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Biomedical subjects

R E Kirsch

Publications and source records attributed to R E Kirsch.

At least 91 records · Page 5Linked to original sources
PubMed1989

Uroporphyrinogen decarboxylase and protoporphyrinogen oxidase in dual porphyria.

The urinary and faecal porphyrin excretory profiles of dual porphyria are said to represent the superimposition of those found in porphyria cutanea tarda on those seen in variegate porphyria. To test this hypothesis the enzymes responsible for these conditions, protoporphyrinogen oxidase (variegate porphyria) and uroporphyrinogen decarboxylase (porphyria cutanea tarda) were measured in vitro in haemolysates and lymphoblasts of 10 subjects with dual porphyria in order to clarify the enzymatic defects. Mean protoporphyrinogen oxidase activity in lymphoblasts from subjects with dual porphyria was decreased by 45% from 0.82 +/- 0.10 to 0.45 +/- 0.09 nmol protoporphyrin/mg protein/h (P less than 0.001). Uroporphyrinogen decarboxylase activity was also significantly reduced from 0.12 +/- 0.05 nmol 7-, 6-, 5- and 4-carboxyl porphyrin/mg protein/h in lymphoblasts from normal subjects to 0.08 +/- 0.02 nmol in lymphoblasts of subjects with dual porphyria (P less than 0.01). There was a similar 27% decrease in mean uroporphyrinogen decarboxylase activity of haemolysates from the same dual porphyria group (P less than 0.01). Mean activity of this enzyme in 5 patients with variegate porphyria did not differ significantly from that in normal subjects. These findings may well provide a rational basis for the abnormal porphyrin excretory profiles found in subjects with dual porphyria.

Carboxy-Lyases↗
PubMed1989

Site-directed inactivation of human lung acidic glutathione S-transferase by 1-chloro-2,4-dinitrobenzene in the absence of glutathione.

Human lung acidic glutathione S-transferase is irreversibly inhibited by 1-chloro-2,4-dinitrobenzene (CDNB) in the absence of the co-substrate glutathione (GSH). The time-dependent inactivation is pseudo-first-order and demonstrates saturation kinetics, suggesting that inactivation occurs from an EI complex. The Ki was 0.14 mM; and kobs was 0.32 min-1 at 0.6 mM CDNB. The enzyme was protected against CDNB inactivation by GSH. The other two classes of glutathione S-transferase, the basic and near-neutral, are not significantly inactivated by CDNB. Incubation with [14C]CDNB indicated covalent binding to all three classes of transferase. One peptide fraction was found to be radiolabelled in both the basic and acidic transferases when these were incubated with [14C]CDNB and GSH, cleaved with cyanogen bromide, and chromatographed by HPLC. Incubation in the absence of GSH yielded one and two additional labelled peptide fractions for the basic and acidic transferases, respectively. Our results suggest that while CDNB arylates all three classes of human transferases, only the acidic transferase possesses a specific GSH-sensitive CDNB binding site, binding to which leads to time-dependent inactivation.

Binding Sites↗
PubMed1989

Diagnostic value of ascites adenosine deaminase in tuberculous peritonitis.

The value of ascitic fluid adenosine deaminase activity in distinguishing tuberculosis from other causes of ascites was examined in a retrospective study of 41 patients with bacteriologically confirmed tuberculous peritonitis and 41 control patients, matched for age and sex, with ascites of other causes (12 alcoholic cirrhosis, 5 cryptogenic cirrhosis, 12 malignant disorders, 3 pancreatitis, and 9 miscellaneous causes). The mean ascites adenosine deaminase activity was 99.8 (SD 49.1) in tuberculous patients and 14.8 (8.4) U/l in control patients (p less than 0.0001). A cutoff of 32.3 U/l had a sensitivity of 95% and specificity of 98% in distinguishing between the two groups. In a subsequent prospective study of 64 patients with ascites, 11 were found to have tuberculosis. Of the others, 23 had cirrhosis (18 alcoholic, 5 cryptogenic), 17 malignant disorders, 3 pancreatitis, 5 cor pulmonale, 3 congestive cardiac failure, 1 systemic mastocytosis, and 1 renal failure and hypothyroidism. The mean ascites adenosine deaminase activity was 112.6 (45.0) U/l in the patients with tuberculous ascites and 16.3 (36.7) U/l (p less than 0.0001) in those with ascites of other causes. In this study, adenosine deaminase had a sensitivity of 100% and specificity of 96% in discriminating tuberculosis from other causes of ascites. These findings suggest that the ascitic fluid adenosine deaminase activity may be used to identify patients in whom the diagnosis of abdominal tuberculosis must be pursued.

Adenosine Deaminase↗
PubMed1989

Single step affinity chromatographic purification of human alpha-amylase from aspirated duodenal juice and its application in the measurement of pancreatic alpha-amylase synthesis rates in man.

Human alpha-amylase was purified from aspirated duodenal juice to electrophoretic homogeneity in a single step by affinity chromatography with the competitive inhibitor acarbose (IC50 = 1.22 mumol/l) as ligand. Duodenal juice was applied to an agarose resin to which acarbose had been coupled covalently via a 1.9 nm spacer group. Pure alpha-amylase, eluted with free acarbose, had a molecular mass of 55,000, and isoelectrofocusing revealed the presence of six isozymes with pI values of 7.3, 6.8, 6.7, 6.5, 6.4 and 6.3, all of which possessed amylase activity based on positive starch/iodine staining. The potential usefulness of this one-step purification procedure in the measurement of pancreatic alpha-amylase synthesis rates was evaluated in two control patients with non-pancreatic disease. Aspirated duodenal juice was obtained during a pulse/continuous intravenous 4 h infusion of [14C]leucine together with secretin and pancreozymin, and alpha-amylase purified using our protocol. Pancreatic alpha-amylase synthesis rates were determined from the rate of incorporation of [14C]leucine into alpha-amylase; values of 4.4 and 5.1 h were obtained for the two control patients.

Acarbose↗
PubMed1988

Catalysis of angiotensin I hydrolysis by human angiotensin-converting enzyme: effect of chloride and pH.

The catalysis of the hydrolysis of angiotensin I, an important natural substrate, by human angiotensin-converting enzyme (ACE) was examined in detail as a function of chloride and hydrogen ion concentration. Chloride was found to be a nonessential activator over the pH range 5.0-10.0, with the chloride dependence increasing with increasing pH: the velocity enhancement at optimal [Cl-] increased from 1.6- to 42-fold; the chloride optimum and Ka' increased from 20 to 520 mM and from 0.22 to 120 mM, respectively, and activity in the absence of chloride decreased from 60.9 to 2.4% (relative to maximal activation). Kinetic analyses at pH 6.0, 7.5, and 9.0 confirmed the nonessential activator mechanism. At all pH values tested chloride was found to be inhibitory (relative to maximal activation) at supraoptimal chloride levels. Depending on the [Cl-] range, both apparent uncompetitive and competitive modes were demonstrated. From pH 6.0 to 9.0 Kis varied between 110 and 1140 mM (apparent). In all cases Ki' much greater than Ka'. We suggest that at high [Cl-] chloride binds to low-affinity inhibitory sites on the free enzyme and on the ES and EP complexes. The pH-rate profile demonstrated a chloride-dependent alkaline shift, with the pH optimum increasing from 7.1 at zero chloride to 7.6 at 400 mM NaCl. At [S] much greater than Km a plot of log nu vs pH revealed pKs of 5.9 and 9.4 in the ES complex in the absence of chloride, while at maximally activating [Cl-] only one ionization at pK = 6.3 was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin I↗
PubMed1988

Halothane decreases albumin and transferrin synthesis: studies in the isolated, perfused rat liver and in the intact rat.

Isolated perfused rat livers exposed to 1.5% halothane (equivalent to 1.35 MAC) in O2/CO2 or to O2/CO2 alone produced urea, as well as albumin and transferrin (both measured by immunodiffusion), at constant rates during a 4.25-h perfusion. Urea production did not differ in the two treatment groups, but halothane depressed albumin and transferrin synthesis 43% and 45%, respectively. Intact rats were also exposed to halothane, after which albumin synthesis was measured by the (14C)carbonate technique. The minimum halothane concentration required to insure sufficient relaxation for ventilation was selected and ranged from 1.0 to 1.5%. Measurements were made in control rats not exposed to halothane (group I) and in halothane exposed rats immediately after 1 h of anesthesia (group II), 24 h after the start of 1 h of anesthesia (group III), and immediately after 1/2 h of anesthesia preceded by a 1-h exposure 24 h earlier (group IV). Single exposures to halothane (groups II and III) resulted in a decrease in albumin synthesis immediately or 24 h later that did not differ significantly from controls (group I). However, halothane given twice to rats at 24-h intervals (group IV) reduced their mean albumin synthesis rate to half that of controls. The early onset and constancy of halothane depression of export protein synthesis by isolated, perfused livers may reflect a response to halothane itself, rather than an effect resulting from the accumulation of halothane metabolites. Similarly, reduction of albumin synthesis in intact rats immediately after a second halothane exposure may indicate a response to halothane, rather than to halothane metabolites.

Albumins↗
PubMed1988

Hepatic stimulator substance in extracts from regenerating porcine liver. Basic physiochemical properties.

Previous studies in rats and dogs have implicated the liver as the source of some of the factors which are responsible for its regeneration. In the present study extracts from regenerating and normal livers were infused into the portal vein of pigs after a 15% partial hepatectomy. Thymidine kinase activity was measured on the 3rd and 4th days as an index of liver regeneration. An intraportal infusion of extracts from regenerating, but not normal, livers resulted in an increased regenerative response. The response was dose-related. The stimulator substance was stable when stored at -20 degrees C and could be precipitated by absolute ethanol.

Animals↗
PubMed1988

Glutathione S-transferase distribution and concentration in human organs.

The concentration of basic, near-neutral and acid GSH S-transferase was measured in 18 organs from each of 9 male human subjects using radial immunodiffusion. Basic transferases were detectable in all tissues studied. Highest concentrations were found in liver, testis, kidney, adrenal and jejunum while low levels were found in bladder, muscle and thyroid. The concentration in liver was 230 times higher than that in thyroid. Near-neutral GSH S-transferase were absent in all tissues in 5 of the 9 individuals studied. When present they were widely distributed, highest concentrations being found in liver, testis, muscle, adrenal and brain and lowest levels in thyroid, lung, duodenum, stomach, heart and kidney. Acid GSH S-transferases were present in every individual studied although they were undetectable in the liver of a single subject. Highest concentrations were present in colon, jejunum, ileum, bladder, spleen and lung while low concentrations were found in liver. Our study provides conclusive evidence of marked inter-individual and inter-organ variation of the three groups of human GSH S-transferase.

Humans↗
PubMed1988

Inhibition of rat liver glutathione S-transferase isoenzymes by acrolein.

The in vitro effect of the toxin and teratogen, acrolein, on the fetal rat liver glutathione S-transferase isoenzyme, YcYfetus, was investigated and compared with acrolein's effect on some of the adult rat liver glutathione S-transferase isoenzymes. Acrolein was found to inhibit all the isoenzymes investigated and double-reciprocal plots suggest that inhibition is either noncompetitive or mixed-type noncompetitive. It is therefore attractive to suggest that should a similar situation arise in vivo, it may provide one mechanism for the teratogenicity of acrolein.

Acrolein↗
PubMed1987

The isolation of a fetal rat liver glutathione S-transferase isoenzyme with high glutathione peroxidase activity.

A previously uncharacterized glutathione S-transferase isoenzyme which is absent from normal adult rat livers has been isolated from fetal rat livers. The enzyme was purified using a combination of affinity chromatography, CM-cellulose column chromatography and chromatofocusing. It is composed of two non-identical subunits, namely, subunit Yc (Mr 28,000) and a subunit (Mr 25,500) recently reported by us to be uniquely present in fetal rat livers and which we now refer to as subunit 'Yfetus'. The enzyme which we term glutathione S-transferase YcYfetus has an isoelectric point of approx. 8.65 and has glutathione S-transferase activity towards a number of substrates. The most significant property of the fetal isozyme is its high glutathione peroxidase activity towards the model substrate cumene hydroperoxide. We suggest that this isozyme serves a specific function in protecting fetuses against the possible teratogenic effects of organic peroxides.

Amino Acids↗
PubMed1987

Porphyria--the UCT experience.

Several forms of porphyria are commonly seen in South Africa. Much of the knowledge accumulated in this field has been due to the meticulous research of Professor Lennox Eales and his colleagues at the University of Cape Town. Professor Eales headed the Porphyria Research Group, founded by the CSIR in 1957 and upgraded to a Unit by the MRC in 1979, until he retired in 1983. Since then porphyria research at UCT has continued within the MRC Liver Research Centre. We present here a description of those forms of porphyria commonly seen in South Africa, based in part on recent work carried out at UCT and in part on the work of Lennox Eales, to whom we dedicate this article.

Acute Disease↗
PubMed1987

Fibrin and fibrinogen-related antigens in patients with stable and unstable coronary artery disease.

Coronary-artery thrombosis may be important in the pathogenesis of unstable angina at rest. To study this possibility, we measured the serum concentrations of fibrin-related antigen, D dimer (the principal breakdown fragment of fibrin), and fibrin monomer (an intermediate product of fibrin formation) in the serum of five groups of subjects. These included 10 healthy controls, 10 controls with noncardiac pain, and three groups of 10 patients each with chronic stable angina, unstable angina at rest, or acute myocardial infarction. The concentration of fibrin-related antigen (normal range, 48 to 184 ng per milliliter) was normal in the control patients with noncardiac pain (63 to 202 ng per milliliter) and in patients with chronic stable angina (95 to 186), but it was increased in patients with unstable angina (401 to 2507) or acute myocardial infarction (470 to 1930) (P less than 0.001). D dimer concentrations in patients with unstable angina (178.3 to 310.6 ng per milliliter) or acute myocardial infarction (103.9 to 321.6) were higher than those in patients with chronic stable angina (28.6 to 52.1), in controls with noncardiac pain (44.7 to 53.1), and in healthy controls (40.4 to 50.3) (P less than 0.001). Concentrations of fibrin monomer were highest in patients with acute myocardial infarction (247.5 to 571.3 ng per milliliter) (P less than 0.001), intermediate in those with unstable angina (54.7 to 241.7) (P less than 0.001), and normal (normal range, 14.5 to 19.8 ng per milliliter) in controls with noncardiac pain (12.0 to 18.4). and patients with chronic stable angina (10.7 to 17.6). These findings suggest the presence of an active thrombotic process in patients with unstable angina at rest or acute myocardial infarction. The data do not prove that the coronary arteries were the site of the thrombotic process, but the observations are consistent with the hypothesis that thrombus formation may have an important role in the pathogenesis of these conditions.

Angina Pectoris↗
PubMed1987

A diazophenylthio-ether-IgG probe for analysing fibrin- and fibrinogen-related antigens.

A technique for identifying and quantitating fibrin- and fibrinogen-related antigens (FRAs) in serum is described. Panspecific antifibrinogen IgG is bound covalently to diazotized aminophenylthio-ether cellulose paper discs. Under conditions of antibody excess, a disc probe extracts 95% of all fibrinogen-related antigen from appropriately diluted serum samples. Bound antigen is then eluted from the washed probe and electrophoresed on 4-11% gradient SDS-polyacrylamide gels. Derivatives of cross-linked fibrin, non-cross-linked fibrin and fibrinogen may thus be identified by their molecular weights, and their relative concentrations determined by densitometric analysis. The total amount of FRA is estimated by a radio-immunoassay which uses labelled D-dimer as the competitive antigen. The probes can extract as little as 0.20 and 0.15 microgram/ml of D-dimer and D-monomer, respectively. Specific FRA binding was confirmed with Western blotting. Potential interfering substances do not influence antigen binding to the discs and, since complete elution of all antigens is achieved, discs may be re-used. This procedure provides estimates of the serum levels of fragments YY, DY, X, DD, Y and D. The procedure is simple to perform and, as a research tool, may help to delineate some of the features of clotting disturbances in clinical states.

Animals↗
PubMed1986

Rapid affinity chromatographic purification of human lung and kidney angiotensin-converting enzyme with the novel N-carboxyalkyl dipeptide inhibitor N-[1(S)-carboxy-5-aminopentyl]glycylglycine.

Human angiotensin-converting enzyme has been purified, in a single chromatographic step, using a novel N-carboxyalkyl dipeptide CA-GlyGly (N-[1(S)-carboxy-5-aminopentyl]glycylglycine) synthesised in our laboratory. CA-GlyGly is a weak competitive inhibitor, Ki = 0.18 mM, and its inhibitory profile is markedly pH-dependent. Human lung and kidney angiotensin-converting enzyme were solubilised with Triton X-100 and after ammonium sulphate fractionation the crude extract was applied to a column containing CA-GlyGly coupled to agarose via a 2.8 nm spacer group. Electrophoretically pure human angiotensin-converting enzyme could be eluted by raising the pH of the chromatography buffer from 7.50 to 9.50. The specific activity of human angiotensin-converting enzyme purified from lung was 104 units/mg, while that from kidney was 88 units/mg. Molecular weight for both enzymes was estimated to be 160,000. The Km with respect to hippuryl-L-histidyl-L-leucine was 1.9 mM in the case of lung angiotensin-converting enzyme and 1.7 mM in that of kidney angiotensin-converting enzyme, while for the substrate angiotensin I Km values were 62 microM and 76 microM, respectively. Hydrolysis of either substrate was chloride-dependent and both enzymes were strongly inhibited by captopril.

Angiotensin I↗