Effect of fermentation conditions of brewing yeasts on folate production

Ann Nutr Metab 2012;61:231–235 DOI: 10.1159/000343112

Published online: November 26, 2012

A History of the Isolation and Identification of Folic Acid (Folate) Irwin H. Rosenberg Jean Mayer USDA Human Nutrition Research Center on Aging and Friedman School of Nutrition Science and Policy, Tufts University, Boston, Mass., USA

Key Words Anemia ⴢ Diet ⴢ Folate ⴢ Folic acid ⴢ Isolation ⴢ Pteroylglutamic acid

Abstract In the 1930s, Lucy Wills identified a ‘new hemopoietic factor’ in yeast and liver which cured tropical macrocytic anemia in humans and experimental anemia in monkeys. Janet Watson and William B. Castle named the unknown substance, which would ultimately become a form of folate, ‘Wills’ factor’. Further studies with this unknown substance showed that it was active against nutritional pancytopenia in monkeys and experimental anemia in chicks, leading to various designations such as vitamin M (monkey) and vitamin Bc (chick). Other factors with growth-promoting activity for microorganisms such as Lactobacillus casei were given the interim names including folic acid – in recognition of extracts from leafy greens. Competing pharmaceutical research groups headed by Robert Stokstad at Lederle Laboratories and Joseph John Pfiffner at Parke-Davis Research Laboratory independently isolated factors bearing the biological properties of Wills’ factor and other unknown related factors including folic acid, Lederle Laboratories from a bacterial culture and Parke-Davis Laboratory from yeast and liver as a

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conjugate of folate. The new vitamin then was crystallized, chemically identified, and synthesized as pteroylglutamic acid and named folic acid between 1943 and 1945. Further studies of the monoglutamic folic acid and the yeast isolate polyglutamyl folate followed through the 1950s and to the present. Copyright © 2012 S. Karger AG, Basel

Introduction

Sixty-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. The synthesis of pteroylglutamic acid by Robert Angier (born 1917) and his associates [1] at Lederle Laboratories 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 anti-anemia factors (table  1). The first of these was a ‘new hemopoietic factor’ in yeast [2] or liver [3] which cured tropical macrocytic anemia in India as well as experimental anemia in monkeys induced by Irwin H. Rosenberg, MD Jean Mayer USDA Human Nutrition Research Center on Aging Tufts University, 711 Washington Street Boston, MA 02111 (USA) E-Mail irwin.rosenberg @ tufts.edu

Table 1. Milestones in the isolation and synthesis of folates Factor

Description

‘Wills’ factor’

yeast extract effective against tropical macrocytic anemia in man and monkeys Vitamin M yeast and liver extract effective against nutritional cytopenia in monkeys Factor U growth factor for chicks found in yeast extract Factors R and S yeast factors required for chick nutrition Vitamin Bc chick anti-anemia factor obtained from liver Norite eluate factor growth factor for L. casei present in yeast and liver ‘Folic acid’ concentrate from spinach leaves, active for Streptococcus faecalis Pure L. casei factor from yeast and liver, microbiologically active Crystalline vitamin Bc from liver, active in chick and microbiological assays Vitamins B10 and B11 from liver, active in chicks, inactive for bacteria Vitamin Bc conjugate from yeast, active in chicks, but active for bacteria only after digestion Crystalline vitamin Bc conjugate from yeast, active in chicks, but active for bacteria only after digestion Pteroylglutamic acid synthetic active for bacteria, chicks, monkeys, and man Pteroylheptaglutamic acid synthetic active for bacteria after deconjugation, active in man

Date

Folate form

References

1931

unknown

2, 3, 12

1938 1938 1940 1939 1940 1941 1943 1943 1943 1944 1945 1945 1968

unknown unknown unknown free free free free free conjugated ? conjugated Pte Glu7 PGA Pte Glu7

5, 6 7 8 9, 10 11, 13 14, 15 16 17 18, 19 20, 21 22, 23 1 24–26

Adapted from Rosenberg and Godwin [27].

similar dietary deficiency. Dr. Lucy Wills (1888–1964) was working in India on leave from the Royal Free Hospital in London. This unknown substance was later named ‘Wills’ factor’ by Janet Watson and William Castle (1897–1990) [4] 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 monkeys [5, 6] or required as growth [7, 8] or anti-anemia factors [9, 10] 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) [6] to vitamin Bc (chick) [10]. The report of vitamin Bc by Albert Hogan (1884–1961) and Ernest Parrott (1903–1994) [10] also provided investigators with a useful tool – the chick anemia assay – for 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 charcoal eluate of yeast served as a growth factor for L. casei [11]. This was also the culmination of a race between a Lederle Laboratories team that included Robert Stokstad (1913–1995) versus a Parke-Davis Research Laboratory team headed by Joseph John Pfiffner (1903–1975). Later, as L. casei factor and vitamin Bc were also found to have anti-anemia potency, a striking inconsistency appeared. Some yeast extracts curative in the macrocytic anemia of chicks and monkeys would not support the 232

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growth of L. casei [20]. Thus, it appeared that the chick anemia assay and the L. casei assay were not measuring the same principle. This apparent paradox was clarified by O.D. Bird et al. [28] who showed that the anti-anemia principle of yeast would support the growth of L. casei if the yeast extract was first digested with the crude-tissue homogenates obtained from animal sources. They gave the name ‘vitamin Bc conjugate’ to the complex form of the anti-anemia principle already known as vitamin Bc, and they named the enzymes that release vitamin Bc from the complex ‘conjugases’. Exploiting the microbiological and chick assays, Stokstad [16] and Pfiffner et al. [17] independently isolated the free vitamin from yeast and liver. Chemical identification of the isolated vitamin was followed quickly by its synthesis as pteroylglutamic acid [1]. Pfiffner et al. [22] then utilized conjugases as an adjunct to microbiological methods and accomplished the isolation and identification of the conjugated vitamin from yeast in 1945. Only in retrospect was it possible to weave the disparate threads into a pattern that indicated that all of the variously named factors were variants of pteroylglutamic acid or its conjugate.

Nutritional Evaluation of Free and Conjugated Folates

Early studies, most of them performed prior to the isolation of pteroylglutamic acid or its heptaglutamate conjugate, indicated that the folate forms in yeast were well utilized by chickens [29], turkeys [30], monkeys [5], and Rosenberg

Fig. 1. Pteroylglutamic acid and its princi-

pal conjugate.

patients with macrocytic anemia [5]. Although the greatest portion of yeast folate is conjugated [31], 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 pteroylglutamic acid permitted studies which quickly demonstrated that this vitamin form is effectively utilized by man when administered either orally or parentally [32, 33]. In fact, daily oral doses of ^1 mg were found to be adequate in the treatment of severe nutritional macrocytic anemia [34]. However, the efficiency of the utilization of conjugated folate remains controversial in humans. The first use in man of the relatively pure heptaglutamic folate conjugate isolated by Pfiffner et al. [22] was reported by Bethell and his coworkers. Three of 9 patients with pernicious anemia in relapse and 1 of 2 patients with macrocytic anemia following subtotal gas-

trectomy 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 the free folate prior to administration, the hematological responses and urinary excretion patterns were similar to those seen after administration of equimolar amounts of crystalline pteroylglutamic acid. 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 pteroylglutamic acid with conjugated folate from yeast in normal subjects were made by Swendseid et al. [35]. Comparison depended upon assay

Isolation and Identification of Folic Acid (Folate)

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of microbiologically active folates excreted in the urine following large (4 mg) doses of pteroylglutamic acid or equimolar amounts of conjugate. When the source of conjugate was a crude yeast extract, or even a partially purified eluate from yeast, the quantity of folate excretion was small when compared with the excretion after molar equivalents of pteroylglutamic acid. However, the apparent absorptive advantage for the free vitamin diminished markedly when a highly purified heptaglutamate from yeast was compared with crystalline pteroylglutamic acid. 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 crude extracts. This concept was supported by in vitro experiments from the same laboratory [36] 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 [37] and Spray and Witts [38] demonstrated that ingested yeast failed to produce an elevation of serum folate, while equal amounts of pteroylglutamic acid 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 was purified by ion exchange chromatography before feeding. The study of Jandl and Lear [39] demonstrated yeast extract in vitro would enhance urinary excretion of folate in normal subjects two-fold but still only to reach 60% of the excretion of an equimolar dose of pteroylglutamic acid. The structures of pteroylglutamic acid and its principal conjugate are shown in figure 1.

Dietary folate consists primarily of conjugates of reduced formyl or methyl folates. Most of the current concepts of folate absorption have been derived from studies utilizing synthetic crystalline pteroglutamic (folic) acid partly because this compound has been the only synthetic, chemically defined folate available in adequate quantities since 1945. Studies have been performed with synthetic reduced folates and with the heptaglutamate conjugate. The large body of valuable information concerning folate absorption and utilization obtained with pteroylglutamic acid forms the basis upon which new insights are developing. Current concepts of the events occurring during folate absorption are outlined as follows: (1) Digestion of ␥-glutamyl peptide conjugates of folate by intestinal enzymes (␥-glutamyl carboxypeptidase, ‘conjugase’) releases monoglutamic folate. The site of hydrolysis, whether intraluminally, at the brush border, or within the intestinal cell, is undetermined. (2) Absorption of monoglutamic folate takes place largely in the proximal small intestine by a process that may be specific and active. (3) Metabolic conversion of monoglutamic folate to reduced methyl folate (largely 5-methyletrahydrofolate) appears to occur within the intestinal cell prior to folate release into the portal circulation. Conversion may be quantitative for reduced folate but only partial for unreduced pteroylglutamic acid because of the substrate specifically of dihydrofolate reductase and the methylation system.

References 1 Angier RB, Boothe JH, Hutchings BL, Mowat JH, Semb J, Stokstad EL, Subbarow Y, Waller CW, Cosulich DB, Fahrenbach MJ, Hultquist ME, Kuh E, Northey EH, Seeger DR, Sickels JP, Smith JM Jr: Synthesis of a compound identical with the L. casei factor isolated from liver. Science 1945;102:227–228. 2 Wills L: Treatment of ‘pernicious anemia of pregnancy’ and ‘tropical anemia’ with special reference to yeast extract as a curative agent. Brit Med J 1931;1:1059–1064. 3 Wills L, Clutterbuck PW, Evans BDF: A new factor in the production and cure of certain macrocytic anaemias and its relation to other haemopoietic principles curative in pernicious anaemia. Biochem J 1937; 31: 2136– 2147.

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4 Watson J, Castle WB: Nutritional macrocytic anemia, especially in pregnancy; response to a substance in liver other than that effective in pernicious anemia. Am J Med Sci 1946;211:513–530. 5 Day PL, Langston WC, Darby WJ: Failure of nicotinic acid to prevent nutritional cytopenia in the monkey. Proc Soc Exp Biol Med 1938;38:860–863. 6 Langston WC, Darby WJ, Shukers CF, Day PL: Nutritional cytopenia (vitamin M deficiency) in the monkey. J Exp Med 1938; 68: 923–940. 7 Stokstad ELR, Manning PDV: Evidence of a new growth factor required by chicks. J Biol Chem 1938;125:687–696.

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8 Schumacher AE, Heuser GF, Norris LC: The complex nature of the alcohol precipitate factor required by the chick. J Biol Chem 1940; 135:313–320. 9 Hogan AG, Parrott EM: Anemia in chicks due to vitamin deficiency. J Biol Chem 1939; 128:46–47. 10 Hogan AG, Parrott EM: Anemia in chicks caused by a vitamin deficiency. J Biol Chem 1940;132:507–517. 11 Snell EE, Peterson WH: Growth factors for bacteria: X. Additional factors required by certain lactic acid bacteria. J Bact 1940; 39: 273–285. 12 Wills L, Clutterbuck PW, Evans BDF: A new factor in the production and cure of certain macrocytic anaemias. Lancet 1937; 1: 311– 314.

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13 Hutchings BL, Bohonos H, Peterson WH: Growth factors for bacteria: XIII. Purification and properties of an eluate factor required by certain lactic acid bacteria. J Biol Chem 1941;141:521–528. 14 Mitchell HK, Snell EE, Williams RJ: The concentration of ‘folic acid’. J Am Chem Soc 1941;63:2284. 15 Mitchell HK: Folic acid: I. Concentration from spinach. J Am Chem Soc 1944;66:267– 268. 16 Stokstad ELR: Some properties of a growth factor for Lactobacillus casei. J Biol Chem 1943;149:573–574. 17 Pfiffner JJ, Binkley SB, Bloom ES, et al: Isolation of the antianemia factor (vitamin Bc) in crystalline form from liver. Science 1943;97: 404–405. 18 Briggs GM Jr, Luckey TD, Elvehjem CA, Hart EB: Studies on two chemically unidentified water-soluble vitamins necessary for the chick. J Biol Chem 1943; 148:163–172. 19 Briggs GM Jr, Luckey TD, Elvehjem CA, Hart EB: Studies on vitamins B10 and B11 and related substances in chick nutrition. J Biol Chem 1945;158:303–312. 20 Binkley SB, Bird OD, Bloom ES, Brown RA, Calkins DG, Campbell CJ, Emmett AD, Pfiffner JJ: On the vitamin Bc conjugate in yeast. Science 1944;100:36–37. 21 Bird OD, Bressler B, Brown RA, Campbell EJ, Emmett AD: A micro-biological assay of vitamin Bc conjugate. J Biol Chem 1945; 159: 631–636.

Isolation and Identification of Folic Acid (Folate)

22 Pfiffner JJ, Calkins DG, O’Dell BL, Bloom ES, Brown RA, Campbell CJ, Bird OD: Isolation of an antianemia factor (vitamin Bc conjugate) in crystalline form from yeast. Science 1945;102:228–230. 23 Pfiffner JJ, Calkins DG, Bloom ES, O’Dell BL: On the peptide nature of vitamin Bc conjugate from yeast. J Amer Chem Soc 1946;68: 1392. 24 Baugh CM, Krumdieck CL: The solid phase synthesis of polyglutamates of folic acid. Fed Proc 1968;27:455 (abstract). 25 Krumdieck CL, Baugh CM: The solid phase synthesis of polyglutamates of folic acid. Biochemistry 1969;8:1568–1572. 26 Godwin HA, Rosenberg IH, Meienhofer J: Absorption of synthetic ‘cold’ and tritiumlabeled pteroylheptaglutamic acid. J Clin Investig 1970;49:35 (abstract). 27 Rosenberg IH, Godwin HA: The digestion and absorption of dietary folate. Gastroenterol 1971; 60:445–463. 28 Bird OD, Binkley SB, Bloom ES, Emmett AD, Pfiffner JJ: On the enzymic formation of vitamin Bc from its conjugate. J Biol Chem 1945;157:413–414. 29 Campbell CJ, Brown JA, Emmett AD: Influence of crystalline vitamin Bc on hematopoiesis in the chick. J Biol Chem 1944; 152:483– 484.

30 Jukes TH, Stokstad ELR, Belt M: Deficiencies of certain vitamins as studied with turkey poults on a purified diet. I. Pteroylglutamic acid, riboflavin, niacin and inositol. J Nutr 1947;33:1–12. 31 Stokstad ELR, Koch J: Folic acid metabolism. Physiol Rev 1967;47:83–116. 32 Spies TD, Wilter CF, Koch MB, Caldwell MH: Observations of the antianemic properties of synthetic folic acid. South Med J 1945; 38:707–709. 33 Moore CV, Bierbaum OS, Welch AD, Wright LD: The activity of synthetic Lactobacillus casei factor (‘folic acid’) as an antipernicious anemia substance. J Lab Clin Med 1945; 30: 1056–1069. 34 Davidson LSP, Girdwood RH: Folic acid as a therapeutic agent. Brit Med J 1947;1:587–591. 35 Swendseid ME, Bird OD, Brown RA, Bethell FH: Metabolic function of pteroylglutamic acid and its hexaglutamyl conjugate. II. Urinary excretion studies on normal persons. Effects of a conjugase inhibitor. J Lab Clin Med 1947;32:23–27. 36 Bird OD, Robbins M, Vandenbelt JM, et al: Observations on vitamin Bc conjugase from hog kidney. J Biol Chem 1946; 163:649–659. 37 Spray GH: The utilization of folic acid from natural sources. Clin Sci 1952;11:425–428. 38 Spray GH, Witts LJ: The utilization of folic acid given by mouth. Clin Sci 1952; 11: 273– 281. 39 Jandl JH, Lear AA: The metabolism of folic acid in cirrhosis. Ann Intern Med 1956; 45: 1027–1044.

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