGA in 1960 represented a most significant advance in the study of folate absorption. For the first time, it became possible to perform balance studies. Anderson et al.,119 first reporting clinical investigations of the compound in man, showed that mean normal absorption (based on fecal excretion of radioisotope) was 79% (range, 41 to 91 %) of an oral dose of 200 f..lg of tritiated PGA. Without a preloading dose of unlabeled PGA, urinary excretion averaged only 6.3% of the 200-f..Lg oral dose, but this increased to a mean of 41% following a preloading injection of 15 mg of PGA. Percentage excretion did not change when the oral dose was increased to 40 f..lg per kg of body weight. Klipstein 98 and Kinnear and co-workers,100 evaluating urinary excretion following an oral dose of 3H-PGA, largely confirmed the earlier observations demonstrating mean excretions of 41 and 48%, respectively, after oral doses of either 40 f..lg per kg or 15 f..lg per kg when preceded by 15-mg "loading" doses of PGA. Halsted et al. 120 noted slightly lower mean excretion (33%) when employing a 30-mg preloading dose. Plasma radioactivity rises have been shown after oral 3H_PGA.110, 120 Peak levels are achieved at 1 hr, which is similar to the experience with microbiologically determined serum rises of folate following unlabeled PGA. Much higher plasma levels are achieved if the patient is preloaded with PGA. The accurate quantitation of folate polyglutamate absorption, as with PGA, requires the use of chemically defined radiolabeled material. In 1968, Krumdieck and Baugh 24,25 synthesized polyglutamates of folic acid using solid phase synthesis techniques. They labeled the compounds with HC either in the pteroyl or the peptide portions of the molecule. Butterworth, Baugh, and Krumdieck 74 employed these HC-Iabeled folates for absorption experiments in 4 patients with chronic lymphocytic leukemia and in 1 with Hodgkin's granuloma. Their experiments clearly confirmed the concepts of deconjugation of heptaglutamic folate during absorption, but their studies were not designed to quantitate absorption fully. In collaboration with Dr. Johannes Meienhofer (Children's Cancer Research Foundation, Boston, Massachusetts), we have employed classical techniques of peptide synthesis to produce an unambiguous pteroylheptaglutamic folate labeled with tritium in the 3'- and 5'-positions of the p aminobenzoic acid portion of the pteroyl moiety. 26 Owing to the weak {3 emission of tritium, we have been able safely to use this preparation in normal subjects and in patients with intestinal diseases and so to compare its absorption efficiency in the same patient with that of equimolar quantities of tritiated monoglutamic folate. In contrast to previous studies, we have given a 15-mg "flushing" dose 4 hr after the oral dose. The timing of the intramuscular dose was designed to displace any absorbed 3H_ folate from tissue binding sites without introducing the problem of intestinal dilution of the labeled test dose by biliary folate excretion; such dilution may occur when the nonradioactive folate is given before or with the oral radioactive dose. Four normal subjects have been found to excrete from 55.1 to 91% (average, 74%) of an oral dose of 3H-PGA (20 to 35 f..lc of tritium, 250-f..Lg PGA dose) in the urine. Six normal subjects, including the 4 tested with 3H-PGA, who had been given equimolar amounts of 3H-Pte Glu7, excreted from 49.3 to 91.4% (average, 62.4%) of the oral dose in the urine. Additional stool radioactivity has accounted for greater than 90% of the total administered dose. Our experience with synthetic pteroylheptaglutamate and synthetic pteroylmonoglutamate leads us to the conclusion that