Biomedical subjects
Publications and source records attributed to F Heinz.
A method for the determination of glucose is described. H2O2, produced by the action of glucose oxidase, is measured from the change in absorbance due to oxidation of NAD(P)H in the presence of catalase, aldehyde dehydrogenase and a high concentration of ethanol. The quality data of the method are equivalent to those of the hexokinase-glucose-6-phosphate dehydrogenase method used as reference.
We prepared highly purified acetylcholine receptor (AChR)-specific T lymphocytes from rats with experimental autoimmune myasthenia gravis (EAMG). Inbred rats were primed with AChR frm 3 different sources: from the electric organs of Electrophorus electricus and Torpedo californica and from denervated rat muscle. After 20 to 30 days, lymphocytes from regional lymph nodes (primary cells) were challenged with soluble AChR in vitro. The activated blast cells were isolated by density gradient centrifugation and allowed to revert back to small secondary lymphocytes in the absence of antigen. These secondary anti-AChR cells were highly responsive to the type of AChR with which they had been primed. Their reactivity critically depended on help by syngeneic accessory cells. Anti-Electrophorus AChR primary and secondary cells cross-reacted detectably with rat AChR and vice versa, whereas anti-Torpedo AChR primary and secondary cells did not significantly cross-react with Electrophorus or rat AChR. Secondary T cells strongly reactive against rat AChR could be selected in vitro from Electrophorus AChR-primed populations by using rat AChR as selecting stimulant. These cells responded equally well against Electrophorus and rat AChR and thus include autoreactive T cell clones.
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A spectrophotometric method especially suitable for biological materials is described for the determination of purine nucleoside phosphorylase activity. In combination with the enzymes xanthine oxidase, catalase and aldehyde dehydrogenase, and in the presence of ethanol and NAD(P), the purines formed by phosphorylysis of purine nucleosides are oxidized and the absorption of the NAD(P)H formed is taken for the calculation of nucleoside phosphorylase activity.
A new spectrophotometric method for the determination of adenosine deaminase is described. Adenosine is deaminated to inosine, the latter is cleaved by an inosine-guanosine specific nucleoside phosphorylase to hypoxanthine and ribose-1-phosphate. Hypoxanthine can be oxidized further to uric acid by xanthine oxidase or to allantoin by xanthine oxidase and uricase. The hydrogen peroxide formed in these reactions is reduced by catalase to water. In the presence of high concentrations of ethanol, equivalent amounts of acetaldehyde are produced. The acetaldehyde is oxidized NAD(P) dependent and the production rate of NAD(P)H is recorded at 334 nm. The new method is suitable for the detection of adenosine deaminase in whole blood, lymphocytes, sera and tissues.
A spectrophotometric method is described for the determination of 5'-nucleotidase. In combination with the enzymes nucleoside phosphorylase and xanthine oxidase, inosine, formed by hydrolysis of 5'-IMP by 5'-nucleotidase, is cleaved phosphorolytically to hypoxanthine, which is oxidized to uric acid. In the presence of ethanol, the hydrogen peroxide formed is reduced by catalase and equivalent amounts of acetaldehyde are produced. The aldehyde is dehydrogenated (NADP-dependent) by aldehyde dehydrogenase and the production rate of NADPH is recorded at 334 nm. The inhibition of the unspecific cleavage of 5'-IMP by phosphatases is examined critically.
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Cerebral lactate, pyruvate, ATP, ADP, creatine and phosphocreatine concentrations were determined in old, 600 g, rats during daily application of 20 mg pemoline (Tradon) (n = 11) and placebo (n = 10). After application for 21 days there were observed no significant differences in ATP, ADP, AMP, creatine and phosphocreatine concentrations between pemoline and placebo treated rats. On the other hand, in pemoline-rats, there was a significant decrease in pyruvate, lactate concentrations and in lactate/pyruvate ratio. On account of an increased dopamine turnover caused by pemoline, a possible increasing aerobic glycolysis and thus a greater energy production are still being discussed.
Sera from 75 patients with Myasthenia gravis were tested for acetylcholine receptor antibodies using acetylcholine receptors from human skeletal muscle. From the crude Triton x-100 extract, which has so far been used for antibody tracing, a pure acetylcholine receptor preparation was obtained by affinity chromatography using alpha-Najatoxin-Sepharose 4B. When the purified 125J-alpha-Bungarotoxin-acetylcholine receptor complex was applied in a radioimmunoassay 80% of the Myasthenia gravis patients had acetylcholine receptor antibodies in contrast to none of the tested control persons. Inspite of using a pure acetylcholine receptor preparation, no clear-cut correlation was found between the amount of serum acetylcholine receptor antibodies and the clinical stage of the disease. When individual antibody titration curves were established, different reaction patterns were observed indicating either different antibody specificities in regard to antigenic determinants on the receptor molecule or differences in the antibody affinity.
Antibodies of IgM and IgG type were detected in the CSF of patients with recent tick-borne encephalitis (TBE) by means of ELISA. No false-positive results were obtained with CSF specimens from patients suffering from meningitis or other illnesses. The ratio of the antibodies in serum to CSF clearly indicated that both IgM and IgG antibodies were produced in the brain itself. In patients who had previously suffered from TBE now with a different inaseptic meningitis, TBE antibodies could also be detected in CSF but only of the IgG class. Again the serum-to-CSF antibody ratio indicated that the antibodies were produced within the central nervous system. For routine diagnosis the micro-ELISA method was found to be useful; antigen-coated plates can be stored as long as three months at +4 degrees C.
An enzyme-linked immunosorbent assay was applied for determining separately IgM and IgG antibodies against tick-borne encephalitis virus. A micro-modification in microtitre plates proved to be at least as sensitive as the HI test. However, more precise information could be achieved by a macrotest using antigen coated polystyrene balls. False positive results in IgM antibody determinations could be caused by a rheumatoid factor. A high content of IgM antibodies in a serum could impair the determination of its IgG antibodies but not vice versa. Titres were expressed in comparison to a positive control serum.
A new method for the determination of xanthine oxidase activity with xanthine or hypoxanthine is described. The hydrogen peroxide produced by the oxidation of the substrates is reduced by catalase in the presence of high concentrations of ethanol. The acetaldehyde formed is further oxidized by aldehyde dehydrogenase NAD or NADP-dependent. The reduction rate of the coenzymes were measured at 334 nm and utilized as indicators for the xanthine oxidase. The sensitivity of the method with xanthine as substrate can be doubled by the addition of uricase, which oxidizes uric acid to allantoin.
A new method for the determination of guanase is described. Xanthine, the product of the guanase reaction, is oxidized by xanthine oxidase, forming uric acid and hydrogen peroxide. Hydrogen peroxide is further reduced to water by catalase in the presence of ethanol. The acetaldehyde formed in this reaction step is dehydrogenated NAD or NADP dependent by aldehyde dehydrogenase. The NADH or NADPH production is measured and utilized for the calculation of the guanase activity. The sensitivity of the method can be doubled by the addition of uricase, which oxidizes uric acid to permit the formation of another mole of hydrogen peroxide.
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