[Impact of small molecules on intermolecular interactions underlying the ligand technologies in laboratory diagnosis].

Hydrogen peroxide has been found to have a distorting effect on the quality of determination of the serological markers of hepatitis B and C, transglutaminase antibodies: an increase in the percent of false higher (anti-HBsAg, HBeAg, anti-HCV) and false lower (anti-HBeAg) values, and on the results of PCR-based diagnosis (PCR inhibition that was more pronounced especially in low viremia). A possibility of interference of measurement results in the blood metabolite pool should be taken into account in the use of high-technology methods of laboratory analysis. In particular, there may be changes in the detection of immunological and molecular biological methods in hyperpyruvatemia and hyperoxaloacetatemia, with elevated peroxide concentrations during pathological processes.

Cadmium affects the expression of heat shock protein 90 and metallothionein mRNA in the Pacific oyster, Crassostrea gigas.

Cadmium (Cd) is a widespread nonessential heavy metal that enters the aquatic environment as a result of natural processes and human activities such as wastewater production, agriculture, and mining. To determine the effects of Cd on organisms, we investigated its time- and dose-related effects on mRNA levels of heat shock protein 90 (HSP90) and metallothionein (MT) in the gill and digestive gland and changes enzyme levels in the hemolymph of the Pacific oyster Crassostrea gigas. Full-length HSP90 cDNA was isolated from C. gigas by rapid amplification of cDNA end (RACE) techniques and found to contain 2154 nucleotides, including an open reading frame, and was predicted to encode a protein of 717 amino acids. BLAST analysis indicated that the HSP90 gene of C. gigas shared high homology with known HSP90 genes of other mollusks. The expression of HSP90 mRNA increased significantly with exposure to 0.01 ppm Cd for 11 days or 0.05 or 0.1 ppm Cd for 7 days. The expression of MT mRNA increased significantly with exposure to 0.01, 0.05, or 0.1 ppm Cd for 11 days. Glutamate oxaloacetate and glutamate pyruvate levels increased significantly with exposure to 0.05 or 0.1 ppm Cd for 7 days. These results indicate that HSP90 and MT play important roles in the physiological changes related to metabolism and cell protection that occur in Pacific oysters exposed to Cd.

Assay of blood and tissue oxaloacetate and alpha-ketoglutarate by isotope dilution gas chromatography-mass spectrometry.

The assay of oxaloacetate and alpha-ketoglutarate in biological samples is complicated by their chemical instability and low concentrations. We present a quantitative assay for physiological concentrations of these metabolites by isotope dilution gas chromatography-mass spectrometry. Samples are spiked with the corresponding internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate prior to their treatment with hydroxylamine. After ethyl acetate extraction and evaporation of the organic phases, the oximes are converted to t-butyldimethylsilyl ethers and analyzed by selected ion monitoring gas chromatography-mass spectrometry of the [M-57]+ ion in electron impact. Although the internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate are not commercially available, they can easily be synthesized in 30 min by reacting [1,2,3,6-13C4]citrate with citrate lyase, and L-[U-13C5]glutamate with pyruvate and glutamate-pyruvate transaminase, respectively. Because of their chemical instability, the internal standards are prepared on the day of the analysis. A stock solution of [1,2,3,6-13C4]citrate is prepared from L-[U-13C4]aspartate using citrate synthase and glutamate-oxaloacetate transaminase and then purified and kept frozen until required. The detection limit of the method is 0.05 nmol in a given sample. The method was applied to measurements of oxaloacetate and alpha-ketoglutarate in human blood and rat liver.

Tricarboxylic acid cycle intermediates in chronic renal failure.

In 21 uraemic subjects not yet requiring dialysis, the serum values of citrate, fumarate, oxalacetate and malate were significantly increased, so that the sum of the concentrations of the TCA-cycle intermediates were increased threefold compared to that of 18 control subjects. The replenishment of TCA intermediates could, in theory, occur by several anaplerotic reactions, mainly those catalysed by pyruvate-carboxylase and malic enzyme. The overall pattern detected in uraemic patients is similar to that observed in rat skeletal muscle during exercise, in which increased acetyl CoA has been found.

[Content of biological oxidation metabolites in the blood and urine of peptic ulcer patients]. / Soderzhanie nekotorykh metabolitov biologicheskogo okisleniia v krovi i moche bol'nykh iazvennoi bolezn'iu.

Content of pyruvic, lactic, citric, acetic, oxaloacetic acids in blood as well as excretion of pyruvic acid with urine were studied in patients with ulcer, chronic gastritis, carcinoma of the stomach and in healthy persons. Concentrations of pyruvic and lactic acids in blood as well as excretion of pyruvic acid with urine were increased in the patients during acute periods of ulcerous disease, chronic gastritis and in carcinoma of the stomach. Level of citric acid was decreased in blood in ulcerous disease and increased in chronic gastritis and, especially, in the carcinoma. During acute period of ulcerous disease concentration of acetic acid was decreased in blood. Content of oxaloacetic acid was similar both in healthy persons and in patients with the disease. The data obtained suggest that oxidative decarboxylation of pyruvic acid is inhibited in patients with ulcerous disease.

Chronic ketosis and cerebral metabolism.

The effects of chronic ketosis on cerebral metabolism were determined in adult rats maintained on a high-fat diet for approximately three weeks and compared to a control group of animals. The fat-fed rats had statistically significantly lower blood glucose concentrations and higher blood beta-hydroxybutyrate and acetoacetate concentrations; higher brain concentrations of bound glucose, glucose 6-phosphate, pyruvate, lactate, beta-hydroxybutyrate, citrate, alpha-ketoglutarate, alanine, and adenosine triphosphate (ATP); lower brain concentrations of fructose 1,6-diphosphate, aspartate, adenosine diphosphate (ADP), creatine, cyclic nucleotides, succinyl coenzyme A (CoA), acid-insoluble CoA, and total CoA; and similar brain concentrations of glucose, malate, calculated oxaloacetate, glutamate, glutamine, adenosine monophosphate, phosphocreatine, reduced CoA, acetyl CoA, sodium, potassium, chloride, and water content. The metabolite data in the chronically ketotic rats demonstrate an increase in the cerebral energy reserve and energy charge. These data also suggest negative modification of the enzymes phosphofructokinase, pyruvic dehydrogenase, and alpha-ketoglutaric dehydrogenase; positive modification of glycogen synthase; and possible augmentation of the hexose transport system. There was no demonstrable difference in brain pH, water content, or electrolytes in the two groups of animals. We speculate that the increased brain ATP/ADP ratio is central to most, if not all, the observed metabolic perturbations and may account for the increased neuronal stability that accompanies chronic ketosis.

Cytochemical identification of oxalosuccinic acid in human leukocytes.

The anthraquinone dye alizarine red S produced a unique red crystalline precipitate with oxalosuccinic acid. By chemical, colorometric and spectrophotometric analysis, this red precipitate was found to be a salt composed of these two substances, and to be insoluble in hydrochloric acid above pH = 3.2. Other biologic substances producing a red color without the formation of precipitates showed a disappearance of the red color and resumption of the yellow color of alizarine red S above pH = 3.8. On the basis of these observations, a cytochemical test was developed that may identify the site of oxalosuccinic acid in the cytoplasm of peripheral blood leukocytes. Because of the initimate association of oxalosuccinic acid with isocitric dehydrogenase, the test may also indicate the site of this enzyme as well.