Search

Peroxyacetyl nitrate was reactive with small molecular-weight sulfur-containing compounds The order of susceptibility was cysteine > reduced lipoic acid = reduced lipoamide > oxidized lipoic acid > oxidized lipoamide > methionine ⪢ cystine. From thiols the predominant product was disulfide. In the early stages of oxidation methionine yielded methionine sulfoxide. Products of oxidation of oxidized lipoic acid and lipoamide were the respective sulfoxides. Cystine was resistant to oxidation, yielding cysteic acid when oxidation took place. Papain was readily inactivated by peroxyacetyl nitrate while lysozyme was resistant. The small amount of inactivation of lysozyme was correlated with methionine oxidation. Papain inactivation was correlated with thiol oxidation and could be reversed by thiol compounds. The oxidation product was judged to be a dimer by methods for determining molecular weight.

1. 1. When dihydrolipoic acid is irradiated by 60Co γ rays at pH 8.0 and in the presence of N2O, it is converted to lipoic acid with a G value of 3.3 ± 0.3, where G is the number of molecules formed per 100 eV energy absorbed. No other products were detectable. The radiation product was identified by its absorption spectrum and thin-layer chromatography. 2. 2. The lipoic acid free radical was studied with pulse radiolysis. Its absorption spectrum has a maximum at 410 nm with a molar extinction coefficient of 7760 M−1 cm−1. The radicals derived from either oxidation of dihydrolipoic acid or reduction of lipoic acid are the same. At pH 8 the radical decays by second order, k = (7.45 ± 0.22) s 10 7 M −1 s s −1 . Ionic strength studies indicate that the radical decay is influenced by a charge of one. 3. 3. The formation of the lipoic acid radical by pulse radiolysis and the rate of its decay are independent of the concentration of dihydrolipoic acid in the solution, which excludes the possibility of an interaction between the radical and its patent compound to form a complex. 4. 4. The lipoic acid radical reduces FAD to form FADHṡ at a rate of k = (2.40 ± 0.18) × 10 8 M −1 s s −1 .

IN A study of the need of turkey poults for unidentified growth factors in natural materials it was necessary to establish that the growth activity of the various natural materials was not caused by any of the nutrients which have been found to be essential for other organisms. Orotic acid is active in promoting the growth of Lactobacillus bulgaricus 09 (Wright et al., 1950). It is widely distributed in natural materials and whey is a particularly rich source. Combs et al. (1954) observed more rapid growth in chicks when orotic acid was added to a practical type diet, containing fish meal. The response was independent of the addition of dried whey or methionine. Fritz et al. (1954) reported approximately 5% increased weight in chicks fed orotic acid which was equal to responses obtained with dried skim milk, dried whey or hydrolyzed whey. Dam et al. (1957) found no response to . . .

SUMMARY Streptococcus faecalis IOCI and 6783 grew anaerobically on glucose in media lacking lipoic acid and acetate, and assays with Streptococcus cremoris KH indicated that extracts of S. faecalis IOCI contained lipoic acid activity equivalent to 200 ng of a-D,L-lipoic acid/mg protein. The Yslllcose value for a continuous culture of strain IOCI grown anaerobically in lipoic acid-deficient medium was 37.2, about the same as that for IOCI in lipoic acid-sufficient medium (35.5). Strains IOCI and 6783 metabolized low concentrations of glucose (I to 8 pmol/ml) non-homolactically and produced per mol of glucose about 1.0 mol lactate, 1-2 mol acetate, and 0.4 mol ethanol. Appreciable amounts of pyruvate, formate, glycerol, acetoin, diacetyl, and 2,3-butylene glycol were not detected. The Yglucoee values for strain IOCI were lower (25, 17 and 21, respectively) if IO-~ M-arsenite or 0.5 % acetate was added or the pH value in the fermentor was decreased to 5.3. With acetate added or the pH value reduced, the glucose was metabolized mostly to lactate. Results indicate that S. Jizecalis IOCI used the pyruvate-dehydrogenase-enzyme complex rather than the phosphoroclastic mechanism in metabolizing anaerobically to acetate approximately half of the pyruvate produced from low concentrations of glucose.

Thioctic acid ( α -lipoic acid) was isolated from milk and found to be associated with the fat globule membrane. The isolation procedure was as follows: Sweet cream buttermilk was hydrolyzed by refluxing for 3 hr with 6.0 n HCl. A benzene extract of the hydrolysate was extracted with 5% NaHCO 3 which, in turn, was acidified to pH less than 1 with 6.0 n HCl. The acidified solution was extracted with ethyl ether, and the extract evaporated to dryness and stored under nitrogen −10C. Identification of thioctic acid was accomplished by UV absorption spectra, thin-layer and gasliquid chromatography techniques.

1. 1. Administration of α-lipoic acid to guinea pigs on a scorbutogenic diet appeared to afford about the same protection as a suboptimal amount of ascorbic acid. When small amounts of ascorbic acid and of α-lipoic acid were given, the combination protected the guinea pigs better than either compound alone. 2. 2. Male and female rats reared since weaning on a diet low in vitamin E did not reproduce. Administration of α-lipoic acid brought about reproduction although not to the same extent as that attained by an optimal amount of α-tocopherol. The offspring, maintained on the α-lipoic acid-supplemented, vitamin E-low diet, reproduced and weaned successfully a third generation. 3. 3. The efficacy of α-lipoic acid in alleviating symptoms of ascorbic acid and of tocopherol deficiencies can best be explained on the basis of a protective action exerted by α-lipoic acid or its dihydro derivative on the two vitamins.

Various compounds, either alone or with pimelic acid, have been studied with respect to their influence on the biosynthesis of biotin by Aspergillus niger growing in synthetic medium. Biotin and/or biotin vitamers synthesized were determined by microbiological assay with Neurospora crassa. Of the more than forty new compounds screened, only lipoic acid and some lipoic acid derivatives showed any effect. Lipoic acid and derivatives did not stimulate biotin biosynthesis in the absence of pimelic acid, but, when added to the synthetic medium in conjunction with pimelic acid, a three- to fivefold enhancement of the pimelic acid stimulation of biotin biosynthesis occurred. 8-Methyl lipoic acid, as well as 5-lipoic acid, antimetabolites of lipoic acid in pyruvate oxidation, were equally active. Growth of A. niger on medium containing lipoic acid- 35 S and pimelic acid did not yield radioactive biotin. The possibility that lipoic acid is stimulatory because of promoting sulfate reduction is suggested.

During the L-malate production through CO2-fixing process by Schizophyllum commune, a blue coloration was seldom seen. When the coloration occurred within 6 days after inoculation, the malate production was markedly reduced, and at the same time the sugar consumption was somewhate lowered. The more earlier the coloration occurred, the more marked was the decrease of L-malate produciton. The addition of thiamine, lipoic acid and pantothenic acid caused the stimulation of sugar utilization and the inhibition of malate production, but the blue coloration caused the lowering of the former and the enhancing of the latter. The inhibitory effect of Cu2+ on the malate production was not affected by the blue pigmentation.The addition of an ethanolic extract of the blue mycelia caused marked decrease of malate production. When the ethanolic extract was converted to a brown red substance during evaporation of ethanol, such effect of addition was not observed.The blue pigment may promote the reverse reaction of phosphoenolpyruvate carboxylation.When mucilage was produced in the medium, an increase of L-malate production was observed.

A method for the histochemical localization of dihydrolipoic dehydrogenase has been developed, using diphospyridine nucleotide (DPN) as a coenzyme and Nitro-blue tetrazolium (Nitro-BT) as the final electron acceptor. With this technique the enzyme, which plays an important role in the synthesis of coenzyme-A linked acyls, was found to be widespread in the tissues of the rat, and its distribution in various organs is presented. The problems involved in the histochemical demonstration of dihydrolipoic dehydrogenase are discussed.

The growth of Streptococcus faecalis is inhibited by propionate, and the inhibition is reversed by lipoic acid or acetate. A study of the role of pyruvate oxidation in S. faecalis showed that propionate inhibited the lipoic acid-dependent aerobic oxidation of pyruvate in resting cells. Pyruvate dehydrogenation with neotetrazolium as a hydrogen acceptor in cell-free extracts also required lipoic acid and was markedly inhibited by propionyl phosphate as well as sodium propionate. Some lipid substances, such as palmitate, oleate, behenate, and lecithin, had a lipoic acid-replacing effect on growth of the organism. Biotin or bicarbonate promoted the lipoic acid-dependent growth. Acetate- 2 - 14 C added to the medium was mainly incorporated into the lipid fraction of the cells. Evolution of 14 CO 2 from pyruvate- 2 - 14 C was not observed in resting cells of the organism, even under aerobic conditions. From the above findings, it is concluded that lipid synthesis through pyruvate oxidation plays a very important role in bacterial growth in medium containing propionate.

A study of the effect of Thiopentone sodium on pyruvate oxidation in brain tissue revealed that the action of this drug could be attributed primarily to an inhibition of pyruvic dehydrogenase, the enzyme which degrades pyruvate to the level of acetate. Of the four cofactors needed by the enzyme, thiamine pyrophosphate (TPP) and lipoic acid (Lp) modified the inhibition when added separately. The inhibition was completely relieved when thiamine pyrophosphate and lipoic acid were added together, suggesting that Thiopentone may be competing not with either alone but with a complex of the two such as "lipothiamide pyrophosphate". Alpha-Ketoglutaric dehydrogenase, which requires the same cofactors as pyruvic dehydrogenase, was somewhat less affected by Thiopentone sodium.

The presence of a lipoic acid activating enzyme has been demonstrated in animal liver and a 50-fold increase in the specific activity of the enzyme was achieved. Some of the general properties of the labile enzyme were determined on the partially purified preparations. The substrate specificity of the enzyme is rather broad and a number of lipoic acid analogues can be converted to the hydroxamate derivative. The enzyme catalyzes a P 32 P-ATP exchange reaction and the cofactor requirements are the same for both the forward and exchange reactions confirming the occurrence of the enzyme in animal liver.

Because the lipoic acid activating systems catalyze reactions which are formally expected for an energy-dependent transport system, an examination of their cellular distribution was made. The pyruvate: lipoate oxidoreductase (acceptor-acetylating) complex (E.C. No. 1.2.4.1) and the lipoic acid activating system in both S. faecalis 10 C1 and E. coli (Crookes strain) were found entirely in the membrane supernatant solution preparations (soluble enzymes). NADH oxidase was located in the solubilized membrane preparation, suggesting that the membrane preparations were representative, and glucose-6-phosphate dehydrogenase was found almost exclusively in the soluble fraction. It appears unlikely from distribution studies that the enzyme(s) of the lipoic acid activating system are involved in the transport of the lipoic acid into the cell.

1. 1. Dihydrolipoic acid readily forms chelates with a number of divalent heavy metals; these differ in stability to acid and other properties. 2. 2. The Co2+-dihydrolipoic acid chelate is oxidized immediately in air with the uptake of 1.5 atoms oxygen per mole chelate. Oxidation is accompanied by an increase in the molecular extinction coefficient from ϵ480mμ = 5.12·103 to ϵ510mμ = 1.45·104 cm2/mmole. Biologically active derivatives of lipoic acid cannot be recovered from the oxidized complex. 3. 3. Comparative studies with model compounds indicate that both sulphydryl groups of dihydrolipoic acid are involved in the chelation of Co2+, and presumably certain other cations. 4. 4. Spectrophotometric measurements, chemical analysis and electrometric titration show that in dilute solution dihydrolipoic acid and Co2+ combine initially in a 2:1 ration, the combination being accompanied by the liberation of one hydrogen ion per mole ligand. 5. 5. Chelates of dihydrolipoic acid with Cu2+ and Ni2+ also undergo oxidation at rates which, whilst similar to one another, are greater than that exhibited by dihydrolipoic acid alone, but considerably less than that of the Co2+ chelate. Oxidation of the Ni2+ chelate is not accompanied by any change in absorption spectrum. 6. 6. Chelates of dihydrolipoic acid with Zn2+ and Cd2+ are stable in air and exhibit no oxygen uptake. These chelates in contrast to those of Co2+ and Cu2+, dissociate in acid solution to yield the dithiol and essentially quantitative recovery of biological activity. 7. 7. It is suggested that the senstivity of the Co2+-dihydrolipoic acid chelate to oxidation may be of significance with regard to the inactivation by Co2+ of biological systems that are dependent upon the reversible interconversion of lipoic acid and dihydrolipoic acid.

Ketoglutarate dehydrogenase has been isolated from pig heart as a soluble complex of enzymes, containing protein bound thiamin pyrophosphate, lipoic acid and FAD in stoichiometric amounts. The flavin constituent of the complex has been shown to be lipoyl dehydrogenase. The complex was resolved into two fractions, one colourless, the other yellow, by fractionation in the presence of 2.5 M urea. The colourless fraction, which contains all of the protein-bound thiamin pyrophosphate and lipoic acid of the complex, was found to catalyse the oxidation of ketoglutarate when ferricyaide was used as electron acceptor. The yellow fraction was shown to be identical with lipoyl dehydrogenase (diaphorase). Neither fraction alone was capable of carrying out DPN+-linked ketoglutarate oxidation, this activity was reconstituted on mixing the two fractions. From chemical analysis, and the stoichiometry of the reconstitution of DPN+-linked ketoglutarate oxidation, the unit molecular weight of the ketoglutarate dehydrogenase complex was calculated to be about 260,000.

During fermentation with Saccharomyces cerevisiae, esters are formed from alcohols and acyl-CoA. The formation of ethyl acetate thus depends on the availability of acetyl-CoA, which is formed partly by activating acetic acid (requiring ATP) and partly by oxidative decarboxylation of pyruvic acid (requiring lipoic acid and thiamine pyrophosphate participation). Formation of ethyl acetate is stimulated by addition of small quantities of DNP (2,4-dinitrophenol), an effect which is enhanced by addition of lipoic acid and counteracted by sodium arsenite (an inhibitor to lipoic acid). These results show that oxidative decarboxylation of pyruvic acid is an important contributive factor to anaerobic formation of acetyl-CoA by Sacch. cerevisiae. Formation of ethyl butyrate from butyric acid is inhibited by DNP in all concentrations; butyrate must first be activated to butyryl-CoA (requiring ATP). Formation of ethyl acetate is inhibited by higher concentrations of DNP, owing to diminished oxidation to acetate in different states, to inhibition of formation of coenzyme A from pantothenic acid (requiring ATP) or to the enzymatic pattern of the cells becoming changed; all these factors may play some part. Formation of esters is stimulated when other reactions requiring acetyl-CoA are inhibited, e.g., the citric acid cycle and carboxylation of acetyl-CoA to malonyl-CoA, which can be achieved by making the medium biotin-free. Malonic acid is also an inhibitor of the citric acid cycle and interacts in lipid synthesis.

Development of Plasmodium circumflexum and P. vaughani in vitro was studied for the first time. P. hexamerium served as the assay organism. The Harvard medium modified by addition of folic acid and B12 was used for controls, and supplemented, singly or in combination, with coenzyme A, ATP, thioctic acid and K-malate (0.01 M; 0.005 M). Success was judged in terms of increase in parasite numbers, staining characteristics, normality of appearance and ability to infect ducklings or canaries. Harvard medium plus coenzyme A, ATP, thioctic acid and K-malate 0.005 M provided good conditions for in vitro growth of P. hexamerium with a growth index of 3.7 after 72 hours incubation. The same combination provided a 2.2 growth index after 48 hours for P. circumflexum. Addition of porcine liver coenzyme concentrate and K-malate 0.005 M produced a 3.5 growth index after 48 hours for P. circumflexum (although individual growth indices as high as 7.9 were obtained). All attempts to obtain in vitro development of Plasmodium vaughani failed, although viability was not affected as shown by relatively short prepatent periods in canaries.

1. Inhibition of pyruvate oxidation in suspensions of Aerobacter aerogenes cells and of isolated mitochondria from rat heart and liver by phenylarsenoxide is prevented by an excess of lipoic acid, whereas inhibition due to certain bivalent cations is not. 2. In both systems inhibition persists when the bacteria and mitochondria are recovered and resuspended in fresh media in the absence of the inhibitor. Persistent inhibition due to preincubation with phenylarsenoxide, but not with the metal ions, is reversed by lipoic acid and by certain other disulphides. 3. 2,3-Dimercaptopropan-1-ol prevents the inhibition of pyruvate oxidation by phenylarsenoxide and by bivalent cations in both mitochondria and bacterial cells. 4. In aerobic suspensions of mitochondria and bacteria disulphides such as lipoic acid are reduced rapidly to dithiols. Reduction is inhibited by Co2+, Ni2+, Cd2+ and Zn2+, but not by phenylarsenoxide. 5. It is concluded that the inability of lipoic acid to prevent the action of the metal ions on pyruvate oxidation is due to the inhibition of its reduction to the effective dithiol.

Solid compounds of α-lipoic acid with Hg2+, and of dihydrolipoic acid and 1,3-dimercaptopropane with Ni2+ and Hg2+ have been prepared. For comparative purposes, qualitative studies have been carried out on the reactions of other metal ions (Zn2+, Cu2+, Co2+ and Pb2+) with these sulfur compounds. With lipoic acid, the product formed readily with Hg2+, but photolytic or thermal cleavage of the disulfide link was necessary before any isolatable products were formed with the other cations. With dimercaptopropane and dihydrolipoic acid, products were formed with all the cations. The importance of these systems is related to the use of lipoic acid in the treatment of heavy metal poisoning.

The growth promoting activity of a little dose of TATD on young small rats and curative effect of the minimum amount of TATD on thiamine deficient rats were examined. And it may well be said that the biological thiamine activity of the minimum amount of TATD is found to be equal to that of equi-molecular amount of thiamine hydrochloride.When a large amount of TATD was given to the rats orally, predominant and continuo us increase of thiamine contents of principal organs and a high urinary excretion ratio were observed. It was assured that absorption rate of TATD from intestine was excellently higher than that of common thiamine hydrochloride.Incubating 35S (lipoic acid labelled)-TATD with heparinized human blood and washing with human plasm, thiamine content and specific radioactivity of blood cells and the rinsings were estimated. As the results, it was supposed that thiamine part of TATD was concentrated and remained in blood cells and lipoic acid derivative part was rapidly reprojected into plasm.When a large amount of 35S-TATD was injected intravenously to the rats, increases of thiamine content and specific radioactivity of several organs were examined. And it was ascertained that TATD devided into thiamine part and lipoic acid derivative part promptly, and thiamine part had a great affinity for liver, kidney and heart, and lipoic acid derivative part had it for liver and kidney, and both parts acted separately and characteristically in the organism, and lipoic acid derivative part was excreted more rapidly than the other part into the urine.

THIOCTIC acid (6,8-dithiooctanoic acid, lipoic acid, protogen) is known to be an important growth factor for protozoa and bacteria, as well as a component of certain enzyme systems in microorganisms and animal tissues (see reviews by Gunsalus, 1954; and Reed and DeBusk, 1954). Despite little evidence for its growth promoting activity in higher animals, thioctic acid has been called a vitamin (Seaman, 1952; Anonymous, 1953; Fischer, 1955) and has been included as a B vitamin in recent reviews on the vitamin B complex (Cheldelin, 1954; Johnson, 1955; Plaut and Betheil, 1956). DeBusk and Williams (1955) reported that thioctic acid added to a purified diet stimulated the growth of White Leghorn chicks and of Wistar rats. However, Stokstad, Broquist and Patterson (1953) had reported earlier that they could not produce a growth response with thioctic acid in chicks or rats fed a purified diet. Recently, Stokstad et al. (1956) and workers . . .

We have observed the interaction of Cd 2+ , Zn 2+ , and AsO 2 2− with a dithiol-substituted polymer ( N -dihydrolipoyl aminoethoxy dextran). Cadmium binding results in an intense uv difference spectrum (λ max = 240 nm). Spectrophotometric titrations with CdCl 2 reveal formation of two Cd-dithiol complexes characterized as 2—SH/Cd 2+ and 3—SH/Cd 2+ . Stability constants were determined by titration of cadmium-saturated polymer with excess EDTA. For the two complexes, K 1 = 2.7 × 10 14 M and K 2 = 7.7 × 10 13 M . NTA is not effective in displacing Cd 2+ . In competition of Cd 2+ and Zn 2+ for dithiol sites, Cd 2+ is bound about 500 times more firmly than Zn 2+ . Arsenite binding is sluggish ( K ∼ 85 M −1 sec −1 ) and yields a single complex (2—SH/AsO 2 2− ). Competition and kinetic data suggest that 10 6 M K AsO 2 − 2 M . We have defined basic criteria for evaluation of enzyme active-site dithiols: (1) the binding order “cadmium stronger than zinc”; (2) relief of Cd 2+ inhibition by 10-fold excess EDTA and no relief by 10-fold excess of NTA; (3) inhibition by arsenite.

Claridge, C. A. (Merck Sharp & Dohme Research Laboratories, Rahway, N.J.)and David Hendlin. Oxidation of glycerol byStreptococcus faecalis. J. Bacteriol.84:1181–1186. 1962.—The nature of the factors in yeast autolysate essential for the oxidation of glycerol byStreptococcus faecalisF24 was examined. Two factors appear to be involved in the oxidation of glycerol. One factor was shown to be an inducer of the enzyme system required for glycerol oxidation; the other was shown to be α-lipoic acid. Minute quantities of glucose will “spark” growth ofS. faecalisin a medium containing glycerol and acetate as carbon sources, probably by supplying sufficient energy for induction of the glycerol-oxidation system.

An energy-requiring, temperature-dependent system capable of concentrating lipoic acid in the cell pool exists in Streptococcus faecalis . This system is easily saturated with lipoic acid and is constitutive. Octanoic acid competes with lipoic acid while the analogues 8-methylthioctic acid, 1,2-dithiolane-3-caproic acid, and 1,2-dithiolane-3-butyric acid are without effect. The cell wall does not influence uptake in any rate-limiting fashion. The system has been demonstrated in Escherichia coli, Staphylococcus aureus , and Aerobacter aerogenes .

1. A reaction of β-lipoic acid with acetic anhydride under mild conditions yielded an unknown product which liberated acetic acid by hydrolysis with NaOH. The product showed almost the same microbiological activity for Streptococcus faecalis 10 C1 and Corynebacterium bovis as α-lipoic acid and β-lipoic acid.2. The results of ultraviolet and infrared spectrophotometry and other tests confirmed the identity of the acetylated product with an acetoxy derivative of α-lipoic acid.3. Deuterium exchange studies revealed the possibility of a proton attracted to the carbon adjacent to the sulfoxide group in β-lipoic acid. A possible steric hindrance caused by the bulky side chain suggests that the acetylated product is 8-C-acetoxy-lipoic acid, the formation mechanism of which is thought to be analogous to that of the Pummerer reaction.4. Associated with the competitive inhibitory effect of 8-C-methyllipoic acid, the results obtained in this experiment would suggest the possible reactivity at carbon 8 of β-lipoic acid which may play an unknown interesting role in biological systems.

Perfusion of the rat heart with lipoic acid inhibited the rise in acetyl-CoA and citrate normally induced by octanoate. With glucose as substrate, lipoic acid reduced cardiac muscle citrate and stimulated phosphofructokinase activity; glucose utilization and glycogenolysis were likewise increased. These effects were observed in normal hearts and in hearts from alloxan diabetic rats. It is suggested that lipoic acid stimulates glycolysis through the lowering of citrate, a known inhibitor of phosphofructokinase.