Formation of meso, N-diphenylprotoporphyrin IX by an aerobic reaction of phenylhydrazine with oxyhemoglobins.

Administration of phenylhydrazine to rabbits resulted in the denaturation of hemoglobins in erythrocytes, causing the formation of intracellular precipitates known as Heinz bodies, severe hemolytic anemia, and reticulocytosis. To elucidate the molecular mechanism of the destabilization, we allowed human oxyhemoglobins to react aerobically with phenylhydrazine. After treatment with acetic acid/HCl and H2SO4/methanol, the chloroform extract contained blue-green pigments of major products accompanied by different minor products. Each product was isolated by column chromatography. By fast-atom-bombardment mass spectrometry (FAB-MS) and proton nuclear magnetic resonance (1H-NMR) spectrometry, dimethyl esters of N-phenylprotoporphyrin IX and meso, N-diphenylprotoporphyrin IX were determined. Other major products also were determined to be dimethyl esters of triphenyl-and tetraphenyl-substituted protoporphyrins by FAB-MS. The formation of meso, N-diphenylprotoporphyrin indicated that the addition of a phenyl radical to the meso-carbon atom of the protoporphyrin ring occurred. Triphenyl and tetraphenyl adducts also indicated the formation of phenyl radicals in the aerobic reaction of phenylhydrazine with oxyhemoglobins. From these results, we suggest that the formation of phenyl radicals and the replacement of heme with phenyl-substituted protoporphyrins cause the destabilization of hemoglobins to induce Heinz bodies and hemolytic anemia with phenylhydrazine.</p

Gas Chromatographic Determination of Sulfuric Acid and Application to Urinary Sulfate

A new gas chromatographic method for the determination of sulfate was developed. In this method, sulfate was quantitatively converted to a volatile derivative, dimethyl sulfate, by a two-step procedure. First, sulfate was converted to silver sulfate by reaction with silver oxide, and then to dimethyl sulfate by reaction with methyl iodide. The derivative was analyzed by gas chromatography. Methyl methanesulfonate was used as an internal standard. The method was applied to the determination of total urinary sulfate. Phosphate and chloride ions, which interfered with the present method, were eliminated with the use of basic magnesium carbonate and an excess of silver oxide, respectively. Recovery was over 96% when 5 to 40 mumol/ml of sulfate was added to human urine samples.</p

Visualization of sialoglycoproteins in polyacrylamide gels by acidic ninhydrin reaction.

A new method for staining sialoglycoproteins in polyacrylamide gel after disc electrophoresis is described. The method utilizes the reaction of sialic acids with an acidic ninhydrin reagent which yields a stable color with an absorption maximum at 470 nm. After electrophoresis, the polyacrylamide gel is placed in a test tube and heated with 5 ml of the acidic ninhydrin reagent for 10 min in a boiling water bath. Sialoglycoproteins are detected as brown bands. No additional procedure such as destaining is necessary. When 20 micrograms fetuin, a sialoglycoprotein, per gel is applied, the band remains visible for at least 2 h. Stained gel can be scanned with a gel scanner at 470 nm. When the stained gel was dried on a sheet of polypropylene filter, the color was stable for at least one month. The present method is superior to the method using Stains-all (3,3'-diethyl-9-methyl-4,5,4',5'-dibenzothiacarbocyanine) in specificity and simplicity for the detection of sialoglycoproteins.</p

Formation of Sulfate from L-Cysteine in Rat Liver Mitochondria

Formation of sulfate in rat liver mitochondria was studied. About 0.1 mumol of sulfate was formed in mitochondria from 1 g of liver in 60 min when 10 mM L-cysteine was used as the substrate. Addition of either 10 mM 2-oxoglutarate or 10 mM glutathione to this system increased sulfate formation 3 to 4 times. The addition of both 2-oxoglutarate and glutathione resulted in a 20-fold increase in sulfate formation. Sulfate formation in the presence of 5 mM L-cysteine was 58% of that with 10 mM L-cysteine. L-Cysteine-glutathione mixed disulfide was not a good substrate, indicating that this mixed disulfide was not an intermediate of sulfate formation in the present system. Incubation of 3-mercaptopyruvate with rat liver mitochondria also resulted in sulfate formation, and the addition of glutathione accelerated it. Formation of sulfite and thiosulfate was also detected. These results indicate that sulfate is produced in mitochondria, at least in part, from L-cysteine through the transamination pathway (3-mercaptopyruvate pathway).</p

A method for determination of total glutathione and total cysteine as S-carboxymethyl derivatives by using an amino acid analyzer, and its application to samples from rat liver, kidney and blood after intraperitoneal administration of 2-(4-carboxy-D-gluco-tetrahydroxybutyl)thiazolidine-4-carboxylic acid.

The effects of intraperitoneal administration of 2-(4-carboxy-D-gluco-tetrahydroxybutyl)thiazolidine-4-carboxylic acid (CGUA), a cysteine derivative conjugated with glucuronic acid, on total glutathione and total cysteine contents in rat tissues were investigated. Total glutathione (GSH and GSSG) and total cysteine (cysteine and cystine) were determined by a new method consisting of preparation of S-carboxymethylglutathione (CMSG) and S-carboxymethylcysteine (CMC), respectively, and subsequent analyses with an amino acid analyzer. CGUA was determined by a coloration method employing an acidic ninhydrin reagent. Total cysteine contents in liver, kidney and plasma rapidly increased to 2.3, 2.7 and 6.5 times the levels of the controls, respectively, after CGUA administration at a dose of 5 mmol/kg of body weight. Total glutathione content did not change significantly in the liver or blood except for the kidney with a significant increase during the first 1-h period after administration. CGUA content increased markedly in these tissues, especially in the kidney, and 30% of administered CGUA was excreted in urine within 2h. These results indicate that CGUA is converted into cysteine in vivo, suggesting the usefulness of this compound for protection of the kidney and the liver.</p

Allelic Variation Investigation of the Estrogen Receptor Within an Australian Multiple Sclerosis Population

Multiple Sclerosis (MS) is a central nervous system (CNS) chronic inflammatory demyelinating disease leading to various neurological disabilities. The disorder is more prevalent for women with a ratio of 3:2 female to male. Objectives: To investigate variation within the estrogen receptor 1 (ESR1) polymorphism gene in an Australian MS case-control population using two intragenic restriction fragment length polymorphisms; the G594A located in exon 8 detected with the BtgI restriction enzyme and T938C located in intron 1, detected with PvuII. One hundred and ten Australian MS patients were studied, with patients classified clinically as Relapsing Remitting MS (RR-MS), Secondary Progressive MS (SP-MS) or Primary Progressive MS (PP-MS). Also, 110 age, sex and ethnicity matched controls were investigated as a comparative group. No significant difference in the allelic distribution frequency was found between the case and control groups for the ESR1 PvuII (P = 0.50) and Btg1 (P = 0.45) marker. Our results do not support a role for these two ESR1 markers in multiple sclerosis susceptibility, however other markers within ESR1 should not be excluded for potential involvement in the disorder

Variation in The Vitamin D Receptor Gene is Associated With Multiple Sclerosis in an Australian Population

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) resulting in accumulating neurological disability. The disorder is more prevalent at higher latitudes. To investigate VDR gene variation using three intragenic restriction fragment length polymorphisms (Apa I, Taq I and Fok I) in an Australian MS case-control population, one hundred and four Australian MS patients were studied with patients classified clinically as Relapsing Remitting MS (RR-MS), Secondary Progressive MS (SP-MS) or Primary Progressive MS (PP-MS). Also, 104 age-, sex-, and ethnicity-matched controls were investigated as a comparative group. Our results show a significant difference of genotype distribution frequency between the case and control groups for the functional exon 9 VDR marker Taq I (p_Gen = 0.016) and interestingly, a stronger difference for the allelic frequency (p_All = 0.0072). The Apa I alleles were also found to be associated with MS (p_All = 0.04) but genotype frequencies were not significantly different from controls (p_Gen = 0.1). The Taq and Apa variants are in very strong and significant linkage disequilibrium (D' = 0.96, P < 0.0001). The genotypic associations are strongest for the progressive forms of MS (SP-MS and PP-MS). Our results support a role for the VDR gene increasing