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H Debuch

Publications and source records attributed to H Debuch.

At least 37 records · Page 2Linked to original sources

Effect of chloroquine treatment on the different phospholipid species of rat liver lysosomes.

The lysosomal phospholipids of rat liver were determined after treatment of the animals with chloroquine for varying periods up to 15 days. During this period, all the measured phospholipids increased. After two weeks of treatment the absolute level of each phospholipid had increased 3 to 6-fold, with the exception of bis(monoacylglycero)phosphate which was increased 25-fold, accounting for one third of the total phospholipids. The fatty acid pattern not only differed between the classes of lipids; it changed during treatment within every lipid class. In addition to other changes, the polyenoic fatty acids of the main glycerophospholipids were markedly reduced between the 3rd and 14th day of chloroquine treatment: to one half in phosphatidylcholine, to about one tenth in phosphatidylethanolamine, and to one quarter in phosphatidylinositol. However, the polyenoic fatty acid content of bis(monoacylglycero)phosphate was doubled, reaching 76% of the total at the end of treatment. Whereas docosahexaenoic acid was not detected in this acidic phospholipid in the first 3 days of treatment, it was present to the extent of 55% of the total fatty acids after 15 days of chloroquine application;

Acid Phosphatase↗

Liberation of free aldehyde from 1-(1-alkenyl)-sn-glycero-3-phosphoethanolamine (lysoplasmalogen) by rat liver microsomes.

We found an enzyme in the microsomal fraction of 21-day-old-rat liver, which liberates a free aldehyde from 1-(1-alkenyl)-sn-glycero-3-phosphoethanolamine (lysoplasmalogen) and which has an activity of about 42 mU/mg protein under the conditions described. Kinetic data are presented. The pH optimum is found around pH 7.1. SH-blocking reagents, as well as deoxycholate, act as strong inhibitors, while Mg2 and Ca2 also inhibit the reaction to some extent. The enzymic activity is specific with respect to the monoradylphospholipid, since the acylated compound 2-acyl-1-(1-alkenyl)-sn-glycero-3-phosphoethanolamine does not serve as substrate. The ether linkage of 1-alkyl-sn-glycero-3-phosphoethanolamine is not hydrolyzed either under these conditions. A similar enzyme activity in liver has only been described for choline-containing lysoplasmalogen.

Animals↗

Production of bis(monoacylglycero)phosphate from phosphatidylglycerol in isolated liver lysosomes of chloroquine-pretreated rats.

Labelled phosphatidylglycerol was incubated with rat liver lysosomes from animals treated for 3 to 20 days with chloroquine diphosphate. The longer the period of pretreatment with the amphiphilic drug, the greater was the increase in the synthesis rate of bis(monoacylglycero)phosphate, both in the absolute values and when related to the lysosomal protein which was also increased. The mechanism of the in vitro conversion of phosphatidylglycerol to bis(monoacylglycero)phosphate was studied by using phosphatidylglycerol labelled with 14C and/or 3H in different positions of the molecule. Assays with rac-1-(1,2-diacyl-[2-3H]glycero-3-phospho)-[U-14C]glycerol clearly demonstrated that the 3H/14C ratio of the substrate was the same as found in the product bis(monoacylglycero)phosphate. Therefore the whole glycerophosphoglycerol backbone of the substrate is used for bis(monoacylglycero)phosphate formation, and recombination of released glycerol moieties can be excluded. Experiments with phosphatidylglycerol labelled in both fatty acids suggest that only one acyl group of the substrate is preserved in bis(monoacylglycero)-phosphate. The analysis of further products formed during incubations of rat liver lysosomes with labelled phosphatidylglycerol showed a rapid degradation of the glycerolipid mainly by the action of phospholipase A and C.

Animals↗

Studies on the properties of phospholipase A1 in liver lysosomes of chloroquine-treated rats.

The influence of chloroquine on phospholipase A1 (acid pH optimum) activity was studied in subcellular fractions of rat liver after intraperitoneal application of the drug for 9, 12 and 21 days. In comparison with other cell fractions lysosomes of treated rats contained the highest enzyme activity with a pH optimum of 4.0. The activity of phospholipase A1 in lysosomes showed a direct relationship to the number of days of chloroquine treatment. The effects of the incubation time, Ca2+, Hg2+, enzyme and substrate concentration on phospholipase A1 activity were studied.

Animals↗

Interactions of chloroquine with different glycerophospholipids.

Amphiphilic drugs are able to accumulate acidic phospholipids in different animal tissues, such as the pineal gland, iris muscle, retina and also in lymphocytes. Chloroquine, an amphiphilic cation, causes a phospholipidosis in liver. Not only is the phospholipid content markedly increased but an unusual acidic glycerophospholipid bis(monoacylglycero)phosphate is enriched many-fold in rat liver lysosomes if the animals have been treated with this drug. Since we found bis(monoacylglycero)-phosphate to be present in these cell organelles of untreated animals in only small amounts, we wanted to prove that chloroquine is able to react with lipids. If this were the case, we were interested in the kind of interactions taking place between the drug and the lipids.

Chemical Phenomena↗

The lipids of the Golgi apparatus subfractions from rat liver.

Golgi apparatus were isolated from untreated rat liver and separated into three fractions. One consisted mainly of vesicles, a second of tubular particles (dictyosomes) and the third was a mixed fraction. Large differences between these fractions could be seen in the electron microscope and by enzyme analysis. The total lipid content of the vesicles was 3.5-times greater than that of the dictyosomes and the neutral lipid value was 7-times greater. The ratio of phospholipids to protein was approximately the same in the three fractions. However, the phospholipid patterns differed between the vesicle and dictyosome fractions.

Animals↗

Studies on the hydrolysis of 1-alkyl-sn-glycero-3-phosphoethanolamine in subcellular fractions of rat brain.

The formation of 1-alkylglycerol from 1-alkyl-sn-glycero-3-phosphoethanolamine in different cell fractions of rat brain is reported. The substrates used were labelled either with 14C or 3H in the alkyl residue or with 14C in the alkyl and 3H in the ethanolamine residue. The examination of the lipid- and water-soluble cleavage products showed that both ethanolamine and phosphoethanolamine are liberated from the substrate in the microsomal fraction of 14-day-old rat brain. The latter product is rapidly hydrolyzed. In comparison with other cell fractions, the microsomes contained the highest enzyme activities, which exhibited a pH optimum of 7.1--7.5. SH-group reagents are inhibitors, whereas diisopropylfluorophosphate has no effect. As the animals age, these enzyme activities decrease in brain homogenates.

Animals↗

Studies on the hydrolysis of 1-alk-1'-enyl-sn-glycero-3-phosphoethanolamine by microsomes from myelinating rat brain.

Microsomal fractions of 14-day-old rat brain were incubated at pH 7.1 with 1-[1'-14C]-alk-1'-enyl-sn-glycero-3-phosphoethanolamine (lysoplasmalogen). 1-[1'-14C]alkenylglycerol was produced by hydrolyzing enzyme activities, which were stimulated by Mg2 and inhibited by SH-group reagents. Hydrolysis of 1-[1'-14C]alkyl-sn-glycero-3-phosphoethanolamine is very similar in this respect, but the Km value is higher in the former case. The 1-alkyl compound acts as a non-competitive inhibitor of the hydrolyzing enzyme activity described, whereas the hydrolysis of the 1-alkyl derivative is not inhibited by the 1-alkenyl compound.

Animals↗

Lymph node excision as a simple diagnostic aid in rare lipidoses.

Autopsy material from a case of Niemann-Pick disease was subjected to lipid analysis. Among the six tissues investigated, lymph nodes exhibited the greatest storage of several lipids. Since lymph nodes are relatively easy to obtain by biopsy, they may be utilized for chemical diagnosis of this type of lipidosis.

Child↗

Lysosomal phospholipids from rat liver after treatment with different drugs.

Rats were treated with 5 different drugs p-ethoxyacetanilide (I), indometacin (II) and nor-amidopyrine-methanesulfonate (III), O,O'-bis(diethylaminoethyl)hexestrol(IV) and choloroquine (V) for 3 - 4 weeks. Liver cell fractions were isolated by discontinuous gradient centrifugation and the specific activity of acid phosphatase was determined in each. Lysosomal fractions contained widely varying amounts of this marker enzyme, indicating that the concentration of lysosomes within these fractions differed. The amounts and patterns of phospholipids reflected this fact. Since we assumed bis(monoacylglycero)phosphate [(MAG)2-P; synonym:lysobisphosphatidic acid] is a marker lipid for secondary lysosomes, we expected and found significant quantities of this acidic phospholipid only in those lysosomal fractions which were also rich in acid phosphatase activity. 12% of the lysosomal phospholipids from animals receiving the hexestrol derivative (IV), and 19% of those from the chloroquine (V) experiment were present as (MAG)2P. The fatty acid compositions of this lysosomal phospholipid were not the same in all lysosome fractions. The more (MAG)2P present in the lysosomes, the more unsaturated are the fatty acids. Thus, after treatment with chloroquine, more than 90% of the fatty acids from (MAG)2P are unsaturated; C22:6 represents about 70% of the total.

Acid Phosphatase↗

Lysosomal bis (monoacylglycero)phosphate of rat liver, its induction by chloroquine and its structure.

Since chloroquine is able to induce phospholipidosis, and in particular formation of bis(monoacylglycero)phosphate [(MAG)2P] in rat liver lysosomes, we tested the influence of the drug on this acidic phospholipid 1) after application of the drug for different periods, 3, 7, 14, 21 and 28 days, and 2) after application of different doses of the drug for 7 days. In both cases, the amounts of the total phospholipids and of (MAG)2P within the lysosomal lipids showed a direct relationship to the quantity of the drug. Lysosomal fractions were identified by acid phosphatase activity and by electron microscopy. After 14 days of treatment, the lysosomal fractions showed an increase in the marker enzyme to more than 30 times the level in the homogenate, and consisted of almost pure "lamellar bodies". (MAG)2P was isolated from those lysosomal lipid fractions by column chromatography. We also isolated this phospholipid from "tritosomal" lipids and tested the two preparations in different ways. They both showed glycerophosphoglycerol as the only P-containing product after mild alkaline hydrolysis, and molar ratios for phosphorus: fatty acid: glycerol close to 1:2:2; and yielded diacetylacyl glycerol after acetolysis. Thus, there is no doubt that (MAG)2P occurs in these secondary lysosomes--as we described in others--and that its production is a consequence of accumulation of lysosomes. There is some indication for the occurrence of an acylated (MAG)2P (which is the same as acylphosphatidylglycerol) within the same cell organelles.

Animals↗