George Wolf

Studies on the function of vitamin A in mucopolysaccharide biosynthesis

An in vitro system of rat colon segments and of rat colon homogenates was elaborated, which incorporated [35S]sulfate and [14C]glucose into mucopolysaccharide. Mucopolysaccharide was identified by paper chromatography and paper electrophoresis along with chondroitin sulfate carrier; by co-precipitation and dialysis with chondroitin sulfate; by hydrolysis and identification of labeled sulfate or labeled glucosamine. The homogenate system required adenosine triphosphate, diphosphopyridine nucleotide, glucose and glutamine. The level of incorporation was lowered to about one-half in colon segments and homogenates from vitamin A-deficient rats. Vitamin A, vitamin A-aldehyde and vitamin A-acid (but no other fat-soluble vitamin tested) restored incorporation to normal. With graded levels of vitamin A added, a maximum of incorporation was reached to 10 μg/6 mg of protein in the homogenates. Using glucosamine or galactosamine instead of glucose and glutamine, or uridine diphosphate acetylglucosamine, uridine diphosphate glucuronic acid, uridine diphosphate glucose and acetylglucosamine, incorporation of labeled sulfate was still dependent on vitamin A. This fact shows that the vitamin may be required either in the polymerisation reaction of the uridine derivatives, or the activation or transfer of sulfate. It was shown that the vitamin A-destroying enzyme lipoxidase can lower or abolish incorporation into mucopolysaccharide.

Studies on the biosynthesis and turnover of carnitine

Abstract To investigate the biosynthesis of carnitine, a number of labeled intermediates, among them acetate, formate, glycine, glucose, some essential amino acids, and 3-hydroxy-4-aminobutyric acid, were injected into rats, and carnitine was isolated after 24 hr. No radioactivity was detectable in that compound. The turnover of carnitine was determined and found to be very slow (half-life, 67 days, body pool about 36 mg./100 g. rat). In long-term experiments (9 days), the methyl groups of methionine were found to be incorporated into the methyl groups of carnitine, and carbon 1 of glycine into the chain to a very small extent. A new synthesis of labeled 3-hydroxy-4-aminobutyric acid is described, as well as methods for degradation, isolation, and assay of carnitine.

Studies on the distribution of free carnitine and the occurrence and nature of bound carnitine

Abstract The distribution of carnitine in various tissues of the rat was investigated, using modifications of existing assays. Muscle showed about 120–180 μ./g. wet weight; brain, 20 μg.; liver 60–90 μg.; testes, 115 μg. Various treatments with organic solvents and acids released no increased amounts of carnitine. All carnitine was dialyzable. No “bound carnitine,” therefore, was detectable in mammalian tissue. This result was confirmed by injection of labeled carnitine, using a radiochemical assay, in combination with the chemical assay. Developing chick embryos showed a great increase in carnitine between the 12th and 16th day (about 175 and 850 μg. per whole egg, respectively). It was found that yolk sac contained about 40–50% of the carnitine of whole egg, and that some of this, about 20–45%, was in a bound form, released by acid treatment and nondialyzable. Specific extraction procedures, followed by hydrolysis, showed this “bound carnitine” to be combined in phospholipid. This finding was confirmed with labeled carnitine. Labeled phospholipid was extractable from the yolk sac, and was purified by silicic acid chromatography. A labeled carnitine-contaming phospholipid emerged with the lecithin fraction. The hypothesis is proposed that this material is phosphatidylearnitine.

The function of vitamin A in carbohydrate metabolism; its role in adrenocorticoid production.

Publisher Summary Rather than study the change in level of a particular metabolite, this chapter uses a more dynamic approach to the problem of vitamin A function by first searching for a metabolic block in the intact animal. In this search for possible leads as to where vitamin A might function in the general metabolic scheme, radioacetate was used as a general metabolic precursor and was administered to A-deficient and pair-fed normal rats. Pair-feeding was used throughout the intact animal work to avoid inanition effects. This work leads directly to a role of vitamin A in carbohydrate metabolism as indicated by a requirement of vitamin A for glycogen synthesis. This proves to be an indirect effect mediated by the glucogenic hormones of the adrenal gland, and it is principally this work which that is presented in the chapter. In many experiments to be reported, work was first done with very severely vitamin A-deficient animals, and at times the results obtained were later compared with those obtainable with more mildly deficient animals to rule out the changes due to general debility and morbidity. Only those changes observable also in the mildly deficient animal are thought to be indicative of a real metabolic function of vitamin A.

Vitamin A and mucopolysaccharide biosynthesis.

Publisher Summary In a search for a biochemical function of vitamin A in metabolism, when looking over the existing literature on the effects of vitamin A deficiency or excess, one is immediately struck by the importance of the influence of vitamin A on mucus formation and on mucosal tissue. Moore, in his monumental work on vitamin A, states: “If we consider the role of vitamin A on the widest possible basis, therefore, we may say that it is necessary for the formation of large molecules containing glucosamine.” Such large molecules are the mucopolysaccharides which, in addition, contain galactosamine, glucuronic acid, sulfate, and in certain cases also sialic acid, mannose, and fucose. Any hypothesis of a systemic function of vitamin A, therefore, has to take into account its effect on the biosynthesis of the mucopolysaccharides. These occur in various forms, and with various functions, in almost all tissues of the mammalian organism, but principally, and in largest quantities, in two locations: in the mucus secreted by mucous epithelium; and in the extracellular matrix of cartilage, mainly as chondroitin sulfate. Attention is focused in this chapter on the influence of vitamin A on these two tissues.

Vitamin A and mucopolysaccharide synthesizing enzymes

Abstract An enzyme fraction was obtained from pig colon mucosal homogenates, after removal of nuclei, mitochondria and microsomes, by precipitation at pH 5.2, which, upon incubation with labeled sulfate or labeled glucose, produced labeled mucopolysaccharide. This enzyme fraction (“pH 5 enzymes”) required glucose, glutamine and diphosphorpyridine nucleotide, but no adenosine triphosphate for mucopolysaccharide synthesis, and was stimulated by uridine triphosphate. Its pH optimum was at 6.8. The [ 35 S]mucopolysaccharide produced by the pH 5 enzymes could be precipitated with carrier chondroitin sulfate by cetylpyridinium bromide. The [ 14 C]mucopolysaccharide obtained from [ 14 C]glucose yielded labeled hexosamine on hydrolysis. The pH 5 enzymes contain about 50% cent of the vitamin A of whole mucosa. Treatment with lipoxidase lowered incorporation, which could be restored by added vitamin A. The pH 5 enzymes from vitamin A deficient pigs showed lowered activity, again restorable by added vitamin A, specifically. Pig colon mucosa catalyzed the activation of sulfate to phosphoadenosine phosphosulfate, and pH 5 enzymes the transfer of sulfate to p -nitrophenol.

Vitamin A and mycopolysaccharide biosynthesis by cell-free particle suspensions☆

In continuation of previous work from this laboratory, a system was developed consisting of particles from colon homogenates sedimenting at 20000 × g (after removal of whole cells and debris at 1000 × g), which could effect net synthesis of mucopolysaccharide-bound hexosamines from glucose 6-phosphate, glutamine, ATP, UTP, DPN and Mg2+. Whereas the whole homogenate had an absolute requirement for, at least, ATP, UTP and DPN, the particles required at least glucose 6-P and Mg2+. The average of mucopolysaccharide-bound hexosamines formed was 2.8 μmoles/100 mg protein for whole homogenate 7.3 μmoles for the 20000 × g particle suspension, and 0.6 μmole for the cell-up and microsome fraction on incubation for 3 h at 37°. Histological examination showed the particles to be cell-free, bacteriological tests (after incubations with chloromyceetin, which gave normal mucopolysaccharide synthesis) revealed no bacterial contamination. The formation of mucopolysaccharide-bound hexosamines showed an approximately linear relationship with the amount of protein in the particles incubated. Mucopolysaccharide synthesis was lower with UDP-glucuronic acid and UDP-acetylglucosamine as substrates in place of glucose 6-P. The level of mucopolysaccharide-bound hexosamines formed by whole homogenates of vitamin A-deficient rats was reduced to 0.27 μmole/100 mg of protein, by vitamin A-deficient particles to less than 0.1 μmole. Complete restoration of the latter to the normal level of mucopolysaccharide synthesis with added vitamin A was not possible, but could be so restored by adding a metabolite derived from vitamin A acid.

Vitamin A and mucopolysaccharide biosynthesis in colon homogenates.

The requirement of vitamin A for the formation in vitro of mucopolysaccharide-bound hexosamines has been studied. Net synthesis of total as well as mucopolysaccharide-bound hexosamines in colon homogenates from glucose 6-phosphate, adenosine triphosphates, uridine triphosphate, glutamine and magnesium chloride, was impaired by vitamin A deficiency, and could be restored specifically by added vitamin A. The minimum effective amount of the vitamin was 1.25·10−2 μmoles per incubation (0.1 μmole/100 mg protein). [14C] Glucose was found to be incorporated into hexosamines bound to mucopolysaccharide under the same conditions. The incorporation was also depressed by vitamin a deficiency and restorable by added vitamin A. It is concluded that vitamin A functions, possibly co-enzymically, in the biosynthesis of mucopolysaccharide.

Metabolic Tranformations of Vitamin A

Publisher Summary The approach to a study of the metabolic transformations of vitamin A has been from two different directions: first, to find a functional form of vitamin A, since circumstantial evidence makes it seems likely that is not vitamin A itself but a derivative thereof which is the active form. As discussed by Moore, the reserve stores of vitamin A disappear long before deficiency symptoms are seen, and much more vitamin A is required for storage than for growth. At least 100 I.U. of vitamin A disappear when administered to a depleted rat, before storage begins. It seems as if vitamin A, when fed to deficient rats, is first transformed into an active derivative which is used for growth promotion and tissue maintenance. When this derivative reaches its proper level, vitamin A itself appears in the blood and is ultimately stored in the liver. An excellent discussion of the present state of knowledge of this unknown derivative and the alleged hidden forms of vitamin A is given by Moore. The second approach is to determine the fate of administered vitamin A with regard to disposal by the organism of excess vitamin, and disappearance after it has fulfilled its function (turnover).

Vitamin A in Adrenal Hormone and Mucopolysaccharide Biosynthesis

W E KNOW OF one function of vitamin A, that in vision, through the researches of Wald and his team. Through their work we know more about what this vitamin does in one particular biochemical reaction sequence than we do about any other fat-soluble vitamin. However, an animal dies of vitamin A deficiency, but not necessarily from blindness. Therefore, vitamin A must have another metabolic function. We began our search for this function in the most generalized l