Paraoxon hydrolysis in human serum mediated by a genetically variable arylesterase and albumin.

Gel filtration chromatography resolves human serum paraoxonase into two fractions: (1) a high molecular weight fraction that is completely inhibited by EDTA and coelutes with arylesterase (E.C.3.1.1.2); and (2) a second fraction that is closely associated with albumin, is only partially inhibited by EDTA, and has relatively little arylesterase activity under the assay conditions used. The activity of the high molecular weight fraction is stimulated by NaCl, whereas the albumin associated activity is partially inhibited by NaCl and is not present in serum derived from an analbuminemic individual. Our data suggest that albumin itself, rather than a protein bound to or cofractionating with albumin, mediates paraoxonase activity. The variation in levels of the activity of the nonalbumin, high molecular weight enzyme is responsible for the observed polymorphism of paraoxonase activity in human serum or plasma. An optimal assay of polymorphic paraoxonase activity should be based on activity measurements of the nonalbumin fraction. It is considered likely that only the nonalbumin fraction is responsible for in vivo hydrolysis of paraoxon.     

Erythrocytenenzyme der Primaten

Zusammenfassung Die Adenosindesaminasen der Primaten zeigen eine genetisch determinierte Variabilität. Bei der Untersuchung von 327 subhumanen Primaten (289 Simiae der Alten Welt, 38 Prosimiae) konnten wir neun Adenosindesaminase-Varianten nachweisen, die auf Grund ihrer unterschiedlichen elektrophoretischen Wandergeschwindigkeit als ADA 6, ADA 4, ADA 2, ADA 2, ADA 1, ADA 3, ADA 5, ADA 5 und ADA 7 bezeichnet werden. Die Verteilung der verschiedenen Phänotypen wurde ermittelt.

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Red cell enzymes of primatesAdenosine deamisnase (EC: 3.5.4.4.)

Summary The polymorphism of adenosine deaminase has been investigated in 327 subhuman Primates (289 Old World Monkeys and 38 Prosimians). Nine adenosine deaminase variants were found to be present, which on the basis of their different electrophoretic mobilities were designated ADA 6, ADA 4, ADA 2, ADA 2, ADA 1, ADA 3, ADA 5, ADA 5 and ADA 7. The distribution of these various phenotypes has been estimated.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Genetisch kontrollierte Varianten der NADH-Diaphorase

Zusammenfassung Die NADH-Diaphorase wurde an 725 gesunden Probanden mit Hilfe der Stärkegelelektrophorese untersucht. Zwei verschiedene Varianten wurden beobachtet: eine heterozygot schnelle (DIA 2-1) und eine heterozygot langsame (DIA 3-1). Die Genhäufigkeiten sind: DIA²=0,0021; DIA³=0,0007.

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Genetically determined variants of NADH-diaphorase

Summary By means of starchgel-electrophoresis a screening for variants of NADH-Diaphorase was carried out within a sample of 725 healthy probands. Two kinds of genetically determined variants have been observed: a heterozygous phenotype with greater mobility (DIA 2-1) and a heterozygous phenotype with slower mobility (DIA 3-1). The gene-frequencies are estimated so far as 0.0021 (DIA²) and 0.0007 (DIA³).

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Population genetics of red cell glyoxalase I (E.C.: 4.4.1.5)

Summary The polymorphism of Glyoxalase I was investigated in a population sample from Southwestern Germany. The frequency of the GLO² allele was determined to be 0.427.

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Zusammenfassung Der Polymorphismus der Glyoxalase I wurde an einer Bevölkerungsstichprobe aus Südwestdeutschland untersucht. Die Genhäufigkeit für GLO¹ beträgt 0,427.

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Supported by the Deutsche Forschungsgemeinschaft.     

Ergebnisse von Bestimmungen menschlicher Serum-Transferrin-Typen

Zusammenfassung Es wird über die Ergebnisse der in Ungarn erstmals durchgeführten Serum-Transferrin-Typen-Bestimmungen an 1007 Personen berichtet. Die Untersuchungen wurden mit der horizontalen Stärkegelelektrophorese durchgeführt. In 5 Fällen wurden Varianten gefunden, die sich als Typ B_0-1C bzw. B₀C erwiesen. Weiterhin wird über die Ergebnisse von Tf-Untersuchungen in Fällen strittiger Vaterschaft berichtet. In einer 2-Männer-Sache gelang es, eine Zuordnung höchstwahrscheinlich zu machen.

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Distribution of serum transferrin groups in Hungrary and their application in paternity proceedings

Summary Results of human serum transferrin-type determinations carried out on 1007 persons the first time in Hungary are reported. The determinations were carried out by way of the horizontal starchgel electrophorese according to Smithies modified by Ashton. In 5 cases variants were found migrating faster than the common type Tf C proving at the identification as type B_0-1 respectively B₀. That is, the frequency of the Tf-variants in Hungary are estimated about 0.5%. The significance of Tf-type determinations in clearing up disputed paternity is discussed. Also the results of determinations of this kind in a juridicial case are reported. It was about a 2-men case. Paternity of the witness was disclosed by way of Tf-type determination, that of the defendant, however, proved.

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Vorstand: Prof. Dr. med. E. Somogyi

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Vorstand: Prof. Dr. J. Márkus     

Organophosphate detoxicating hydrolases in different vertebrate species

Phosphorylphosphatase activities in various organs of vertebrate species from different classes were determined using a spectrophotometric assay for paraoxonase (EC 3.1.1.2) and a potentiometric assay with a fluoride sensitive electrode for DFPase (EC 3.8.2.1). Temperature-dependent inactivation experiments, an extended interpretation of mixed substrate studies and activity distribution patterns confirm that in vertebrate tissue at least two different enzymes are responsible for hydrolytic detoxication of paraoxon and DFP. Total organophosphate detoxicating phosphorylphosphatase activity of a certain animal species is shown to be the major determinant for differences between the inhibitory potency of organophosphorus compounds on the animal's target enzymes in vitro and organophosphate toxicity in vivo.     

A sandwich enzyme-linked immunosorbent assay for human serum paraoxonase concentration

Serum paraoxonase (PON) is associated with plasma high density lipoproteins, and prevents the oxidative modification of low density lipoproteins. We have developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA), using two monoclonal antibodies against PON, to measure serum PON concentration. The concentration of PON in healthy Japanese subjects was 59.3 +/- 1.3 microgram/mL (mean +/- SEM; n = 87). Serum PON concentrations in relation to the PON 192 genetic polymorphism were: 69.5 +/- 2.9 microgram/mL in the QQ genotype; 63.0 +/- 1.9 microgram/mL in the QR genotype; and 52.8 +/- 1.7 microgram/mL in the RR genotype. Concentrations were significantly lower in the RR than in the QQ genotype (P < 0.01). Serum paraoxonase specific activity was higher in RR than in QQ subjects (18.6 +/- 0.40 vs. 2. 56 +/- 0.05 nmol/min/microgram, P < 0.01), but arylesterase specific activity was unrelated to genotype. PON concentration was positively associated (P < 0.001) with both serum arylesterase activity and, after adjusting for the effect of the position 192 polymorphism, with serum paraoxonase activity. Subjects with angiographically verified coronary heart disease had significantly lower PON concentrations than the healthy controls (52.0 +/- 2.3 microgram/mL; n = 35, P < 0.01). This association was independent of the position 192 genotype. Our new ELISA should be of value for epidemiologic and clinical studies of serum PON concentration. immunosorbent assay for human serum paraoxonase concentration.     

Disk-elektrophoretischer Nachweis des Lp(a)-Proteins in Lipoproteinfraktionen

Zusammenfassung Die Untersuchung der Serumlipoproteinfraktion HDL₂ (1,063< <1,125 g/ml) von 35 Personen mit Hilfe der Polyacrylamid-Gelelektrophorese zeigte, daß auch bei Anwendung dieser empfindlichen Technik eine eindeutige Einteilung in Lp(a+)- und Lp(a-)-Typen nicht möglich ist. Von den 20 Seren, die durch Agar-Gel-Doppeldiffusion als Lp(a-) bestimmt wurden, zeigten 19 in ihrer HDL₂-Fraktion in der für das Lp(a)-Lipoprotein typischen Position eine Bande. Dieser Befund bestätigt die Annahme, daß der Faktor Lp(a) als ein quantitatives genetisches Merkmal angesehen werden muß.Die -Lipoproteine des HDL₂-Bereiches erwiesen sich als disk-elektrophoretisch heterogen.

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Determination of the Lp(a)-protein by disc-electrophoresis of lipoprotein-fractions

Summary Gelelectrophoretic studies of the serum lipoprotein fraction HDL₂ (1.063< <1.125 g/ml)_of 35 individuals showed, that using this technique no clear classification into Lp(a+) and Lp(a-)-types could be made. 19 out of 20 sera typed Lp(a-) by double diffusion contained in their HDL₂-fraction a lipoprotein with the electrophoretic mobility characteristic of the Lp(a)-lipoprotein. This observation confirmes previous suggestions, that Lp(a) indeed is a quantitative genetic trait.The -lipoproteins of the HDL₂-fraction are heterogeneous in gelelectrophoresis.

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Untersuchung mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Interspecific variability of red cell phosphoglycerate kinase in primates

Summary The genetic polymorphism of red cell phosphoglycerate kinase was investigated in primates. Five variants of the enzyme were detected.

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Zusammenfassung Für die erythrocytäre Phosphoglyceratkinase der Primaten konnte eine genetisch determinierte Variabilität (5 Enzymvarianten) nachgewiesen werden.

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Supported by the Deutsche Forschungsgemeinschaft.     

Transspecific variability of glutamic oxaloacetic transaminase (E.C. 2.6.1.1.) in Primates

Summary The genetic polymorphism of soluble and mitochondrial glutamic oxaloacetic transaminase has been investigated in Primates. 3 variants of the soluble enzyme and 2 variants of the mitochondrial enzyme could be demonstrated. The distribution of the various phenotypes has been estimated.

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Zusammenfassung Die Glutamat-Oxalacetat-Transaminasen der Primaten, zeigen eine genetisch determinierte Variabilität. Es konnten 3 cytoplasmatische und 2 mitochondriale Enzymvarianten nachgewiesen werden. Die Verteilung der Phänotypen wurde ermittelt.

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Direktor: Prof. Dr. Dr. H. Ritter

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Supported by the Deutsche Forschungsgemeinschaft.     

The determination of Q192R polymorphism of paraoxonase 1 by using non-toxic substrate p-nitrophenylacetate

CONTEXT:

The human serum paraoxonase 1 (PON1) is calcium-dependent esterase and associates with the high density serum lipoproteins. PON1 plays a major role in oxidation of high density lipoprotein and low density lipoprotein and prevention of atherogenesis in coronary heart disease. PON1Q and R allele hydrolyses number of substrates like paraoxon (PO) (diethyl p-nitrophenyl phosphate) and phenylacetate.

AIMS:

The aim of the study is to the determination of Q192R polymorphism of PON1 by using non-toxic substrate p-nitrophenylacetate and compares it with the phenotype determined by using PO as substrate.

MATERIALS AND METHODS:

The study group consists of 60 healthy normal patients. Paraoxonase activity was measured using the procedure described by Eckerson (Reference method) and for phenotyping; the ratio of hydrolysis of PO in the presence of 1 M NaCl (salt-stimulated PON1, SALT) to the hydrolysis of phenylacetate (PA) is calculated. In new method (Haagen et al.) arylesterase activity measured using p-nitrophenylacetate and for phenotyping arylesterase, the ratio of inhibition of enzymatic hydrolysis of p-nitrophenylacetate (substrate) by phenyl acetate to non-inhibited hydrolysis of p-nitrophenylacetate (inhibited arylesterase activity (IA-IA₀)/non-inhibited arylesterase activity (NIA).

RESULTS:

It was found that paraoxonase activity is trimodally distributed in both the methods. There is no significant difference in the distribution of PON1 phenotypes of both reference method and new method being frequencies 0.946 and 0.376 respectively and there was no significant difference for phenotypic polymorphism for an individual by both methods (χ²= 0.15 and P = 0.9262).

CONCLUSION:

The Q192R polymorphism of PON1 by using non-toxic substrate p-nitrophenylacetate showed trimodal distribution of QQ (homozygous), QR (heterozygous), and RR (homozygous) phenotype and it is comparable with reference method. This method can be used for PON1 phenotype in different pathological and complex disease conditions.     

On the genetics of the human serum paraoxonase (EC 3.1.1.2)

Summary Incubation of the sera of 799 nonrelated persons with paraoxon led to varying degrees of inhibition of the serum cholinesterase (EC 3.1.1.8) with residual activity between 0% and 67.4% of the initial activity. This is the result of a differing paraoxonase (EC 3.1.1.2) activity. The residual activities show a trimodal distribution. The results of studies of 99 families with children show that an autosomal dominant heredity factor is most likely. Consideration of the constellations of the activity values within the families can thus yield a stochastic external criterion. This, together with the shape of the distribution of the individual values, gives good statistical estimates for the distributions and frequencies of the three groups obtained by an iteration technique. Tests of association that take account of group membership show that residual activity does not depend on the blood groups A, B, 0, and Rh, or on age. A conclusive argument for our assumption of three activity groups is that the resulting group frequencies are consistent with the Hardy-Weinberg rule.     

Genetic polymorphism of mannosephosphate isomerase in Primates

Summary Genetic polymorphism of mannosephosphate isomerase has been demonstrated in Primates. Three variants of the enzyme were detected. The distribution of MPI phenotypes has been estimated.

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Zusammenfassung Für die Mannosephosphat-Isomerase der Primaten konnte eine genetisch determinierte Variabilität (3 Enzymvarianten) nachgewiesen werden. Die Verteilung der Phänotypen wurde ermittelt.

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Supported by the Deutsche Forschungsgemeinschaft.     

The effect of several diphosphonates on acid phosphohydrolases and other lysosomal enzymes.

Diphosphonates are known to inhibit bone resorption in tissue culture and in experimental animals. This effect may be due to their ability to inhibit the dissolution of hydroxyapatite crystals, but other mechanisms may be important. Since lysosomal enzymes have implicated in the process of bone resorption, we have examined the effect of several phosphonates and of a polyphosphate (P20,2) on lysosomal hydrolases derived from rat liver and rat bone. Dichloromethylene diphosphonate strongly inhibited acid beta-glycerophosphatase (EC 3.1.3.2) and acid p-nitrophenyl phosphatase (EC 3.1.3.2) and to a lesser degree (in descending order) acid pyrophosphatase (EC 3.1.3.-), arylsulfatase A (EC 3.1.6.1), deoxyribonuclease II(EC 3.1.4.6) and phosphoprotein phosphatase (EC 3.1.3.16) of rat liver. Inhibition of acid p-nitrophenyl phosphatase and arylsulfatase A was competitive. Ethane-1-hydroxy-1, 1-diphosphonate did not inhibit any of these enzymes, except at high concentrations. Neither dichloromethylene diphosphonate nor ethane-1-hydroxy-1, 1-diphosphonate had any effect on beta-glucuronidase (EC 3.2.1.31), arylesterase (EC 3.1.1.2) and cathepsin D (EC 3.4.23.5). Of several other phosphonates tested only undec-10-ene-1-hydroxy-1, 1-diphosphonic acid inhibited acid p-nitrophenyl phosphatase strongly, the polyphosphate (P20, I) had little effect. Acid p-nitrophenyl phosphatase in rat calvaria extract behaved in the same way as the liver enzyme and was also strongly inhibited by dichloromethylene diphosphonate, but not by ethane-1-hydroxy-1, 1-diphosphonate. It is suggested that the inhibition of bone resorption by dichloromethylene diphosphonate might be due in part to a direct effect of this diphosphonate on lysosomal hydrolases.     

Daten zur Populationsgenetik der 6-Phosphogluconat-Dehydrogenase

Summary Four Malaysian racial groups were typed for red cell adenylate kinase: 324 Malays, 300 Chinese, 256 Indians, and 483 West Malaysian Aborigines. The AK² gene frequencies found were 0.015, 0.0, 0.086, and 0.013, respectively. All 244 Malays, 170 Chinese, 153 Indians, and 132 West Malaysian Aborigines examined had the common cytoplasmic malate dehydrogenase phenotype.

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Zusammenfassung Vier Rassengruppen aus Malaysia wurden auf Adenylatkinase-Varianten hin untersucht: 324 Malayen, 300 Chinesen, 256 Inder und 483 Eingeborene von West-Malaysia. Die Genhäufigkeiten des Allels AK² waren: 0,015, 0,0, 0,086 und 0,013. Alle 244 Malayen, 170 Chinesen, 153 Inder und 132 west-malaysischen Eingeborenen, die daraufhin untersucht werden konnten, hatten den häufigen Phänotyp der cytoplasmatischen Malat-Dehydrogenase.

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This work was supported by the University of Maryland and University of California International Centers for Medical Research and Training with research grants AI-10049-12, AI-10051, and HE 10486, all from the National Institutes of Health, U.S. Public Health Service.     

Transspecific variability of mitochondrial NAD-malate dehydrogenase (E.C.: 1.1.1.37) in Primates

Summary The genetic polymorphism of mitochondrial NAD-malate dehydrogenase has been investigated in Primates. 3 enzyme variants could be demonstrated.

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Zusammenfassung Die mitochondriale NAD-Malatdehydrogenase der Primaten zeigt eine genetisch determinierte Variabilität. Es konnten 3 Enzymvarianten nachgewiesen werden.

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Direktor: Prof. Dr. Dr. H. Ritter

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Supported by the Deutsche Forschungsgemeinschaft.     

GPT, 6-PGD,PGM and AK phenotyping inone starch gel

Summary A method is described for simultaneous electrophoretic separation of GPT (EC 2.6.1.2), 6-PGD (EC 1.1.1.44), PGM (EC 2.7.5.1) and AK (EC 2.7.4.3) enzyme variants.

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Zusammenfassung Beschreibung einer Methode zur gleichzeitigen elektrophoretischen Auftrennung der Enzymvarianten von GPT (EC 2.6.1.2), 6-PGD (EC 1.1.1.44), PGM (EC 2.7.5.1) and AK (EC 2.7.4.3).

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Abbreviations GPT Glutamic Pyruvic Transaminase - 6-PGD 6-Phosphogluconate Dehydrogenase - PGM Phosphoglucomutase - AK Adenylate Kinase Supported by the Deutsche Forschungsgemeinschaft, the Stiftung Volkswagenwerk and the Fonds der Chemischen Industrie.     

Populationsgenetische Untersuchungen über die Verteilung von Hämoglobin S und Glucose-6-Phosphat-Dehydrogenasemangel im Bodrogköz (Nordostungarn)

Zusammenfassung An einer Stichprobe von n=363 bzw. n=233 Individuen verschiedener Altersstufen im Bodrogköz (Nordostungarn) wurden Untersuchungen Bezüglich der Frequenz von Hb S und G-6-PD-Mangel durchgeführt. Die Frequenz von Hb S beträgt 0,0%, diejenige des G-6-PD-Mangels 3,9%. Bei dem zwischen Bodrog und Theiss gelegenen Bodrogköz handelt es sich um eine Sumpflandschaft, die bis zur Drainage in den ersten Jahrzehnten dieses Jahrhunderts malaria verseucht war. Die Population des Bodrogköz hat sich offenbar über den G-6-PD-Polymorphismus selektiv an die Malariabelastung ihrer Umwelt angepaßt. — Weiterhin wird über eine Sippe mit G-6-PD-Mangel berichtet.

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The authors report on an investigation concerning the frequencies of Hb S and Glucose-6-Phosphate Dehydrogenase Deficiency (G-6-PDD) in a population sample from Bodrogköz (NE-Hungary). The frequency of Hb S (n=363) was found to be 0,0%, that of G-6-PDD (n=233) was calculated to be 3,9%. Bodrogköz, situated between the rivers Bodrog and Theiss, is a swamp which was contaminated with malaria until its drainage within the first decades of our century. The Bodrogköz population seems to have been adapted to this malaria contamination by developing a G-6-PDD-polymorphism. — Furthermore a family with G-6-PDD is presented.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Contribution to the PGM3 polymorphism phenotype frequencies in Prague

Summary The genetic polymorphism of D-fructose-1,6-diphosphate-1-phosphohydrolase has been investigated in Primates. 3 enzyme variants could be demonstrated.

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Zusammenfassung Die D-Fructose-1,6-Diphosphat-1-Phosphohydrolase der Primaten zeigt eine genetisch determinierte Variabilität. Es konnten 3 Enzymvarianten nachgewiesen werden.

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Supported by the Deutsche Forschungsgemeinschaft.     

The frequencies of Gm(1), Gm(2), Gm(4), Gm(12), and Inv(1) in Hessen (Germany)

Summary The serum groups Gm(1) [Gm(a)], Gm(2) [Gm(x)], Gm(4) [Gm(f)]. Gm(12) [Gm(b)] and Inv(1) [Inv(1)] of 2000 sera of healthy blood donors from the land Hesse were examined. The results obtained were compared with those known until now. Three persons, not related to each other, possessed the extremely rare phenotype Gm(-1, 2, 4, 12) [Gm (a-x+b+f+)]. In 0.75% of the cases we found a discordant behaviour of the factors Gm(4) and Gm(12) [Gm(f) and Gm(b)].

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Zusammenfassung 2000 Seren von gesunden Blutspendern aus Hessen wurden bezüglich der Gamma-Globulin-Serumgruppen Gm(1) [Gm(a)], Gm(2) [Gm(x)], Gm(4) [Gm(f)]. Gm(12) [Gm(b)] und Inv(1) [Inv(1)] untersucht. Die gefundenen Resultate wurden mit den bisher bekannten verglichen. Drei miteinander nicht verwandte Personen wiesen den äußerst seltenen Phänotyp Gm(-1, 2, 4, 12) [Gm(a-x+b+f+)] auf. In 0.75% der Fälle fanden wir ein diskordantes Verhalten der Faktoren Gm(4) und Gm(12) [Gm(f) und Gm(b)].

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Director: Prof. Dr. W. Wachsmuth

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Director: Prof. Dr. W. Spielmann

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The nomenclature suggested by WHO at a round-table conference over genes, genotypes and allotypes of immunglobulins is used. The conference took place in Geneva on the 1965 31. 5. to the 5. 6. [5].

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With technical assistance of S. Mohs.