Effects of some aldehyde binding agents on the in vitro synthesis of L-ascorbic acid.

Abstract: Three different fractions of d -glucuronolactone dehydrogenase were isolated from rat liver supernatant by means of CM and DEAE Sephadex ion exchange chromatography. These fractions also had acetaldehyde dehydrogenase activity. Intraperitoneal administration of phenobarbital, p , p ′-DDT and 3-methylcholanthrene increased the enzyme activity in two of the fractions, while the third remained unaffected by these drugs. Enhanced activity was obtained with both substrates, but it varied in degree within the group of animals. The rats could be divided into poor, medium and high response groups. A change in the ratio of the d -glucuronolactone and acetaldehyde dehydrogenase activities was also found after the drug treatment. Spironolactone, which is also an inducer of drug metabolism, did not alter the activity of any of the enzyme fractions, at the dose used.

Abstract: 1. 1. Microsomal fractions of tissues from various species that are known to produce l -ascorfoic acid—namely, amphibians, reptiles, mammals, and birds—were found to synthesize the vitamin from d -glucurono-1,4-lactone semicarbazone irrespective of whether the enzyme system is present in the kidney or in the liver. 2. 2. The rate of synthesis of l -ascorbic acid from d -glucurono-1,4-lactone semicarbazone is much fatter than that obtained from sodium glucuronate. 3. 3. The significance of the presence of a separate pathway of synthesizing l -ascorbic acid from d -glucurono-1,4-lactone conjugate has been discussed.

Abstract: Recent studies are elucidating the genetic basis for the loss of the ability to synthesize ascorbic acid in animals subject to scurvy.

Abstract: d -Glucofuranosiduronolactone conjugates, namely, semicarbazone, oxime, cyanohydrin, and thioacetal of d -glucurono-1,4-lactone, were converted to l -ascorbic acid by an enzyme system present in rat, goat, or rabbit liver microsomes. Guinea pig liver microsomes were ineffective. Neither methyl- d -glucofuranosiduronolactone nor any glucopyranosiduronic acid conjugates examined were converted into l -ascorbic acid. Methods of preparation and properties of d -glucurono-1,4-lactone semicarbazone and d -glucurono-1,4-lactone oxime have been described and a tentative mechanism of enzymic reduction of the conjugates into l -gulono-1,4-lactone has been suggested. A conjugate of d -glucurono-1,4-lactone and imidazole, which is a substrate for the microsomal enzyme leading to synthesis of l -ascorbic acid, has been identified in the urine of rat treated with chloretone, barbital, and 1,2-benzanthracene. It has been indicated that the enhanced synthesis of l -ascorbic acid after administration of various drugs and toxic chemical compounds is due to the induced formation of this endogenous substrate.

Abstract: In exploring the mechanism of biosynthesis of ascorbic acid by the rat, King and his coworkers (Musulin, Tully, Longenecker & King, 1939; Longenecker, Musulin, Tully & King, 1939; Longenecker, Fricke & King, 1940) observed that rats treated with Chloretone (1:1:1-trichloro-2methylpropan-2-ol) excreted more ascorbic acid than did the controls. By employing Chloretonized rats and isotopically labelled precursors they (Jackel, Mosbach, Burns & King, 1950; Horowitz, Doerschuk & King, 1952; Horowitz & King, 1953a, b) presented evidence for the following route of the synthesis: D-Glucose -+ D-glucurono-y-lactone -* L-ascorbic acid. Subsequently, Isherwood, Chen & Mapson (1954) and Mapson, Isherwood & Chen (1954) came to the conclusion from an extensive series of experiments that in plant and animal systems respectively the following sequences were involved: (1) D-Galactose -+ D-galacturonic acid methyl ester -+ L-galactono-y-lactone -+ L-ascorbic acid. (2) D-Glucose -+ D-glucurono-y-lactone -+ Lgulono-y-lactone --. L-ascorbic acid. Hassan & Lehninger (1956) reported that ratliver extracts containing the microsomes and soluble supernatant could convert D-glucuronolactone, L-gulonolactone and also the corresponding free acids into L-ascorbic acid in the presence of cofactors. In experiments previously carried out in this Laboratory it had been observed that rat-liver tissue incubated with sodium pyruvate showed a higher ascorbic acid value than did the controls incubated without the substrate (Roy, Roy & Guha, 1946). But in no experiment was the value so obtained higher than the original ascorbic acid content of the tissue before incubation, and it was possible therefore that pyruvic acid was not a direct precursor. In a search for a system in which a net synthesis over and above the original tissue ascorbic acid level takes place, Chatterjee, Ghosh, Ghosh & Guha (1957a) found, in later experiments, that a marked synthesis of ascorbic acid occurred in homogenates of rat-liver tissue, containing potassium cyanide, from D-glucurono-ylactone, the increase in ascorbic acid value being sometimes two to three times that originally present. No other substrate, including sodium Dglucuronate and sodium pyruvate, could be so converted into ascorbic acid. Subsequently, it was observed that L-gulonolactone, but not sodium L-gulonate, could also act as the substrate and this conversion took place in the absence of cyanide. Liver tissues of all the mammals investigated could bring about this cyanide-stimulated synthesis, but homogenates of the brain (rat), kidney (rat and goat) or adrenal gland (cow) were ineffective in this respect. Extracts from cow and goat livers were found to be the most active, and goat liver, being more freshly available, was chosen for further studies on the enzyme system concemed. The enzyme system was separated from goat-liver homogenates and obtained in the microsomes and the soluble fraction; it could be further concentrated from the extract by precipitation with ammonium sulphate (Chatterjee, Ghosh, Ghosh & Guha, 1958a). The present paper deals with detailed studies of this enzyme preparation.

Abstract: On incubating l-gulonolactone with rat liver microsomes, l-ascorbic acid is formed. l-Ascorbic acid, once formed, acts as a catalyst for the formation of a 2-thiobarbituric acid-reacting substance(s), tentatively lipid peroxide. The lipid peroxide, on the other hand, irreversibly inactivates the l-gulono dehydrogenase. Hence, in such a system, the amount of l-ascorbic acid formed does not give a true value of the enzymic activity. However, when a chelating agent or an antioxidant is added to the system, the formation of lipid peroxide is completely inhibited and the amount of l-ascorbic acid formed represents the true activity of the enzyme system. The use of a chelating agent has a further advantage. In the presence of 0.001 M KCN, it completely prevents the oxidation of l-ascorbic acid, so that the l-ascorbic acid synthesized can be estimated by simple titration with 2·6-dichlorophenol indophenol.