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Biomedical subjects

R C Reitz

Publications and source records attributed to R C Reitz.

At least 19 recordsLinked to original sources

Regulation of sex pheromone biosynthesis in the housefly, Musca domestica: relative contribution of the elongation and reductive steps.

The regulation of production of the sex pheromone (Z)-9-tricosene (Z9-23:Hy) in the housefly, Musca domestica, was studied by examining the chain length specificity of the fatty acyl-CoA elongation reactions and the reductive conversion of fatty acyl-CoAs to alkenes in 1- and 4-day-old male and female houseflies. Microsomal preparations from 4-day-old female insects produced as the predominant alkene Z9-23:Hy when incubated with malonyl-CoA, NADPH, and [9,10-3H2]oleoyl-CoA (18:1-CoA), whereas microsomal preparations from 4-day-old male insects produced predominantly (Z)-9-heptacosene (Z9-27:Hy). These are the major alkenes produced in vivo by Day 4 females and males, respectively. Microsomes prepared from both Day 1 males and Day 1 females produced Z9-27:Hy as the major alkene from labeled 18:1-CoA. This is the major alkene produced in vivo by both sexes at Day 1. An examination of the chain length specificity of the elongation reactions showed that microsomes prepared from Day 4 male insects readily elongated both 18:1-CoA and 15-[15,16-3H2]tetracosenoyl-CoA (24:1-CoA) to 28-carbon moieties, whereas microsomes from Day 4 female insects did not efficiently elongate either substrate beyond 24 carbons. With high substrate concentrations, microsomes prepared from male insects converted 24:1-CoA to Z9-23:Hy more efficiently than did those from females, whereas under lower and presumably more physiological substrate concentrations, microsomes from females had slightly higher activity than did those from males. Taken together, these data show that the regulation of the chain length of the alkenes, and thus sex pheromone production, in the housefly resides predominantly in the elongation reactions and not in the step which converts the fatty acyl-CoA to hydrocarbon.

Acyl Coenzyme A

Effects of high alpha-linolenate and linoleate diets on erythrocyte deformability and hematological indices in rats.

Rats were fed either a high alpha-linolenate diet or a high linoleate diet from weaning to 4 mon of age. Soybean oil was used as a control. Phospholipid compositions of erythrocytes from the three dietary groups were not significantly different. However, the difference in the alpha-linolenate (18:3n-3)/linoleate (18:2n-6) ratio of the diets was reflected in the n-3/n-6 ratios of the 20 and 22 carbon highly unsaturated fatty acids except for docosahexaenoic acid (22:6n-3) in the phospholipids. Despite the significant differences in the fatty acid compositions of phospholipids, no measurable differences were detectable in erythrocyte deformability, whole blood viscosity and hematological indices of the three dietary groups. These results indicate that the beneficial effects of the high alpha-linolenate diet, as compared with the high linoleate diet, are exerted without significant changes in these parameters.

Animals

Phosphatidylethanolamine N-methyltransferase in human red blood cell membrane preparations. Kinetic mechanism.

The successive methylations of phosphatidylethanolamine to form phosphatidylcholine were measured using exogenously added intermediates and membrane preparations from human red blood cells. The addition of phosphatidylethanolamine resulted in no increase in methylation rate over that with endogenous substrate; however, the addition of monomethylphosphatidylethanolamine (PME) and dimethylphosphatidylethanolamine (PDE) markedly increased the reaction rate and allowed studies into the kinetic mechanism for the second and third methylation reactions. The data are consistent with catalysis of the last two methylations being by a single enzyme with a random Bi-Bi sequential mechanism. Analysis of PDE:phosphatidylcholine product ratios indicates that the enzyme can conduct multiple methylations of enzyme-bound phospholipid. The nature of the acyl chain (16:0 versus 18:1) of the phospholipid had only a small effect on the value of the kinetic constants. The maximal velocities obtained with the 18:1 substrate were less than 5% lower than those obtained with the 16:0 substrate. The Km values for the two phospholipids were 20-45 and 10-14 microM for the methylation of PME and PDE, respectively. The Km for S-adenosylmethionine (AdoMet) was 5-9 microM with PME and 4 microM with PDE as substrates. Depending on the acyl chain and the phospholipid, the Ki(AdoMet) varied from 8 to 19 microM, the Ki(PME) from 41 to 82 microM, and the Ki(PDE) from 35 to 61 microM. The Ki for S-adenosylhomocysteine (AdoHcy) was between 1.0 and 1.4 microM depending upon the variable substrate. The endogenous concentrations of PME and PDE in red blood cell membranes were estimated to be 0.49 and 0.24 mumol/liter packed cells, respectively. The product from the utilization of AdoMet, S-adenosylhomocysteine (AdoHcy), was shown to be a competitive inhibitor of its precursor, AdoMet, and a noncompetitive inhibitor of the two phospholipid substrates.

Adult

Effects of dietary fish oil on human mammary carcinoma and on lipid-metabolizing enzymes.

The growth rate of a human mammary carcinoma, MX-1, was significantly reduced in athymic "nude" mice fed fish oil. Tumors from the fish oil-fed animals also showed a greater sensitivity to two anti-neoplastic agents, mitomycin C and doxorubicin. Mitochondria were isolated from control livers, host livers and tumors from fish oil- and corn oil-fed animals, and increased levels of 20:5n-3 and 22:6n-3 were found in mitochondrial lipids in all three tissues from the fish oil-fed animals. To investigate the effect of dietary n-3 fatty acids on lipid metabolism, the activity of the acyl-CoA:carnitine acyltransferase and three acyl-CoA desaturases were measured. Carnitine acyltransferase activity toward all four acyl-CoA substrates tested was markedly increased in mitochondria from liver by feeding fish oil. In mitochondria from tumors, feeding fish oil resulted in an increased activity toward only 18:3n-3. These data suggest that fish oil may induce an increase in the oxidation of fatty acids. The delta 9-desaturase activity was decreased in microsomes from liver and tumor from fish oil-fed animals. However, both the delta 6 and delta 5 desaturases were increased in tumor and in control liver as a result of feeding fish oil. The delta 5 desaturase was not altered in microsomes from the host animals. The effect of fish oil on the delta 5 and delta 6 desaturases may involve alterations to metabolism of specific polyunsaturated fatty acids especially in the tumor tissue.

Acyltransferases

Tissue and chain length specificity of the fatty acyl-CoA elongation system in the American cockroach.

The elongation of fatty acyl-CoAs, reactions involved in hydrocarbon biosynthesis, was examined in the cockroach, Periplaneta americana. Products were analyzed by radio-HPLC and radio-GLC. The majority of the elongation activity was observed in microsomes prepared from abdominal epidermal tissue. Linoleoyl-CoA (18:2-CoA) was elongated most efficiently followed by stearoyl-CoA (18:0-CoA), linolenoyl-CoA (18:3-CoA; n-3) and oleoyl-CoA (18:1-CoA). The products of 18:2-CoA elongation included all even numbered acyl groups up to 28 carbons, and the products of 18:0-CoA included all even numbered acyl groups to 26 carbons. The 18:3-CoA was elongated only to 20 and 22 carbons. Radioactivity from both 18:2-CoA (5.4%) and 18:0-CoA (1.2%) was recovered in the hydrocarbon fraction. Analysis of this hydrocarbon fraction showed that the radio-activity from 18:2-CoA was present in (Z,Z)-6,9-heptacosadiene and that the radioactivity from 18:0-CoA was present in n-pentacosane. These data demonstrate for the first time in an in vitro insect system that the fatty acid elongation reactions are coupled with the conversion of the elongated product to hydrocarbon. Thus, each of the expected intermediates in the conversion of 18:0 and 18:2 to 25 and 27 carbon hydrocarbons, respectively, was observed, and the results demonstrate high tissue, substrate, and product specificity.

Acyl Coenzyme A

Ethanol-induced alterations in rat synaptosomal plasma membrane phospholipids. Relationship to changes in the phospholipid methyltransferases.

The effects of ethanol ingestion on the lipids of the synaptic plasma membrane (SPM) have been measured and correlated with the time frame for the development of physical dependence. Alterations were observed in three of the phospholipid fractions: phosphatidylcholine (PC) increased, and the phosphatidylethanolamine (PE) and phosphatidylserine (PS) plus phosphatidylinositol (PI) fractions decreased. These alterations occurred after the animals showed signs of dependence. Because PC can be synthesized from PE by the methyltransferase pathway, synaptosomal methyl group incorporation was measured. Rats were fed ethanol for 6 days before an increase was observed in methyl incorporation, a shorter length of time than was necessary to demonstrate physical dependence or phospholipid alterations (10 to 14 days). After ethanol withdrawal, 7 days of control diet feeding were required for methyl group incorporation to return to control values. In vitro ethanol (10-250 mM) additions to the methyltransferase incubations resulted in a slight increase in methyl incorporation. These data suggest that synaptic membrane lipid alterations may be related to ethanol dependence and that changes in the PC/PE ratio may be the result of an increase in the incorporation of methyl groups into synaptosomal phospholipids.

Animals

The acyl-CoA desaturases of microsomes from rat liver and the Morris 7777 hepatoma.

We have investigated the role of the microsomal oxidative desaturase in defining the aberrant phosphoglyceride fatty acid composition of hepatomas. The microsomal delta 9-stearoyl-CoA, delta 6-oleoyl(linolenoyl)-CoA, and delta 5-eicosatrienoyl-CA desaturase activities were studied in control and host liver and in the poorly differentiated Morris 7777 hepatoma. The delta 9-stearoyl-CoA desaturase of the hepatoma was significantly decreased (42%) relative to control liver, yet the hepatoma specific activity was twice that of host liver. Additionally, the specific activity of the delta 9-stearoyl-CoA desaturase of the tumor was found to decrease with increasing tumor weight. Also this desaturase was inactivated by freezing and thawing. The delta 6-oleoyl(linolenoyl)-CoA and delta 5-eicosatrienoyl-CoA desaturases of the hepatoma were 39% and 4% of control, respectively. The electron transport components involved in the desaturase system were reduced, although this did not appear to be rate-limiting. In addition, two competing metabolic reactions which could lower the observed desaturase activities, hydrolysis of the thioester and incorporation of substrate acyl-CoA molecules into glycerides, did not appear to be responsible for the lowered desaturase activities of the tumor. Thus, it appears that reduced levels of the desaturases themselves may be responsible for the observed activities. These results indicate that the capacity of the hepatoma to biosynthesize polyunsaturated fatty acids is greatly reduced and this is consistent with the decreased polyene content observed in many neoplasms.

Animals

A possible mechanism for the increased oxidation of choline after chronic ethanol ingestion.

An attempt has been made to determine the location of the site at which the metabolism of ethanol interacts with that of choline to produce an increase in the oxidation of choline. The first enzyme in the oxidation pathway for choline, choline dehydrogenase, was assayed using a newly developed spectrophotometric assay and freshly isolated intact rat liver mitochondria. No changes were observed in either 'apparent' V or the 'apparent' Km values of choline dehydrogenase for choline after ethanol ingestion. However, when the choline oxidase system was assayed, a 28% decrease in 'apparent' Km for choline and a 53% increase in 'apparent' V was observed. The effects of ATP on choline oxidase were studied further, and a 29.4% decrease was observed in mitochondrial ATP levels from freshly isolated mitochondria from the ethanol-treated rats. In vitro aging of mitochondria further decreased the level of ATP, and the rate of decrease was considerably faster during the first hour in the mitochondria from the ethanol-treated animals. The decreases in ATP from both control and experimental mitochondria were accompanied by increases in choline oxidase activity. The initial decrease in ATP was correlated with an increase in mitochondrial ATPase activity which may be related to an increase in mitochondria Mg2+. Because chronic ethanol ingestion has resulted in decreased oxidation rates of succinate and beta-hydroxybutyrate while at the same time increasing the oxidation rates of choline, the studies reported here suggest that the effect of chronic ethanol ingestion is primarily on a step that is unique to choline and which probably exists prior to the electron transport chain.

Adenosine Triphosphatases

Arachidonic acid and other free fatty acid changes during abortion induced by prostaglandin F2alpha.

Serum free fatty acids (FFA's) were measured after intra-amniotic injection of prostaglandin F2alpha (PGF2alpha) for the induction of abortion in eight healthy women in the midtrimester of pregnancy. The total FFA levels increased in all cases during the period between PGF2alpha administration and abortion. This lipolysis most likely is secondary to the effect of the catecholamines as indicated by the temporal relationship between increased plasma and urine catecholamine and serum FFA levels. The percentage of arachidonic acid in the total amount of FFA's decreased after administration of PGF2alpha. This proportional decrease in arachidonic acid (p less than 0.05) may be due to selective utilization for the production of endogenous prostaglandins.

Abortion, Induced

Mitochondrial and microsomal phospholipids of Morris hepatoma 7777.

The phospholipids of both mitochondrial and microsomal membranes from normal liver, host liver, and Morris hepatoma 7777 were isolated, separated, and quantitated. The total as well as the individual fatty acid concentrations and compositions were determined. The total phosphlipids isolated from tumor mitochondria were idly altered, compared with mitochondria from other normal or host liver. The polyenoic acids were decreased, and there was a concomitant increase in the monoenes. When the respiratory control was determined, the tumor mitochondria exhibited a significant decrease in this parameter. The tumor microsomal membrane fraction, on the other hand, contained about 50% less phospholipid than the controls. The fatty acid patterns of the total as well as the individual phospholipids were quite similar to those observed in the mitochondria. The species of phosphatidylcholine from both membrane fractions were separated by argentation chromatography of the intact molecules, and, as predicted by the fatty acid compositions, the major species of the tumor was the monoenoic/dienoic fraction. The acyl coenzyme A:1-acyl glycerophosphorylcholine acyltransferases, which aid in controlling the fatty acid composition of phospholipids, were measured. The very marked increase in activity of these enzymes toward polyenoic as well as monoenic fatty acids suggested that the polyenoic acids were not available for use in the resynthesis of the phosphatidylcholines in the tumor.

1-Acylglycerophosphocholine O-Acyltransferase

Studies on the mechanism of the NADPH-catalyzed peroxidation of endogenous microsomal lipid.

The importance of metal chelation in the mechanism of microsomal lipid peroxidation has been studied using both phosphate- and sulfhydryl-containing compounds. The optimal concentration for maximum stimulation by each of these compounds has been determined, and the decrease in stimulation observe at concentrations above the maxima has been related to the ability of these compounds to form stable chelation complexes with non-heme iron. Of the compounds tested, only ADP and ATP facilitated the cooperative binding of NADPH to the membrane and thus suggested the possibility of three binding sites for NADPH. Neither of the other two phosphate-chelating agents (Pi or PPi) and neither of the two thiols (cysteine or dithiothreitol)facilitated cooperative binding of NADPH. These data suggested that the adenine ring of ADP or ATP is directly involved in the cooperativity of NADPH binding. They also emphasized that the binding of the chelation complex to the protein is an important parameter in the mechanism of the NADPH-catalyzed peroxidation of endogenous microsomal lipids. Furthermore, stimulation of the rat of lipid peroxidation by sulhydryl-containing compounds, by freezing thawing the microsomal protein, and by treatment of the protein with detergent may be due to a decrease in this cooperative binding effect. Since cysteine and deoxycholate as well as freezing and thawing alter membrane structure, the stimulation of lipid peroxidation seems to involve some alteration to the structure of the microsomal membrane prior to the onset of enzymatic lipid peroxidation.

Adenosine Diphosphate

A possible mechanism for the peroxidation of lipids due to chronic ethanol ingestion.

The effects of chronic ethanol ingestion on NADPH-oxidase and on the NADPH-catalyzed peroxidation of lipids in rat liver microsomes have been studied. It was demonstrated that the rates of NADPH oxidation, of oxygen consumption, and of malondialdehyde formation increased significantly above control values after one month of ethanol ingestion. Further, the fatty acid composition of these microsomes revealed a decrease in arachidonate and in the C22 polyenes. Also, the energies of activation for the formation of malondialdehyde increased in the microsomes from the ethanol-treated animals. These results were interpreted to mean that ethanol ingestion had induced changes in the microsomal membranes such that additional or alternate, possibly abnormal, pathways for lipid peroxidation were functional. Finally, these data suggest a mechanism whereby chronic ethanol ingestion inhances the production of lipid peroxides via the microsomal-catalyzed oxidation of NADPH.

Aldehydes