Experimental isovalthinuria. III. Induction by bile acids, and hypocholesterolemic agents

Some bile acids (dehydrocholic, cholic, chenodeoxycholic, ursodeoxycholic, and deoxycholic acids), and some hypocholesterolemic agents (22, 25 diazacholestanol, 20,25-diazacholesterol, triparanol, and SKF 525-A) are the inducers of isovalthinuria in guinea pig. Administration of methionine appears to increase the pool of sulfur compound which participates in the formation of isovalthine. Cholesterol appears to have no enhancing effect on the induction activity of isovalthinuria inducers. The mechanism of isovalthine formation and the role of sulfur amino acids in lowering blood cholesterol are discussed.</p

Metabolism of L-cysteine in rats fed low and high protein diets.

L-Cysteine (5.0 mmol per kg of body weight) was intraperitoneally injected into rats fed a 25% casein or 5% casein diet. Concentrations of acidic and neutral amino acids in various tissues were determined 2 h later. In the rats fed the 25% casein diet there was a tendency for tissue amino acid and glutathione levels to be slightly lower than controls. In the 5% casein diet group, however, concentrations of tissue amino acids and glutathione generally increased after L-cysteine administration. S-(2-Hydroxy-2-carboxyethylthio)cysteine (HCETC,3-mercaptolactate-cysteine disulfide), though in trace amounts, was detected in kidney and blood plasma in the 5% casein diet group. Increases in cysteine-glutathione disulfide in liver, kidney and erythrocytes in the 5% casein diet group were considerable. These results indicate that L-cysteine was rapidly metabolized in the 25% casein diet group through the oxidative pathway, while in the 5% casein diet group, in which liver cysteine dioxygenase activity is supposed to be quite low, the oxidative metabolism of L-cysteine decreased and part of the L-cysteine was metabolized through the transaminative pathway. Administration of 15.0 mmol L-cysteine per kg of body weight to rats fed the 25% casein diet resulted in an increase in cysteine-glutathione disulfide in liver, kidney and erythrocytes, and the appearance of HCETC in blood plasma.(ABSTRACT TRUNCATED AT 250 WORDS)</p

Determination of Sialic Acids by Acidic Ninhydrin Reaction

A new acidic ninhydrin method for determining free sialic acids is described. The method is based on the reaction of sialic acids with Gaitonde's acid ninhydrin reagent 2 which yields a stable color with an absorption maximum at 470 nm. The standard curve is linear in the range of 5 to 500 nmol of N-acetylneuraminic acid per 0.9 ml of reaction mixture. The reaction was specific only for sialic acids among the various sugars and sugar derivatives examined. Some interference of this method by cysteine, cystine and tryptophan was noted, although their absorption maxima differed from that of sialic acids. The interference by these amino acids was eliminated with the use of a small column of cation-exchange resin. The acidic ninhydrin method provides a simple and rapid method for the determination of free sialic acids in biological materials.</p

Reduction of 3-mercaptopyruvate in rat liver is catalyzed by lactate dehydrogenase.

It has been assumed that the in vivo reduction of 3-mercaptopyruvate, an intermediate of cysteine metabolism, to 3-mercaptolactate is catalyzed by lactate dehydrogenase (EC 1.1.1.27) though no definitive evidence has been presented. In order to examine this assumption, reduction of 3-mercaptopyruvate and its inhibition were studied using rat liver homogenate, lactate dehydrogenase purified from rat liver and anti-lactate dehydrogenase antiserum. Reduction of 3-mercaptopyruvate was actively catalyzed by rat liver homogenate and by the purified lactate dehydrogenase. This reducing activity was completely inhibited by anti-lactate dehydrogenase antiserum. These results indicate that the reduction of 3-mercaptopyruvate to 3-mercaptolactate in rat liver is catalyzed by lactate dehydrogenase.</p

Experimental isovalthinuria IV. Incorporation of S35-Methionine or S35-Cystine into urinary isovalthine

In the course of experimental isovalthinuria induced by cholic acid, S35-methionine or S35-cystine administered was incorporated into urinary isovalthine in guinea pigs. Sulfur atom of cysteine seems to be utilized much better for isovalthine synthesis than that of methionine.</p

Effect of cystathionase on isovalthine

In the course of studies on the cleavage reaction of S-(isopropylcarboxymethyl) glutathione (GSIV) into isovalthine in kidney homogenate or glutathionase preparation, it has sometimes been observed that the amount of isovalthine formed is far less than that of GSIV decomposed&#185;. Furthermore, when such reaction mixture is analyzed on an automatic amino acid analyzer, prominent peak corresponding to the reasonable amount of S-(isopropy1carboxymethyl)cysteinylglycine which is an expected intermediate of the GSIV cleavage reaction cannot be found up to 400 effluent ml. Though several reasons may be considered for the explanation of the above curious phenomenon, the effect of cystathionase on isovalthine is at first examined here. But the result was negative. L- and L-Alloisovalthineused as substrate were prepared by the method of OHMORI&#178;. Homoserine and purified cystathionase in ammonium sulfate solution prepared according to the method of GREENBERGB&#179; were kindly furnished by Prof. M. Suda of Osaka University. Incubation mixture contains 0.1 ml of enzyme solution, 1.0 ml of 0.2 M borate buffer (pH 8.0) containing 2×10-&#179;M cysteine, 0.lml of 0.1 M substrate, and 0.8ml of deionized water containing 5×10-4M EDTA. The mixture was shaken at 37°C for 30 minutes in the air. The reaction was terminated by adding 2ml of 10% trichloroacetic acid and the &#945;-keto acids formed were determined by the method of FRIEDEMANN and HAUGEN4 with a following modification: toluene extract was washed once with 8 ml of 10% sodium sulfate. The results obtained are summarized in Table l. When the reaction mixtures are analyzed before or after incubation on an automatic amino acid analyzer, the amount of L- or L-Alloisovalthine is found to be unchanged. Furthermore, as indicated in Table 1, L-isovalthine showed no inhibitory effect on the homoserine cleavage by cystathionase. Since amino acid oxidases have already been reported to have no effect on isovalthine&#179;, the curious phenomenon above cited may have to be explained by other reaction mechanism such as transpeptipation reaction.</p

Experimental beta-alaninuria induced by (aminooxy)acetate

Experimental beta-alaninuria was induced in rats by injection of (aminooxy)acetate (AOA), a potent inhibitor of aminotransferases, in order to elucidate the pathogenesis of hyper-beta-alaninemia. A 27-fold increase of beta-alanine (BALA) excretion was induced by subcutaneous injection of 1 5 mg of AOA per kg of body weight. A 13-fold and a 9-fold increase of beta-aminoisobutyric acid (BAIBA) and gamma-aminobutyric acid (GABA), respectively, were also induced simultaneously by the AOA injection. Identification of BALA and BAIBA isolated from the rat urine was performed by chromatographic and mass spectrometric analyses. The effects of AOA injection on the tissue levels of these amino acids were also studied. Contents of BALA in the liver and kidney and GABA in the brain increased significantly in response to AOA injection. The present study indicates that BALA transaminase is involved in hyper-beta-alaninemia.</p

Tissue contents and urinary excretion of taurine after administration of L-cysteine and L-2-oxothiazolidine-4-carboxylate to rats.

&lt;p&gt;Tissue contents and urinary excretion of taurine were studied in rats after the administration of L-cysteine and its derivatives. Average taurine content in the liver of rats fed a 25% casein diet for 7 days increased 2-fold 2h after the intraperitoneal administration of 5 mmol of L-cysteine per kg of body weight, whereas that in rats fed a 5% casein diet for 2 days increased only slightly. The difference in the liver taurine contents between these two groups was discussed in relation to cysteine dioxygenase. Taurine contents in the heart, brain and blood did not differ significantly between these two groups or between the control and the group of rats which received L-cysteine. The increase in liver taurine concentrations after L-cysteine administration was much higher than that after L-cystine administration, suggesting a difference in their absorption. The intraperitoneal administration of 5 mmol/kg of L-2-oxothiazolidine-4-carboxylate (OTCA) resulted in a 3-fold increase in liver taurine content. The average increase in taurine excretion in the 24-h urine after OTCA administration corresponded to about 6.0% and that in the next 24-h urine to about 2.6% of OTCA administered, suggesting that nearly 10% of OTCA was metabolized to taurine and excreted in the urine.&lt;/p&gt;</p

Determination of hypotaurine and taurine in blood plasma of rats after the administration of L-cysteine.

A method for the simultaneous determination of hypotaurine and taurine was developed. The method consisted of the elimination of urea, which interfered with the determination of hypotaurine, by immobilized urease, and determination of hypotaurine and taurine with an amino acid analyzer. The analyzer equipped with a cation-exchange column was operated at 32 degrees C with 0.2 M sodium citrate buffer, pH 2.8. Using this method, the dynamics of hypotaurine and taurine in blood plasma of rats was studied after the intraperitoneal injection of L-cysteine. The concentration of cysteine reached the maximum 1 h after L-cysteine loading. The concentration of hypotaurine and taurine increased in parallel and reached the maximum 2 h after L-cysteine loading. These changes seem to indicate the precursor-product relationship of these substances and the rapid conversion of hypotaurine to taurine in vivo.</p

Transaminative metabolism of L-cysteine in guinea pig liver and kidney.

Transaminative metabolism of L-cysteine was investigated using homogenates of guinea pig liver and kidney. L-Cysteine was transaminated in the presence of 2-oxoglutarate and the homogenate of either liver or kidney. S-(2-Hydroxy-2-carboxyethylthio)cysteine (HCETC) (3-mercaptolactate-cysteine disulfide) was formed by liver homogenate, but the amount was very small. On the other hand, a relatively large amount of HCETC was formed in the presence of kidney homogenate. Transamination between 3-mercaptopyruvate and certain amino acids was catalyzed actively by both liver and kidney homogenates in the presence of L-glutamate. However, more half-cysteine was formed by liver than kidney, and more HCETC was produced by kidney than liver. L-Glutamate was the most potent amino donor, and L-aspartate strongly inhibited the reaction. Results indicate that L-cysteine can be transaminated both in liver and kidney of the guinea pig, and that kidney is more active than liver. 2-Oxoglutarate is the most active 2-oxo acid for cysteine transamination. Oxaloacetate (and aspartate in the reverse reaction) is inhibitory to the reaction. These results are in agreement with the previous conclusion that cysteine aminotransferase is identical with aspartate aminotransferase.</p