Esmond E. Snell

Vitamin B6 Group. XV. Urinary Excretion of Pyridoxal, Pyridoxamine, Pyridoxine, and 4-Pyridoxic Acid in Human Subjects.*:

Summary The known metabolic products of vitamin B6—pyridoxal, pyridoxamine, pyridoxine and pyridoxic acid—were measured in normal human urine and in the urine of human subjects each fed one of the 3 forms of the vitamin. The chief product found, regardless of the form fed, was pyridoxic acid. Pyridoxal gave rise to significantly higher amounts of this product than did pyridoxine or pyridoxamine. No evidence could be obtained showing the conversion of pyridoxal or pyridoxamine to pyridoxine. When pyridoxal or pyridoxine was fed, the chief form in which the vitamin oc- curred in the urine was the form fed. However, when pyridoxamine was fed both pyridoxal and pyridoxamine were excreted in approximately equal amounts. Ingestion of pyridoxine also greatly increased the amount of pyridoxal and pyridoxamine excreted. The excretion of all products was very rapid. The largest amounts of each of the compounds were found in samples collected 2 and 5 hours after ingestion of the dose. The levels of pyridoxic acid returned to normal values after 12 hours, while the vitamin levels had returned to normal within 8 hours. The amount of the dose recovered varied with the form fed. The highest recovery, 70%, was obtained when pyridoxal was fed; 45% of the pyridoxine was recovered, while only 31% of the pyridoxamine could be recovered. Together with published data which indicate that complete absorption of large doses of vitamin B6 occurs, these findings suggest that a large proportion of the vitamin B6 was converted to products still unknown.

The nature of the requirement of Saccharomyces carlsbergensis for vitamin B6

Abstract Saccharomyces carlsbergensis 4228, an organism widely used for determination of vitamin B6, grows well without this vitamin if thiamine is also omitted from the basal medium, and an inoculum grown in a thiamine-low medium is used. Thiamine inhibits growth when added to such a medium. The thiazole moiety of thiamine, but not the pyrimidine, is also inhibitory, but less so than thiamine itself. Growth inhibition by thiamine is prevented by vitamin B6. At low concentrations of thiamine, the amount of vitamin B6 required for growth increases with the thiamine concentration; at concentrations of thiamine above 1 μg./10 ml. the vitamin B6 requirement for growth remains essentially constant. Since these higher concentrations of thiamine have been used in methods that utilize this organism for determination of vitamin B6 (1,2), the validity of these methods is confirmed. In the presence of thiamine, growth was also permitted by additions of the thiamine antagonist, neopyrithiamine. In this case, however, the relationship was fully competitive; i.e., the amount of neopyrithiamine required for growth increased regularly with the thiamine concentration. At concentrations considerably higher than those required for growth, neopyrithiamine again inhibited growth, and this inhibition was prevented by an increase in the thiamine concentration. Thus neopyrithiamine acts by lowering the effective thiamine concentration to subinhibitory levels; if excessive amounts are used, it prevents essential metabolic functions of thiamine and itself becomes toxic. The mechanism by which vitamin B6 prevents thiamine toxicity is not known. The appearance of a requirement for certain growth factors because of inhibitory effects of other metabolically important compounds, rather than because of an intrinsic inability of the organism to synthesize the growth factor, may be much more common than the few recorded instances of this phenomenon indicate.

Vitamin B6 antagonists and growth of microorganisms. I. 4-Desoxypyridoxine

Abstract 4-Desoxypyridoxine is relatively ineffective as an antagonist of vitamin B 6 for organisms that grow in the absence of the latter vitamin, and for many lactic acid bacteria that require the vitamin. It is an effective antagonist of vitamin B 6 for certain yeasts and molds that require an external source of vitamin B 6 for growth. It is shown that the same organism may be sensitive to the inhibitor when dependent upon an external supply of vitamin B 6 for growth, but insensitive when cultured under conditions that permit it to synthesize this vitamin. The comparative effectiveness of various forms of vitamin B 6 in counteracting growth inhibition by desoxypyridoxine varies from organism to organism. For Saccharomyces carlsbergensis the order of effectiveness was pyridoxine > pyridoxamine = pyridoxal. For Neurospora sitophila this order was pyridoxine ≧ pyridoxal > pyridoxamine.

Reversal of aminopterin inhibition in the chick embryo with desoxyribosides.

Summary Aminopterin, injected prior to, or early in, the incubation period, is highly toxic to the chick embryo. Ten micrograms or more of this compound produce 100% mortality; 100 μg or more per egg completely inhibit embryonic development. The inhibitor becomes relatively less toxic as embryonic development progresses; at nine days or more, twenty micrograms is non-toxic. The same amount injected at three days results in almost immediate embryonic death. Thymidine partially counteracted the inhibitory effects of aminopterin. A combination of hypoxanthine desoxyriboside and thymidine was more effective than thymidine alone. In the absence of thymidine, hypoxanthine desoxyriboside was without effect. An enzymatic digest of desoxyribonucleic acid also counteracted aminopterin inhibition partially. Thymine, hypoxanthine, folic acid, vitamin B12, and concentrates of the Leuconostoc citrovorum factor, alone and in various combinations, were ineffective in counteracting aminopterin inhibition. These experiments indicate that aminopterin acts in animal tissues in part by preventing synthesis of thymidine and one or more of the purine desoxyribosides, and that these compounds are immediately essential for embryonic growth. Since aminopterin is thought to act by creating a deficiency in folic acid, the necessity for folic acid in synthesis of these compounds in animal tissues is implied.

Vitamin B6 antagonists and growth of microorganisms. II. 5-Desoxypyridoxal and related compounds.

Abstract Three 5-desoxy analogs of vitamin B6 were compared with 4-desoxypyridoxine and ω-methylpyridoxine as antagonists of vitamin B6. Except for minor variations, the order of decreasing effectiveness as antagonists of vitamin B6 for Saccharomyces carlsbergensis was: ω-methylpyridoxine > 4-desoxypyridoxine > 5-desoxypyridoxine ≧ 5-desoxypyridoxal > 5-desoxypyridoxamine. Pyridoxine was more effective than pyridoxal in preventing the inhibitory effects of these antagonists; pyridoxal in turn was more effective than pyridoxamine. For Streptococcus faecalis, 5-desoxypyridoxamine was an effective antagonist of vitamin B6; 5-desoxypyridoxal was somewhat less active; the remaining compounds were inactive. Pyridoxamine was more effective than either pyridoxal or pyridoxamine phosphate in preventing growth inhibition by these antagonists. The antagonists did not inhibit growth when vitamin B6 was made nonessential by the addition of d -alanine to the medium. For Lactobacillus helveticus, only 5-desoxypyridoxal proved effective as an antagonist of vitamin B6. The inhibitory effects on Saccharomyces carlsbergensis of 4-desoxypyridoxine and ω-methylpyridoxine were additive, indicating that these two compounds inhibited the same reaction in this organism. In contrast, the effects of 4-desoxypyridoxine (or of ω-methylpyridoxine) and of 5-desoxypyridoxal were exerted independently of one another; these inhibitors, therefore, appear to act by independent mechanisms.

ION ANTAGONISM IN BACTERIA AS RELATED TO ANTIMETABOLITES

In 1882, Ringer’ showed that a solution of sodium chloride would not maintain the beat of a heart perfused with it unless additions of calcium and potassium chlorides were made. This initial observation of what is now called “ion antagonism” was extended to intact animals by Loeb12 to plants by Osterhout? and to bacteria by Flexner? E i ~ l e r , ~ and Lipmann.e Subsequently, results of a large number of investigators (cf. Falk7) have emphasized the importance of maintaining the proper ratio of ions for the proper functioning of biological systems, and a host of “antagonistic” or “synergistic” relationships between individual ions in particular biological systems has been observed. The trend of thought concerning ion antagonism can be summarized in the statement by Falk in 19227 that “the effect exerted by electrolytes appears to be primarily an effect upon external or internal membrane surfaces and upon surface interphases in colloidal systems.” Few attempts a t a more exact delineation of the mechanism of ion antagonism in living organisms have been made. Since knowledge of the inorganic requirements of the biological systems studied was either limited or nonexistent, no attempts to relate ion antagonism to nutritional requirements of the organism were made. Indeed, Loebs discounted the possibility that physiologically balanced ion solutions had nutritional significance, on the grounds that certain fish studied by him survived for long periods of time either in distilled water or in a solution containing correct proportions of NaC1, KCI, and CaCL, but not in “unbalanced” solutions of these ions. I t is the thesis of the present paper that many cases of ion antagonism can be explained on nutritional grounds, ie., that an ion which suppresses growth frequently does so by interfering with one or more of the essential metabolic roles played by an ion required for growth. Since the nutritionally essential trace elements function at least in part as necessary components of metabolically essential enzymes, a more exact picture of the mechanism of action of antagonistic ions might be to visualize a competition between the antagonists for an enzyme surface. An enzymatically active metalloprotein (MEE) results from the normal combination of the nutritionally essential ion (ME) and the apoenzyme (E) ; an enzymatically inactive metalloprotein (MTE) results when the “toxic” ion (MT) is thus combined. If combination of both metals with the protein is readily reversible, as diagrammed here, then the extent to which the enzyme can function (and growth of the organism proceed where the functional enzyme is required for growth) will depend onlyupon the ratio of ME to MT and not upon the absolute concentration of either, and a true competitive type of inhibition will result. Noncompetitive and intermediate types of inhibition could also result where reaction (2) was irreversible or only partially reversible.

Reversal of Aminopterin Inhibition in the Chick Embryo with the Leuconostoc citrovorum Factor.

Summary Concentrates of the Leuconostoc citrovorum factor (CF) and of “folinic acid” partially counteract the inhibitory action of aminopterin for the chick embryo. Folic acid and formylfolic acid were ineffective or only slightly effective under the same conditions. The magnitude of the effects obtained with CF were similar to those obtained previously with mixtures of thymidine and hypoxanthine desoxyriboside 1). The viewpoint is developed that conversion of folic acid to CF is a necessary preliminary to the catalytic action of the former compound, that synthesis of thymidine and one or more of the purine desoxyribosides are among the synthetic reactions for which CF or folic acid) is required, and that these synthetic reactions are those inhibited in the chick embryo by small amounts of aminopterin. The great variation that exists in the ability of CF to counteract aminopterin inhibition in various organisms is pointed out.

MICROBIOLOGICAL METHODS IN AMINO ACID ANALYSIS

For many years, investigators interested in microbial nutrition have utilized the growth responses of microorganisms to detect the presence of substances favorable or necessary for growth of such organisms on laboratory media. In many instances, such responses could be controlled to yield results sufficiently quantitative to permit isolation or identification of the active substances involved. As a result of such investigations, the nutritional requirements of a large number of microorganisms are now known. Once the growth requirements of an organism are known, it becomes pos-, sible to devise media which are deficient in a single substance only and to employ the response of the organism to additions of that substance, for the quantitative estimation of the substance in natural materials. Such procedures depend upon the observed fact that, in the presence of limiting amounts of a substance essential for growth, the amount of growth obtained (or the amount of some metabolic product used to follow growth) is a function of the concentration of essential nutrilite and increases to a maximum as the concentration of the nutrilite is increased. A curve relating the amount of growth to the concentration of the pure essential substance is thus obtained, and can be used to assess the amount of the essential substance present in mixtures of varying complexity. Such procedures have met with widespread success in the estimation of many vitamins. Similar techniques are now being successfully applied to the estimation of the amino acids. While any organism which specifically requires a given amino acid for growth can, theoretically, be used in this manner for its determination, only a few have so far proved useful. Foremost among these are various species of lactic acid bacteria. These organisms possess a number of characteristics which make them especially valuable for this type of work. Their nutrition is complex. A variety of vitamins and amino acids is required, among other substances, for growth. Extensive study has elucidated these requirements, so that it is possible to grow many of these organisms on synthetic media. Media deficient in any one of the numerous

Specificity of the response of various assay organisms to nicotinic acid.

Summary Kynurenine and hydroxyanthran-ilic acid, which appear to be intermediates in the conversion of tryptophan to nicotinic acid by Neurospora, neither replace nicotinic acid nor enhance the growth response to sub-optimal amounts of nicotinic acid for L. arabinosus, Leuc. mesenteroides, S. jaecalis, Proteus vulgaris, or Torula cremoris. These substances do not, therefore, interfere in microbiological assays for niacin which employ these test organisms.

Growth-Promotion by Lyxoflavin. II. Relationship to Riboflavin in Bacteria and Chicks.

Summary 1. L-lyxoflavin, D-galactoflavin and isoriboflavin markedly increase the growth response of Lactobacillus casei to suboptimal amounts of riboflavin, but do not promote growth in the absence of this vitamin. Appropriate differential assays indicated that lyxoflavin was not deposited in cells of L. casei under conditions where it stimulated growth, and actually decreased the concentration of riboflavin deposited in such cells. Thus it does not serve as a partial substitute for riboflavin in L. casei, but appears to enhance the efficiency with which limited supplies of riboflavin are utilized. When riboflavin is present in excess, lyxoflavin does not increase the cell yield or the growth-rate. At higher concentrations, lyxoflavin and galactoflavin inhibit growth by acting as competitive antagonists of riboflavin. Dichlororiboflavin and isoriboflavin are ineffective as inhibitors of this organism. 2. L-lyxoflavin promotes maximum growth of Lactobacillus lactis in the absence of riboflavin, and such growth continues indefinitely upon subculture. Differential assay with L. casei and L. lactis showed that cells of the latter organism grown with lyxoflavin contain no riboflavin, but do contain lyxoflavin; i.e., in this organism lyxoflavin can fill the essential metabolic roles normally played by riboflavin. Similar assays of tissues from rats grown on natural rations indicate that lyxoflavin does not occur naturally in significant quantities. 3. In chicks, lyxoflavin stimulated growth when fed at levels 2.5 to 10 times the amount of riboflavin in the diet. At ratios of lyxoflavin to riboflavin higher than 40 to 1. lyxoflavin inhibited growth. Amounts of lyxoflavin that inhibited growth on diets of low riboflavin content were non-toxic when the amount of riboflavin was increased. 4. Although the growth-responses observed in chicks when L-lyxoflavin is added to diets low in riboflavin may result in part from a sparing action similar to that found with L. casei, such a sparing action does not explain the growth effects of the analog observed in rations of high riboflavin content.