Nicotinamide coenzyme content in the normal and regenerating liver of rats

The content of NADH and NADPH was measured in the intact and regenerating rat liver. In the intact rat liver, the content of NAD+, NADH, NADP+ and NADPH was 235 +/- 6.4, 66.6 +/- 4.3, 73.3 +/- 2.5 and 148.0 +/- 4.6 micrograms/g crude liver weight, respectively. Seasonal alterations in the rat liver content of coenzymes were established. No changes were found in the content of nicotinamide coenzymes in the regenerating liver 4 and 18 h after operation. Twenty-four hours after operation, a 25.6% increase in the content of NAD+ and a 57.8% reduction in the NADH content were recorded in the liver of hepatectomized animals. At the same time the total content of NAD+ plus NADH changed but insignificantly (14.7%). The total content of NADP+ plus NADPH dropped by 29.8% (within the above period). Thirty-two hours after operation the content of all the nicotinamide coenzymes returned to the initial level.     

Effect of tuftsin, a neuropeptide and its derivatives on the content of nicotinamide coenzymes and the activity of cytochrome- c-oxidase of brain tissue

Tuftsin, a neuropeptide, and its derivatives are studied for their influence on the content of oxidized and reduced forms of nicotinamide enzymes as well as on the activity of cytochrome-c-oxidase of the cortex and limbic system. Peptides under study are shown to increase the content of nicotinamide coenzymes, mainly due to the oxidized forms of NAD and enhance the activity of cytochrome-c-oxidase as well mainly in the emotiogenic brain structures. Under these conditions tuftsin and its derivatives intensify cellular respiration of the neurons.     

Effect of dipyroxime on the concentration of nicotinamide coenzymes and adenylate nucleotides in the myocardium and liver of rats poisoned with phthalophos]

Experiments were conducted on rats; in depression of blood cholinesterase activity by 68.6 percent phthalafos proved to decrease the myocardial nicotinamide coenzymes content on account of reduction in the amount of the oxidized forms. In the liver phthalafos diminished the content of oxidized and reduced forms of nicotinamide coenzymes, decreased the level of adenylic nucleotides chiefly at the expense of ATP. Diproxim prevented the changes caused by phthalafos in blood cholinesterase reactivation to 47.5 percent. It is supposed that the capacity of diproxim to normalize the oxidative processes in the cell by acting upon the nicotinamide coenzymes and adenylic nucleotides underlies its antidote action.     

Formation of taurine and amino acid pools in rat tissues upon stimulation of NAD synthesis

A single intraperitoneal injection of nicotinamide (500 mg/kg) to mongrel albino rats causes a 6-hour increase in the 2-oxoglutarate level and the free NAD+/NADH ratio in liver mitochondria. The levels of taurine and taurocholates as well the activity of cysteine oxidase in liver tissues remains thereby unchanged, whereas the cysteine transaminase activity diminishes. In the heart and brain of experimental animals the activity of both enzymes is decreased. In the liver, blood plasma and heart of experimental animals, the Ala and Ser levels are low, whereas the taurine content is elevated both in blood plasma and brain. Nicotinamide administration eliminates positive correlations between the levels of taurine, its precursors and metabolically bound amino acids. In the liver the negative correlations between the activities of cysteine oxidase and cysteine transaminase observed in the control group disappear in the experimental group. Apparently, one of regulatory mechanisms of the taurine pool formation in the liver is the ratio of activities of the both enzymes as well as their competition at the substrate level. This emphasizes the importance of the transamination reactions in the metabolism of sulphur-containing amino acids.     

Tryptophan metabolism through the kynurenine pathway in rat brain and liver slices

We hypothesized that hyperbaric oxygen (HBO) enhances tryptophan (TRP) flux through the kynurenine (KYN) pathway because oxygen is a substrate for four pathway enzymes. Our objective was to compare the biosynthesis of KYN pathway intermediates by rat brain and liver slices with air or HBO as the gas phase. One-millimeter thick liver and brain slices were obtained from male Sprague-Dawley rats and incubated individually in chambers containing Hanks'-HEPES- buffer with (3)H-TRP (30 Ci/mmol) for 2 h (37 degrees C) in either room air or oxygen (1.2 or 5.2 atmospheres absolute [ATA] oxygen). After incubation, tissue was snap-frozen and analyzed for protein content while medium was extracted for high-performance liquid chromatography analysis. Radiolabeled nicotinamide adenine dinucleotide (NAD) was produced by brain and liver; liver (with air as the gas phase) also produced quinolinic acid (QA). HBO at 1.2 and 5.2 ATA caused increased QA and NAD from liver slices. HBO did not affect KYN metabolism in brain slices, although there was decreased production of NAD during high oxygen. We conclude that rat brain and liver contain the complete KYN pathway and that HBO enhances KYN flux in liver tissue.     

Histotopography of changes in the enzyme activity of rat brain exposed to fluorophenazine]

The influence of fluorophenazine on succinic-, citric acid-, NAD-H2-and NADP-H2-dehydrogenases in different structures of rat brain was investigated by histochemical methods.Three hours after a single subcutaneous injection of fluorophenazine (1 and 5 mg/kg) the enzymatic activity was the greatest in the limbic and the frontal regions of the cortex. The inhibitory effects of fluorophenazine in these structures were greater than those of the neuroleptics trifluoroperazine, chlorpromazine.     

Postnatal development of the actual and total activity of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues

Actual and total activities of the branched-chain 2-oxo acid dehydrogenase complex were determined in homogenates of quadriceps muscle, heart, liver, kidney and brain from rats of 0-70 days age. All rat tissues except quadriceps muscle showed a marked increase of total activity between 0 and 21 days, heart and kidney also after weaning. The actual activity rose after birth in liver, kidney and brain and after weaning in liver, kidney and heart. The activity state was always about 100% in liver and varied between 40-60% in kidney and brain, 10-23% in heart and 6-12% in quadriceps muscle. The actual activities measured indicate, that the degradation of branched-chain 2-oxo acids mainly takes place in the liver of the newborn, suckling and young-adult rat.     

Purification and subunit structure of phosphoglycerate dehydrogenase from rabbit liver.

The nicotinamide nucleotide dimers (NAD)2 and (NADP)2, obtained by electrochemical reduction of NAD+ and NADP+, are able to reduce such single-electron acceptors as the proteins cytochrome c, azurin and methaemoglobin, though at different rates. Under the same conditions the reduced nicotinamide coenzymes NADH and NADPH are not able to reduce these proteins at measurable rates unless a catalyst (phenazine methosulphate or NADH-cytochrome c reductase in the case of cytochrome) is present. The redox mechanism seems to involve the formation of an NAD(P). radical that in the presence of O2 gives rise to superoxide (O2.-), since superoxide dismutase inhibited these reactions.     

Properties and Structure of a Novel Peptide Antibiotic No. 1907

There are three NAD biosynthetic pathways: the nicotinic acid-NAD, nicotinamide-NAD, and quinolinic acid (derived from tryptophan)-NAD pathways. To discover the main pathways of NAD biosyntheses in various tissues of the rat, the tissue distribution of nicotinamidase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase, nicotinamide phosphoribosyl-transferase, nicotinamide mononucleotide adenylyltransferase, and NAD⁺ synthetase were investigated. All of the tissues could synthesize NAD from nicotinamide, judging from that the activities of nicotinamide phosphoribosyltransferase and NMN adenylyltransferase detected in all of the tissues. From nicotinic acid, only liver, kidneys, and heart could. Liver and kidney can also synthesize NAD de novo from quinolinic acid.     

Nicotinamide nucleotide synthesis in regenerating rat liver

1. The concentrations and total content of the nicotinamide nucleotides were measured in the livers of rats at various times after partial hepatectomy and laparotomy (sham hepatectomy) and correlated with other events in the regeneration process. 2. The NAD content and concentration in rat liver were relatively unaffected by laparotomy, but fell to a minimum, 25 and 33% below control values respectively, 24h after partial hepatectomy. NADP content and concentration were affected similarly by both laparotomy and partial hepatectomy, falling rapidly and remaining depressed for up to 48h. 3. The effect of injecting various doses of nicotinamide on the liver DNA and NAD 18h after partial hepatectomy was studied and revealed an inverse correlation between NAD content and DNA content. 4. Injections of nicotinamide at various times after partial hepatectomy revealed that the ability to synthesize NAD from nicotinamide was impaired during the first 12h, rose to a peak at 26h and fell again by 48h after partial hepatectomy. 5. The total liver activity of NAD pyrophosphorylase (EC 2.7.7.1) remained at or slightly above the initial value for 12h after partial hepatectomy and then rose continuously until 48h after operation. The activity of NMN pyrophosphorylase (EC 2.4.2.12) showed a similar pattern of change after partial hepatectomy, but was at no time greater than 5% of the activity of NAD pyrophosphorylase. 6. The results are discussed with reference to the control of NAD synthesis in rapidly dividing tissue. It is suggested that the availability of cofactors and substrates for NAD synthesis is more important as a controlling factor than the maximum enzyme activities. It is concluded that the low concentrations of nicotinamide nucleotides in rapidly dividing tissues are the result of competition between NAD synthesis and nucleic acid synthesis for common precursor and cofactors.     

The distribution of nicotinamide nucleotides during the life cycle of the blowfly Lucilia cuprina Wied

1. Variations in the amounts and distribution of the nicotinamide coenzymes during the life cycle of the blowfly Lucilia cuprina have been investigated. 2. The concentrations of NAD and NADP declined to a minimum in mid-pupal life, then increased threefold from this to about 600 and 40mmumoles/g. fresh wt. respectively in the adult. 3. The NADH/NAD ratio was relatively constant (0.15) except in mid-pupal life (0.09); the NADPH/NADP ratio was lower (0.6) in larvae and mated adults than at other stages (0.90). 4. The increase in NAD content during development was greatest in the thorax, which finally contained about 75% of the total coenzyme. 5. Incorporation of NAD into the thoracic sarcosomes began about 2 days before ecdysis. Initially, most (77%) of the coenzyme was in particles 0.2-1mu in diameter, at a concentration of 11.1mmumoles/mg. of protein. During development there was a gradual appearance of NAD in larger particles (1-10mu) containing 2.8mmumoles/mg. of protein. 6. Similar but less marked changes occurred with NADP, which had a final concentration in the large particles of 0.01mmumole/mg. of protein.     

Effect of apressin, obsidan, diprazine and their combination on the concentration of NAD and NADH2 in the liver and brain of hypoxic rats]

Apressin (2.5 mg/kg), obsidan (10 mg/kg), and diprazine (10mg/kg) caused an increase in the content of NAD + NAD.H2, without affecting their ratio, in the liver and brain of intact animals. These drugs, taken in the same doses, especially when used together, caused an increase in the NAD + NAD.H2 level; as to NAD/NAD.H2 ratio--it decreased in the state of hypoxia. The authors believe the antihypoxic action of apressin, obsidan, and diprazine to be connected with the rise in the total nicotinamide adenine denucleotide content and with increase of its oxidized form.     

NAD metabolism and induction of tyrosine aminotransferase in the rat

The relationship between the NAD-metabolism and the induction of the tyrosine aminotransferase was studied. The content of NAD+ + NADH differs markedly from organ to organ. The highest values can be found in the liver. In intact animals tryptophan leads to an increase of NAD in liver and kidney, but not in brain and spleen. Nicotinamide, on the other hand, induces NAD synthesis in all the organs tested. In adrenalectomized animals, however, there is practically no rise of the NAD content after application of tryptophan contrary to the effect of nicotinamide. The enzyme tyrosine aminotransferase can be induced in intact animals by nicotinamide and tryptophan. This effect is much less pronounced in adrenalectomized animals. In adrenalectomized animals the induction of the tyrosine aminotransferase by tryptophan is markedly elevated by caffeine and theophylline. Under these conditions there is a significant increase of the NAD content as well. The tryptophan promoted induction of the tyrosine aminotransferase is influenced by inhibitors of the ADPR-transferase. The data presented give further evidence that the NAD adenoribosylation metabolism is involved in the induction of the tyrosine aminotransferase.     

Regulation of gluconeogenesis during exposure of young rats to hypoxic conditions

1. Two-day-old rats were exposed at constant temperature to atmospheres containing air and nitrogen with the air content varied in steps from 100 to 0%. By using this system of graded hypoxia a comparison was made between rates of gluconeogenesis from lactate, serine and aspartate in the whole animal and the concentrations of several liver metabolites. 2. Gluconeogenesis, expressed as the percentage incorporation of labelled isotope into glucose plus glycogen, proceeds linearly for 30min when the animals are incubated in a normal air atmosphere, but is completely suppressed if the atmosphere is 100% nitrogen. 3. Preincubation of animals for between 5 and 30min under an atmosphere containing 19% air results in the attainment of a new steady state with respect to gluconeogenesis and hepatic concentrations of ATP, ADP, AMP, lactate, pyruvate, beta-hydroxybutyrate and acetoacetate. 4. When lactate (100mumol), aspartate (20mumol) or serine (20mumol) was injected, it was shown that the more severe the hypoxia the greater the depression of gluconeogenesis. Under conditions when gluconeogenesis was markedly inhibited there were no changes in the degree of phosphorylation of hepatic adenine nucleotides, but free [NAD(+)]/[NADH] ratios fell in both cytosol and mitochondrial compartments of the liver cell. 5. Measurements of total liver NAD(+) and NADH showed that the concentrations of these nucleotide coenzymes changed less with anoxia, in comparison with the concentration ratio of free coenzymes. 6. Calculations showed that the difference in NAD(+)-NADH redox potentials between mitochondrial and cytosol compartments increased with the severity of hypoxia. 7. From the constancy of the concentrations of adenine nucleotides it is concluded that liver of hypoxic rats can conserve ATP by lowering the rate of ATP utilization for gluconeogenesis. Gluconeogenesis may be regulated in turn by the changes in mitochondrial and cytosol redox state.     

Study of the role of nicotinamide coenzymes in the regulation of glyceroneogenesis from pyruvate in rat epididymis fat tissue

The paper deals with a regulatory effect of the redox state of nicotinamide coenzymes on glyceroneogenesis in the epididymal fatty tissues involving incorporation of [2-14C] pyruvate into synthetized de novo blood glucose, glycerol and fatty acids of triacyglycerines. Large values of the NAD+/NADH and NADP+/NADPH ratios in cytoplasm and mitochondria promote a high rate of lipogenesis and glucose oxidation processes, which is pronounced in a more intense 14C incorporation into fatty acids than in triacylglycerol glycerols. A decrease in the NAD+/NADH ratio and an increase in the reducing ability of NAD-pairs under fasting intensify glyceroneogenesis in the fatty tissue. The incorporation of [14C] pyruvate into blood glucose in 3.6 times as high, the radioactivity of fatty acids lowers. Nicotinamide administered to animals after fastening inhibits glyceroneogenesis in the fatty tissue, lowering considerably the incorporation of [14C] pyruvate into triacylglycerol glycerol and blood glucose.     

The relationship between the (NAD+)/(NAD-H) ratio and the adenylate system in the cytoplasm of loach oocytes and embryos

The dependence was found between the phosphorylated state of the adenylic system (ATP)/(ADP) (HPO42-) and the ratio (NAD+)/(NAD-H) during the maturation of the loach (Misgurnus fossilis L.) oocytes which is accompanied by the cessation of gluconeogenesis and onset of gycogenolysis. The twofold decrease of the value of phosphorylated state of the adenylic system is correlated with the twofold decrease of the value of the ratio (NAD+)/(NAD-H).     

Features of poly-ADP-ribosylation and chromatin structure under conditions of varying NAD content in the rat liver

It is shown that nicotinamide-induced in vivo stimulation of NAD biosynthesis in the liver nuclei of rats causes a decrease of the DNA sensitivity to treatment with DNAse I under conditions of weak hydrolysis. When rats are given synthetic vitamin PP-deprived food, the NAD level in the liver falls down to 40% and a great number of DNAse I-hypersensitive chromatin sites appear. A 24% decrease in the level of poly-ADP-ribosylation of total histones in comparison with the control has been observed with hypovitaminosis. Under conditions of vitamin PP deficiency nicotinamide administered to animals has increased the 14C NAD incorporation into histones 2 times (as compared with the control). These variations occur primarily due to increase of the label incorporation to histone H1. Fractionation of chromatin by solutions of different ionic strength has confirmed that vitamin PP deficiency and NAD amount decrease in the liver are accompanied by a relative increase of the NAD-pyrophosphorylase and poly-ADP-ribose polymerase activities in the fraction extracted by the low ionic solution.     

Metabolic Changes in Nicotinamide Adenine Dinucleotide in Response to Anthrax Toxin

Bacillus anthracis produces a toxin both in vitro and in vivo which, when injected intravenously into rats, brings about the death of the animals accompanied by gross pulmonary edema. Lung tissue removed prior to death showed, in vitro, a 30% reduction in overall oxidative metabolism (Q(o2)), whereas the nicotinamide adenine dinucleotide (NAD)-independent succinic dehydrogenase remained unaffected. The NAD concentration in the lungs of injected animals was reduced by 50%. Upon addition of NAD, the Q(o2) of lung tissue from injected animals rose to control values. At 45 min after toxin injection, the serum lactate concentration began to rise, showing about a 3.5-fold increase over controls after 75 min. No changes occurred in the pyruvate concentration. These changes may be explained by increased use of the pyruvate for glycolytic energy production with further loss of NAD. Additional experiments with liver, spleen, kidney, and brain tissues showed that the toxin-induced reduction of Q(o2) is an effect specific for lung tissue. Brain tissue showed a significant increase in oxidative metabolism upon the addition of the toxin, whereas the other tissues remained unaffected. It is suggested that a principal effect of the toxin is to inhibit, in lung tissue, the regeneration of NAD in the respiratory chain.     

Nucleic acids in the tissues of white rats with toxic liver damage following protein starvation and parenteral nitrogen nutrition]

The authors studied the content of nucleic acids in the tissues (the skeletal muscle and myocardium, the liver) in albino rats with toxic affection of the liver caused by ccl4, in protein deficiency and parental administration of amino acid mixture of moriamin S-2 and "improved" caseine hydrolysate. Protein deficiency in albino rats with toxic hepatitis was accompanied by a considerable increase of RNA and DNA in the skeletal muscle and the myocardium with a simultaneous reduction of their level in the liver. The RNA/DNA ratio changed. The RNA content in the hepatocytes diminished on account of reduction of the nuclear RNA fraction closely bound with chromatin. Parenteral administration of nitrogen preparations led to normalization of the nucleic acid content in all the tissues under study.     

Combined coenzyme-substrate analogues of various dehydrogenases. Synthesis of (3S)- and (3R)-5-(3-carboxy-3-hydroxypropyl)nicotinamide adenine dinucleotide and their interaction with (S)- and (R)-lactate-specific dehydrogenases.

Two diastereomeric nicotinamide adenine dinucleotide (NAD+) derivatives were synthesized in which the substrates of (S)-and (R)-lactate-specific dehydrogenases are covalently attached via a methylene spacer at position 5 of the nicotinamide ring. The corresponding nicotinamide derivatives were obtained stereospecifically by enzymatic reduction of 5-(2-oxalylethyl)nicotinamide. (3S)-5-(3-Carboxy-3-hydroxypropyl)-NAD+ undergoes and intramolecular hydride transfer in the presence of pig heart lactate dehydrogenase, forming the corresponding coenzyme-substrate analogue composed of pyruvate and NADH. No cross-reaction products resulting from an intermolecular reaction are observed. Two (R)-lactate specific dehydrogenases, however, do not catalyze a similar reaction in either one of the two diastereomers. A possible arrangement of the substrates in the active centers of these enzymes is proposed. 5-Methyl-NAD+ and 5-methyl-NADH are active coenzymes of pig heart lactate dehydrogenase in contrast to reports in the literature. (S)-Lactate binds to this enzyme in the absence of coenzyme, exhibiting a dissociation constant of 11 mM.