Jörg-Michael Chemnitius

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 animals target enzymes in vitro and organophosphate toxicity in vivo.

Rapid preparation of subsarcolemmal and interfibrillar mitochondrial subpopulations from cardiac muscle

1. Subsarcolemmal and interfibrillar mitochondria were prepared with complete recovery from rabbit and porcine heart muscle by upward-flotation during 60 sec of Percoll density gradient centrifugation. 2. Mitochondrial subpopulations were identified and characterized according to buoyant density, electron-microscopy, marker enzyme activities and respiratory performance. 3. ADP-induced state 3-respiration related to latent citrate synthase activity as a marker for structurally intact mitochondria was not significantly different in both mitochondrial subtypes.

Identification of isoenzymes in cholinesterase preparations using kinetic data of organophosphate inhibition

Abstract Commercial preparations of acetylcholinesterase (EC 3.1.1.7) and of cholinesterase (EC 3.1.1.8) were characterized by organophosphate inhibition. Cholinesterase activities were inhibited by varying organophosphate concentration and time of inhibition. Bimolecular rate constants were determined by plotting log activity vs inhibitor concentration or inhibition time. Inhibition of acetylcholinesterase from bovine erythrocytes by diethyl p-nitrophenyl phosphate (Paraoxon), diisopropylphosphorofluoridate (DFP), and N,N′-diisopropylphosphorodiamidic fluoride (Mipafox) in semilogarithmic plots showed a linear decay of activity. Inhibition of acetylcholinesterase from electric eel (Electrophorus electricus) and of cholinesterases from horse serum and from human serum did not show linear characteristics, indicating the presence of more than one single enzyme in these preparations. The corresponding inhibition curves were resolved by subtraction of exponential functions. In each case two different activity components were identified and characterized in respect to partial activity, substrate specificity, and reactivity with organophosphorous compounds. The suitability of the method for application on crude homogenates is discussed.

Carboxylesterases in primate brain: characterization of multiple forms

Carboxylesterase activity of primate brain (Macaca mulatta) was determined using phenyl valerate (PV) as substrate. Eight carboxylesterases of primate brain were characterized in respect to PV-hydrolysing activity and to their inhibition rate constants for the reaction with organophosphorus compounds. Carboxylesterase III was identified as neurotoxic esterase (NTE). Organophosphate inhibition data of primate acetylcholinesterase (EC 3.1.1.7) and of primate cholinesterase (EC 3.1.1.8) were determined and are compared to corresponding data of primate brain carboxylesterases. Physiological functions, clinical and toxicological significance of primate brain carboxylesterases are discussed.

Preparation of two neurotoxic esterases from the chick central nervous system

A standard procedure for lipid-extraction of lyophilized hen brain material is described. Nine carboxylesterase isoenzymes (EC 3.1.1.1) are identified in lipid-extracted lyophilized material (LELM) using kinetic analysis of organophosphate inhibition. Total phenyl valerate (PV) hydrolysing carboxylesterase activity in LELM is 43.3 U X g-1. Two carboxylesterase isoenzymes of LELM are classified as neurotoxic esterases (NTEA and NTEB). Using n-octylglucoside 51% of the water-insoluble neurotoxic esterase activity from LELM are solubilized. Six carboxylesterase isoenzymes including NTEA (6.5 U X 1(-1] and NTEB (4.2 U X 1(-1] are present in the solubilized preparation. Throughout purification and separation steps carboxylesterase isoenzymes are identified by their rate constants for the reaction with organophosphorus inhibitors.

Zur altersabhängigen Verminderung von Lipoproteinen hoher Dichte (HDL) bei Frauen nach der Menopause

BACKGROUND The decline in the concentration of high density lipoproteins (HDL) observed in postmenopausal women is thought to contribute to the increasing incidence of coronary artery disease (CAD) after menopause. Human serum arylesterase (EC 3.1.1.2) is exclusively associated with HDL. We therefore investigated possible differences in the decline of HDL-levels and of HDL-subfractions HDL2 and HDL3 between postmenopausal women without and with angiographically documented CAD. PATIENTS AND METHODS HDL-, HDL2-and-HDL3- concentrations were studied in postmenopausal women with angiographically documented CAD (n = 24; 51 to 72 years mean: 62 years) and compared to HDL-parameters of women without CAD (n = 22; 51 to 81 years, mean: 58 years). Arylesterase activities of HDL2-and HDL3-subfractions and HDL2-cholesterol concentrations were determined after differential precipitation with polyethylene glycol (4.7 mM PEG). Phenotyping of HDL-arylesterase was achieved in CAD patients and in women without CAD after determining hydrolysis of arylesterase substrates paraoxon (PO) and phenylacetate (PA) by calculating paraoxonase/arylesterase activity ratios R (R = [PO]/[PA] x 1000): phenotype A (n = 26) with R < 2.5, phenotype AB (n = 16) with 5.0 < R < 10.7, and phenotype B (n = 4) with R > 13.5. RESULTS In postmenopausal women with documented CAD, as compared to women without CAD, HDL-cholesterol (55 +/- 3 mg/dl vs. 69 +/- 3 mg/dl HDL2-arylesterase (25 +/- 1 kU/l vs. 33 +/- 2 kU/l), and HDL3-arylesterase (89 +/- 4 kU/l vs. 106 +/- 5 kU/I) were found to be significantly reduced. Analysis of the correlation of lipid parameters and age revealed in CAD patients, but not in postmenopausal women without CAD, a significant increase of total cholesterol (r = 0.42), and significant reductions of both HDL2-arylesterase (r = -0.47) and HDL3-arylesterase (r = 0.74) with increasing age. In contrast, HDL-cholesterol (r = -0.14) and HDL2-cholesterol (r = -0.06) of CAD patients showed only slight and non-significant reductions with age. Since HDL3-arylesterase was found to be age-dependently reduced in women without CAD (r = 0.17), HDL2-arylesterase of postmenopausal women, among all lipid parameters showed the most pronounced differences between women without CAD and CAD patients. The age-dependent decrease of HDL2-arylesterase in postmenopausal women with CAD does not result from an increased frequency of B-allele carriers in the subgroup of CAD patients with an age above the median (64 years). CONCLUSION Genetically determined serum HDL-arylesterase is well suited to quantify HDL in postmenopausal women without and with CAD. HDL2-arylesterase of postmenopausal women should be evaluated as a screening parameter for both primary and secondary CAD prevention.Zusammenfassung□ HintergrundBei postmenopausalen Frauen trägt die Abnahme der Konzentration von Lipoproteinen hoher Dichte (HDL) zur steigenden Inzidenz der koronaren Herzkrankheit bei. Die Arylesterase (EC 3.1.1.2) des menschlichen Serums ist ausschließlich mit HDL assoziiert. Es sollte daher untersucht werden, ob HDL und die HDL-Subfraktionen HDL2 und DHL3, quantifiziert über die HDL-Arylesterase, bei postmenopausalen Frauen ohne und mit koronarer Herzkrankheit unterschiedlich stark vermindert sind.□ Patienten und MethodenBei postmenopausalen Frauen mit angiographisch gesicherter koronarer Herzkrankheit (n=24; Alter 51 bis 72 Jahre, im Mittel 62 Jahre) wurde die altersabhängigkeit der Konzentrationen von HDL, HDL2 und HDL3 untersucht und jeweils mit derjenigen von koronargesunden Frauen (n=22; 51 bis 81 Jahre, im Mittel 58 Jahre) verglichen. Nach fraktionierter Präzipitation mit Polyäthylenglykol (4,7 mM PEG) wurden die Arylesterase-aktivitäten der HDL2-und der HDL3-Subfraktion und das HDL2-Cholesterin bestimmt. Der Phänotyp der HDL-Arylesterase wurde bei Patientinnen mit koronarer Herzkrankheit und koronargesunden Frauen nach Bestimmung des Umsatzes der Arylesterasesubstrate Paraoxon (PO) und Phenylacetat (PA) über die Berechnung des Paraoxonase/Arylesterase-Aktivitätsquotienten R ermittelt (R=[PO]/[PA]×1000): Phänotyp A (n=26) mit R<2,5, Phänotyp AB (n=16) mit 5,0<R<10,7 und Phänotyp B (n=4) mit R> 13,5.□ ErgebnisseBei postmenopausalen Patientinnen mit koronarer Herzkrankheit waren gegenüber koronargesunden Frauen nach der Menopause HDL-Cholesterin (55±3 mg/dl vs. 69±3 mg/dl), HDL2-Arylesterase (25±1 kU/l vs. 33±2 kU/l) und HDL3-Arylesterase (89±4 kU/l vs. 106±5 kU/l) jeweils signifikant vermindert. Bei Untersuchung der Korrelation der Lipidparameter mit dem Lebensalter ergab sich bei Patientinnen mit koronarer Herzkrankheit, nicht dagegen bei koronargesunden Frauen nach der Menopause für das Gesamtcholesterin ein signifikanter Anstieg (r=0,42), für die HDL2-Arylesterase (r=−0,47) und die HDL3-Arylesterase (r=−0,74) jeweils mit zunehmendem Lebensalter eine signifikante Verminderung. Dagegen waren bei Patientinnen mit koronarer Herzkrankheit HDL-Cholesterin (r=−0,14) und HDL2-Cholesterins (r=−0,06) jeweils altersabhängig vermindert, ohne daß statistische Signifikanz erreicht wurde. Da die HDL3-Arylesterase auch bei apostmenopausalen koronargesunden Frauen mit dem Lebensalter abfiel (r=−0,17), zeigte von allen untersuchsten Lipidparameter nach der Menopause die HDL2-Arylesterase die deutlichsten Unterschiede zwischen koronargesunden Frauen und Patientinnen mit koronarer Herzkrankheit. Die altersabhängige Abnahme der HDL2-Arylesterase-Aktivität bei postmenopausalen Frauen mit koronarer Herzkrankheit beruht nicht auf einem höheren Anteil von Frauen mit dem B-Allel der HDL-Arylesterase in der Altersgruppe oberhalb des Altersmedians (64 Jahre).□ SchlußfolgerungDie genetisch determinierte HDL-Arylesterase des Serums ist zur Quantifizierung von HDL bei postmenopausalen Frauen ohne und mit koronarer Herzkrankheit geeignet. Die HDL2-Arylesterase sollte bei Frauen nach der Menopause als Screening-Parameter für die Primär- und Sekundärprävention der koronaren Herzkrankheit evaluiert werden.Summary□ BackgroundThe decline in the concentration of high density lipoproteins (HDL) observed in postmenopausal women is thought to contribute to the increasing incidence of coronary artery disease (CAD) after menopause. Human serum arylesterase (EC 3.1.1.2) is exclusively associated with HDL. We therefore investigate possible differences in the decline of HDL-levels and of HDL-subfractions HDL2 and HDL3 between postmenopausal women without and with angiographically documented CAD.□ Patients and MethodsHDL-, HDL2- and HDL3-concentrations were studied in postmenopausal women with angiographically documented CAD (n=24; 51 to 72 years, mean: 62 years) and compared to HDL-parameters of women without CAD (n=22; 51 to 81 years, mean: 58 years). Arylesterase activities of HDL2-and HDL3-subfractions and HDL2-cholesterol concentrations were determined after differential precipitation with polyethylene glycol (4.7 mM PEG). Phenotyping of HDL-arylesterase was achieved in CAD patients and in women without CAD after determining hydrolysis of arylesterase substrates paraoxon (PO) and phenylacetate (PA) by calculating paraoxonase/arylesterase activity ratios R (R=[PO]/[PA]×1000): phenotype A (n=26) with R<2.5, phenotype AB (n=16) with 5.0<R<10.7, and phenotype B (n=4) with R<13.5.□ ResultsIn postmenopausal women with documented CAD, as compared to women without CAD, HDL-cholesterol (55±3 mg/dl vs 69±3 mg/dl), HDL2-arylesterase (25±1 kU/l vs 33±22 kU/1), and HDL3-arylesterase (89±4 kU/l vs 106±5 kU/I) were found to be significantly reduced. Analysis of the correlation of lipid parameters and age revealed in CAD patients, but not in postmenopausal women without CAD, a significant increase of total cholesterol (r=0.42), and significant reduction of both HDL2-arylesterase (r=−0.47) and HDL3-arylesterase (r=−0.74) with increasing age. In contrast, HDL-cholesterol (r=−0.14) and HDL2-cholesterol (r=−0.06) of CAD patients showed only slight and non-significan reductions with age. Since HDL3-arylesterase was found to be age-dependently reduced in women without CAD (r=−0.17), HDL2-arylesterase of postmenopausal women, among all lipid parameters showed the most prononced difference between women without CAD and CAD patients. The age-dependent decrease of HDL2-arylesterase in postmenopausal women with CAD does not result from an increased frequency of B-allele carriers in the subgroup of CAD patients with an age above the median (64 years).□ ConclusionGenetically determined serum HDL-arylesterase is well suited to quantify HDL in postmenopausal women without and with CAD. HDL2-arylesterase of postmenopausal women should be evaluated as a screening parameter for both primary and secondary CAD prevention.

Computerized analysis of covalent inhibition kinetics for identification of heart muscle cholinesterase and brain carboxylesterase isoenzymes. Design of differential inhibition assays

The kinetics of time- and concentration-dependent covalent organophosphorus inhibition of carboxylesterase isoenzymes (EC 3.1.1.1) and cholinesterase isoenzymes (EC 3.1.1.7 and EC 3.1.1.8) were investigated using a wide range of organophosphate inhibitor concentrations (10(-10)-10(-3) mol/l) and different inhibition times. Computerized analysis of inhibition curves by weighted non-linear least-squares curve fitting was compared to graphic analysis by iterative elimination of exponential functions. Possible experimental errors due to inhibitor saturation kinetics and enzymatic organophosphate hydrolysis were thoroughly investigated. In mammalian heart muscle, three different cholinesterase isoenzymes were identified. High sensitivity and specificity of the classic differential inhibition test for carboxylesterase activity of hen brain neuropathy target esterase (NTE) could be confirmed independently with both methods of inhibition curve analysis.