Protective effect of the new calcium antagonist anipamil against isoprenaline-induced cardionecrosis in rats.

The new calcium antagonist anipamil (1,7-bis-(3-methoxyphenyl)-3-methylaza-7-cyano-nonadecane) exhibited a pronounced protective effect against isoprenaline-induced myocardial necrosis in rats. Anipamil was administered in single doses of 10 or 20 mg/kg daily for 4 days. 30 mg/kg isoprenaline was given by subcutaneous injection on the 3rd and 4th days of the study. The protective effect of anipamil was assessed by histological investigations, and its effect on the activity of the enzymes succinate dehydrogenase, NADH-NBT reductase, acid phosphatase and glucose-6-phosphate dehydrogenase in experimentally-induced myocardial damage was assessed quantitatively by microphotometry. The protective effect of anipamil against isoprenaline-induced myocardial necrosis was definitely dose-dependent: 10 mg/kg anipamil exhibited a partial protective effect, whilst 20 mg/kg anipamil protected the heart completely.

Post-mating non-infectious hydrometra in BALB/c: Bom mice.

Post-mating non-infectious hydrometra and hydrovagina of unknown aetiology, leading to a scrotum-like swelling of the perineum, was observed during an experiment on BALB/c: Bom mice. There was a closure of the vulva with accumulation of secretions of uterine glands under high pressure in both uterus and vagina. The mice were otherwise clinically healthy, gained body weight normally, but were incapable of breeding. The morbidity was approximately 1% and the disease could not be transmitted to other females.

[Development of volatile compounds from radicals arising during intermediate steps of the lipoxygenase-reaction (author's transl)]. / Untersuchungen über die Entstehung flüchtiger Verbindungen aus radikalischen Reaktionszwischenstufen bei der Lipoxygenase-Reaktion.

The development of volatile compounds arising from radicals during intermediate reaction steps of the lipoxygenase-linoleic-acid-reaction (from soy) was investigated in model experiments with defined conditions. The results indicate as follows: Among the volatile compounds formed, hexanal has a special position. Its development is closely connected to the enzymatic formation of the 13-linoleic-acid-hydroperoxide because it is formed from the 13-linoleic-acid-peroxy-radical, which is a direct precursor of the 13-linoleic-acid-hydroperoxide. The other volatile products are apparently formed in the same way as by autoxidation. Their development is favoured by lack of oxygen, when the primarily formed linoleic-acid-radicals cannot react rapidly with the oxygen-radicals to form hydroperoxides. A small part of the volatile compounds is formed by autoxidation which always accompanies the enzymatic reaction.

[On lipoxygenase and enzymes which decompose linoleic acid hydroperoxides in rye (author's transl)]. / Uber Lipoxygenase und Linolsäurehydroperoxid-abbauende enzyme in Roggen.

An enzyme fraction from rye containing lipoxygenase activity was investigated. The molecular weight of lipoxygenase was found to be about 102000. Two bands groups with isoelectric points between 5.1-5.5 and 5.8-6.4 were obtained by isoelectric focusing. Three isoenzymes could be separated by ion exchange chromatography. Lipoxygenase has optimum activity at pH 7.3-7.5 and predominantly forms 13-hydroperoxy-9-cis, 11-trans-octadecadienoic acid (13-LHPO). In rye the 13-LHPO is converted to alpha-ketols by a high molecular protein fraction. This isomerase converts the LHPO formed by rye lipoxygenase predominantly to 12,13-ketohydroxy acids. The Michaelis Constant of isomerase is 3-5 X 10(-5), using LHPO as substrate. At low protein concentrations the reaction velocity of LHPO-conversion increases linearly with protein concentration.

[Hydroperoxide degradation by oat isomerase. Investigation of the reaction mechanism (author's transl)]. / Hydroperoxid-Abbau durch Isomerase aus Hafer. Untersuchungen zum Reaktionsmechanismus.

Oat isomerase is inhibited by hydroxyoctadecadienoic acids (monohydroxy acids) to a degree comparable with inhibition by linoleic acid hydroperoxides (LHPO). Hydroxy acids seem to combine with the enzyme like LHPO do. In an experiment on LHPO breakdown by isomerase 1-14C-hydroxy acids were added and it was examined whether the epoxyhydroxy acids are formed by an intermolecular or intramolecular mechanism. In this experiment 1-14C-labeled trihydroxy acids were formed; they arise from the hydrolysis of epoxyhydroxyoctadecenoic acids formed on their part by isomerase effected LHPO-breakdown. It was determined that at least 70% of LHPO are converted by intermolecular reaction.

[On the lipoperoxidase-isomerase-system from oat. Experiments for enzyme separation (author's transl)]. / Untersuchung über das Lipoperoxidase-Isomerase-System von Hafer. Versuche zur Trennung der Enzyme.

It was attempted to separate isomerase and lipoperoxidase activity from oat by means of gel and ion exchange chromatography and by isoelectric focusing. The molecular weight of the enzyme fraction tested was found to be of the order of 3 X 10(6) daltons. By ion exchange chromatography and isoelectric focusing the enzyme fraction with lipoperoxidase and isomerase activity could be separated into isoenzymes. These isoenzymes continued to exhibit both enzymatic activities. The lack of success in achieving of separation and the high molecular weight lead to the conclusion of the asistence of a close interrelation between the two enzymes or possibly of a bifunctional enzyme complex.

[Enzymatic breakdown of linoleic acid hydroperoxides to volatile carbonyl compounds by isomerase from barley (author's transl)]. / Enzymatischer Abbau von Linolsäurehydroperoxiden zu flüchtigen Carbonylverbindungen durch Isomerase aus Gerste.

Barley protein fractions with active isomerase, purified by means of gelchromatography were incubated at room temperature with linoleic acid hydroperoxides (LHPO), containing 9-hydroperoxy-10-trans,12-cis-octadecadienoic acid (9-LHPO) and 13-hydroperoxy-9-cis,11-trans-octadecadienoic acid (13-LHPO) in the ratio of about 1:1. The volatile compounds resulting from the reaction have been isolated, concentrated and investigated by means of gas- and radio-gaschromatography. In the case of incomplete LHPO-breakdown remaining hydroperoxides and nonvolatile breakdown products have been separated before gaschromatographic analysis. In addition to hexanal as main product, traces of 2-tr-heptenal and 2-tr-octenal were found; about 6% of the converted hydroperoxides were transformed to carbonyl compounds. By numerous additional experiments it was confirmed that the volatile compounds are formed by enzymatic catalysis.

[On characterization of lipoxygenase from barley (author's transl)]. / Zur Charakterisierung der Lipoxygenase aus Gerste.

Lipoxygenase from brewing barley of Nordbaden has optimum activity at alkaline pH and predominantly forms 9-hydroperoxy-10-trans, 12-cis-octadecadienoic acid (9-LHPO) from linoleic acid. While at pH 7 90% 9-LHPO is produced, its proportions drops during incubation at more alkaline pH (pH 7,75) to 70%. This positional specifity is not caused by isoenzymes. While linoleic acid methylester is converted only to a lesser degree, trilinolein is not accepted as substrate.

[Carbonyl compounds from the reaction of barley isomerase with linoleic acid hydroperoxides. Their development from the 9- or the 13-hydroperoxide-isomer (author's transl)]. / Flüchtige Carbonylverbindungen aus der Reaktion von Gerstenisomerase mit Linolsäurehydroperoxiden. Ihre Entstehung aus den 9- oder 13-Hydroperoxid-Isomeren.

UNLABELLED: Hydroperoxid isomerase from barley was incubated with linoleic acid hydroperoxides (LHPO), containing nearly exclusively the 13-LHPO or the 9-LHPO isomer; the volatile reaction products were isolated, concentrated and investigated by means of gas and radio-gaschromatography. Thus it was possible to establish the precursors of the volatile compounds hexanal, 2-trans-heptenal and 2-trans-octenal, which develop during formerly described reactions of isomerase with substrates, containing 9- and 13-LHPO in equal amounts. 13-LHPO was found to be a precursor of hexanal and 2-tr-octenal, while the 9-LHPO isomer in the barley isomerase LHPO breakdown reaction obviously cannot be accepted as precursor of volatile components. The origin of 2-tr-heptenal could not be clarified; it occured neither in the experiments with predominating 9-LHPO nor in those with predominating 13-LHPO. Perhaps 2-tr-heptenal is only produced in the presence of a defined ratio of both isomeric hydroperoxides. ABBREVIATIONS: 13-LHPO = 13-hydroperoxy-9-cis, 11-trans-octadecadienoic acid. 9-LHPO = 9-hydroperoxy-10-trans, 12-cis-octadecadienoic acid.

[Histological and biochemical studies on the postnatal development of the guinea pig liver (author's transl)]. / Histologische und biochemische Untersuchungen zur postnatalen Entwicklung der Leber beim Meerschweinchen

PROBLEM: In newborn guinea pigs the structural development of the liver was studied with emphasis on changes in the lipid content as related to biochemical parameters. MATERIAL AND METHODS: The livers of 149 guinea pigs (strain PIRBRIGHT) up to an age of 21 days were examined. The mothers were grouped in cages and kept as usual (room temperature 22 degrees C, air humidity 60-70%, natural light-dark rhythm and commercial standard diet ad libitum, sawdust and straw as bedding). Pregnant animals were placed in individual cages being left there together with their offspring until weaning or sacrification. In this period additional uptake of solid diet was allowed to the guinea pigs. The animals were bleeded to death in anesthesia (by head stroke). For histological examination tissue samples from the Lobes sinister lateralis were fixed in Barker's formol or absolute alcohol. The following staining reactions were applied (paraffin embedding or frozen sections): haemalum-eosin stain; PAS-reaction; Sudan III; Sudan black; Best's carmine (for details on the techniques see ROMEIS 1968). In biochemical analysis the total lipids of the liver were determined by the isolation methods reported by FOLCH et al.(1957). Moreover, in modification of the analytical procedures earlier described (SALLMANN 1972 a, b) several fractions of the liver total lipids could be isolated. By dialysis against a rubber membrane a dialysable lipid component (hydrocarbons, triglycerides, incomplete glycerides, cholesterol and free lipid acids) was isolated from the phosphatide fractions. Further separation of the "dialysable" lipids on Florisil columns yielded purified glyceride fractions. The total lipids as well as all components isolated from these were determined gravimetrically after removal of the solvents concerned. In biochemical analysis the livers of fetuses 10-12 days ante partum and of young animals up to 53 days were included. Results (see also table 1): The relative liver weight--related to the body weight reduced for that of the gastrointestinal tract--is highest at birth. It is lowered independent of the development of the gastrointestinal tract within a short period, then it remains fairly constant during the rest of the time of observation. On the 1st day abundant lipids are regularly distributed throughout the whole liver lobule (fig. 1). Beginning with the 3rd day the central parts of the lobules are nearly free from lipids, only in the peripheral zones of the lobules lipid augmentation was still observed. Consequently the marked decrease in liver weight is due to the rapid reduction of lipids within the first 3 days of life. These observations are in accordance with the biochemical findings: about 10 days ante partum noticeable storage of lipids occurs in the liver reaching its peak 2 to 3 days after birth (physiological fatty infiltration of the liver during the last days of intrauterine life). In fetal liver tissue predominantly tryglycerides are augmented.

[Investigations on the development of volatile substances during lipoxygenase-linolic-acid-reaction (author's transl)]. / Untersuchungen über Entstehungswege flüchtiger Substanzen bei der Lipoxygenase-Linolsäure-Reaktion

During incubation of soja-lipoxygenase with linolic acid, volatile compounds are formed the development of which can be seen in two possible ways:from preformed linolic-acid-hydroperoxides splitproducts arise or volatile substances of different chemical nature are built depending on the reaction conditions like temperature, O2-pressue, partner-concentration etc. By trials with hydroperoxyde-decomposing enzymes (peroxidase) and by means of radio-active labelled linolic-acid-hydroperoxides the pathways mentioned above were investigated. The results indicate that the volatile compounds are built from by-products; n-hexanal was formed from these by-products as well as from decomposed hydroperoxide. The previously proposed reaction-scheme has this been ascertained by experimental means.

[Analysis of volatile aldehydes by radio-gaschromatography (author's transl)]. / Radiogaschromatographische Analyse flüchtiger Aldehyde, die bei der mit Soja- und Hafer-Lipoxygenase katalysierten Linolsäure-Oxidation entstehen

Soya- and oats-lipoxygenase (E.C. 1.13.1.13.) are incubated by 14C-marked linoleic acid. The volatile aldehydes arising thereby are isolated. The activity of the components separated by gaschromatography is written down by a printing indicator and the impulses/min are registered and printed out by a ratemeter. Thus the aldehydes which are produced by the enzymatic oxydation with lipoxygenase from the molecule of the linoleic acid can be determined. The composition of the mixture of aldehyde is calculated in mol-% from the measured impulses per peak. A possible origin of pathway is indicated for the main reaction products hexanal (soya-lipoxygenase) and non-trans-2-enal (oats-lipoxygenase).

[About the formation of volatile products during lipoxigenase-linoleic-acid reaction (author's transl)]. / Uber die Bildung flüchtiger Produkte bei der Lipoxigenase-Linolsäure-Reaktion

Soya-lipoxigenase (E.C. 1.13.1.13) is incubated by linoleic acid at room temperature; the arising volatile products are isolated by different methods, concentrated, and investigated by means of gas chromatography. If the non-volatile hydroperoxides, formed during the incubation are also injected, 15-20% are decomposed to volatile products superposing the primarily enzymatically formed volatile components as artefacts and consequently falsifying the result. Without separating the linoleic acid hydroperoxides (LHPO) the quantitative proportion Hexanal/Decadienal is approximately in accordance with the proportion 13-linoleic acid hydroperoxide/9-linoleic acid hydroperoxide (this means for soya-lipoxigenase and pH 7 about 1:1). After separating the linoleic acid hydroperoxides only these volatile products are found which are arising during the lipoxigenase reaction. These are 1-2% in relation to the LHPO. In this case, the quantitative ratio Hexanal/Decadienal is not corresponding to the 13-/9-linoleic acid hydroperoxide proportion. Nearly the only product formed by this process is Hexanal.