Long-term biopermanence of ceramides, cholesteryl esters, and ether-linked triglycerides with very-long-chain PUFA in the cadmium-damaged testis

Cadmium is known to harm rat testis by causing the dose-dependent apoptotic or necrotic death of seminiferous epithelium cells. Here we investigated how this affects the lipids with long-chain (C18-C22) and very-long-chain (C24-C32) polyunsaturated fatty acids (VLCPUFA) typical of spermatogenic and Sertoli cells. A severe acute inflammatory reaction resulted from the massive necrotic death of these cells two days after a single high (4 mg/kg) dose of CdCl2. This led to the conversion of most testicular glycerophospholipids to diradylglycerols (DRG) and free fatty acids (FFA) and of most sphingomyelins to ceramides (Cer). By day 30 the testis weight had decreased three fold. The DRG and FFA had been metabolized but, unexpectedly, ceramides persisted. Also slow to disappear were VLCPUFA-containing triacylglycerols from former germ cells and ether-linked triglycerides and cholesteryl esters (CE) from former Sertoli cells. Similar results were observed 30 and 45 days after administering repeated small non proinflammatory CdCl2doses (1 mg/kg). At day 30 after both treatments, an amorphous material replaced the original seminiferous tubules and testicular macrophages populated the interstitium. Species of CE and etherlinked triglycerides containing fatty acids other than VLCPUFA steadily accumulated in the irreversibly damaged testis, a manifestation of the activity of phagocytic cells. The long-term permanence of original VLCPUFA-containing neutral lipids, especially ceramides, indicates that these cells were slow to clear out the acellular material contained in seminiferous tubules, pointing to a form of silent chronic inflammation as an additional outcome of the multifactorial commotion caused in the testis by experimentally administered cadmium.Fil: Zanetti, Samanta Romina. Universidad Nacional del Sur; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET Bahía Blanca. Instituto de Investigaciones Bioquímicas Bahía Blanca (i); ArgentinaFil: Aveldaño, Marta I.. Universidad Nacional del Sur; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET Bahía Blanca. Instituto de Investigaciones Bioquímicas Bahía Blanca (i); Argentin

Experimental and computational studies on lipoprotein lipolysis at acidic pH

Atherosclerosis is a disease of the arteries; its characteristic features include chronic inflammation, extra- and intracellular lipid accumulation, extracellular matrix remodeling, and an increase in extracellular matrix volume. The underlying mechanisms in the pathogenesis of advanced atherosclerotic plaques, that involve local acidity of the extracellular fluid, are still incompletely understood. In this thesis project, my co-workers and I studied the different mechanisms by which local extracellular acidity could promote accumulation of the atherogenic apolipoprotein B-100 (apoB-100)-containing plasma lipoprotein particles in the inner layer of the arterial wall, the intima. We found that lipolysis of atherogenic apoB-100-containing plasma lipoprotein particles (LDL, IDL, and sVLDL) by the secretory phospholipase A2 group V (sPLA2-V) enzyme, was increased at acidic pH. Also, the binding of apoB-100-containing plasma lipoprotein particles to human aortic proteoglycans was dramatically enhanced at acidic pH. Additionally, lipolysis by sPLA2-V enzyme further increased this binding. Using proteoglycan-affinity chromatography, we found that sVLDL lipoprotein particles consist of populations, differing in their affinities toward proteoglycans. These populations also contained different amounts of apolipoprotein E (apoE) and apolipoprotein C-III (apoC-III); the amounts of apoC-III and apoE per particle were highest in the population with the lowest affinity toward proteoglycans. Since PLA2-modification of LDL particles has been shown to change their aggregation behavior, we also studied the effect of acidic pH on the monolayer structure covering lipoprotein particles after PLA2-induced hydrolysis. Using molecular dynamics simulations, we found that, in acidity, the monolayer is more tightly packed laterally; moreover, its spontaneous curvature is negative, suggesting that acidity may promote lipoprotein particles fusion. In addition to extracellular lipid accumulation, the apoB-100-containing plasma lipoprotein particles can be taken up by inflammatory cells, namely macrophages. Using radiolabeled lipoprotein particles and cell cultures, we showed that sPLA2-V-modification of LDL, IDL, and sVLDL lipoproteins particles, at neutral or acidic pH, increased their uptake by human monocyte-derived macrophages.Ateroskleroosin komplikaatiot ovat länsimaissa suurin kuolinsyyn aiheuttaja ja tauti yleistyy maailmanlaajuisesti erityisesti vanhusväestön keskuudessa. Ateroskleroottinen plakki kehittyy hitaasti vuosikymmenten aikana. Lopulta pitkälle edennyt plakki voi revetä ja johtaa verihyytymään ja siitä seuraavaan sydän- tai aivoinfarktiin. Tiedetään että ateroskleroosin synty on seurausta aktiivisesta tulehdusprosessista, jossa valtimon seinämän sisäkerrokseen eli intimaan kertyy solunulkoisia ja solunsisäisiä rasvapisaroita, jotka ovat peräisin veriplasman apolipoproteiini B-100:aa sisältävistä lipoproteiineista, erityisesti LDL-hiukkasista. Pitkälle edenneen plakin syntyyn liittyy myös paikallinen hapen puute ja siitä seuraava kudoksen happamuus, jonka vaikutusmekanismit ateroskleroosiin ovat kuitenkin vielä suurelta osin tuntemattomat. Tutkimustyömme keskeisenä tavoitteena on ollut selvittää intimassa mahdollisesti vallitsevan solunulkoisen happamuuden vaikutusta useaan ateroskleroosia edistävään mekanismiin käyttäen sekä kokeellisia että laskennallisia biokemiallisia menetelmiä. Tutkimme kuinka lipoproteiinien entsymaattinen käsittely sekretorisella fosfolipaasi A2 tyyppi V (sPLA2-V)-entsyymillä vaikuttaa lipoproteiinien sitoutumiseen intiman soluväliaineeseen ja sisäänottoon syöjäsoluihin. Intiman soluväliaine koostuu pääasiallisesti proteoglykaaneista, joihin apoB-100:aa sisältävät lipoproteiinit voivat sitoutua. Ateroskleroottisessa valtimon seinämässä on myös sPLA2-V-entsyymiä, jonka eritys lisääntyy runsasrasvaisen ruokavalion seurauksena, ja lisäksi sPLA2-V-entsyymin on todettu kasvattavan ateroskleroottisten plakkien kokoa hiirimallissa. Tutkimuksemme keskeinen ensihavainto oli, että happamassa pH:ssa lipoproteiinien pintakalvon fosfolipidien lipolyysi sPLA2-V-entsyymillä lisääntyy, mikä johtaa lisääntyneeseen lysoPC- ja rasvahappomolekyylien vapautumiseen ja kerääntymiseen lipoproteiinien pintakalvoon. Tutkimuksemme seuraava tärkeä löydös oli, että happamassa pH:ssa sPLA2-V-entsyymillä käsitellyt lipoproteiinit sitoutuivat voimakkaasti intiman proteoglykaaneihin. Havaitsimme myös, että pienet apolipoproteiinit (apoE ja apoC-III) estävät sVLDL-hiukkasten sitoutumista proteoglykaaneihin. Tarkastelimme myös lipoproteiinien pintakalvon rakennetta molekyylitasolla ja havaitsimme, että happamassa pH:ssa lipolysoitu fosfolipidikalvo on järjestäytyneempi ja kalvon spontaani kaarevuus on negatiivinen. Nämä kalvomuutokset saattavat edesauttaa muuntuneiden lipoproteiinien aggregoitumista, fuusioitumista ja kertymistä solunulkoiseen tilaan suurina rasvapisaroina. Ateroskleroosin edetessä intimaan kertyy monia immuunipuolustuksen soluja, erityisesti syöjäsoluja, jotka tunnistavat muuntuneet lipoproteiinit ja pyrkivät poistamaan ne soluvälitilasta endosytoosivälitteisesti. Totesimme myös, että lipolyysi sPLA2-V-entsyymillä lisäsi lipoproteiinien sisäänottoa näihin soluihin sekä normaalissa että happamassa pH-ympäristössä. Yhteenvetona totean, että väitöskirjatyössäni olemme selvittäneet molekyyli- ja solutasolla happaman pH:n vaikutusta tekijöihin, jotka vaikuttavat ateroskleroosin kehittymiseen. Ateroskleroosin mekanismien tunteminen luo uusia mahdollisuuksia ehkäisevän lääkehoidon kehittämiselle. Fosfolipaasi A2-entsyymien toiminnan estäminen vaikuttaisi lipoproteiinien lipolyysiin ja siten rasvajuosteen, vaahtosolujen ja tulehduksen syntyyn, sekä muihin ateroskleroosin keskeisiin patofysiologisiin tapahtumiin, ja saattaisi siten tarjota uuden ateroskleroosin hoitomuodon, jolla saatetaan tulevaisuudessa ehkäistä tämän taudin kehittymistä

Working note-book for the practical classes on biochemistry

БИОХИМИ

Role of the regulation of cell lipid composition and membrane structure in the antitumor effect of 2-hydroxyoleic acid

El ácido 2-hidroxioleico (2OHOA) es un fármaco antitumoral diseñado para regular la estructura y composición de los lípidos de membrana y la función de importantes proteínas de membrana. El objetivo principal de este trabajo fue estudiar cómo el 2OHOA modula la composición lipídica y la estructura de membrana en las células tumorales. Se observó que el 2OHOA indujo profundas alteraciones en el contenido de fosfolípidos, aumentando el contenido de esfingomielina y disminuyendo el contenido de fosfatidiletanolamina y fosfatidilcolina. Este efecto fue específico contra las células cancerosas, ya que el tratamiento no afectó la composición lipídica de las células no tumorales MRC-5 de fibroblastos humanos. El aumento de SM se debió a una activación rápida y específica de las SM sintasas. Como consecuencia de la activación sostenida de la SMS, todo el metabolismo de los esfingolípidos se vio afectado. Finalmente, se evaluó el impacto de todos estos cambios sobre las propiedades biofísicas de membrana mediante espectroscopia de fluorescencia2-Hydroxyoleic acid (2OHOA) is a potent antitumor drug that was designed to regulate membrane lipid composition and structure and the function of important membrane proteins. The main goal of this work was to study how 2OHOA modulates the membrane lipid composition and structure of tumor cells. 2OHOA induced dramatic alterations in phospholipid content, increasing sphingomyelin mass, and decreasing phosphatidyl-ethanolamine and phosphatidylcholine. This effect was specific against cancer cells as it did not affect non-tumor MRC-5 cells. The increased SM mass was due to a rapid and highly specific activation of SM synthases. As a consequence of the sustained activation of SMS, the whole sphingolipid metabolism was affected. Then, the impact of all these changes on membrane biophysical properties was evaluated by fluorescence spectroscop

Nutritional status of a nereidid polychaete cultured in sand filters of mariculture wastewater

This study examined the nutritional composition of the intertidal marine polychaete Perinereis helleri (Nereididae)when artificially cultured in sand filters treating mariculture wastewater. Moisture levels in harvested P. helleri ranged from 758 to 855 g kg1, and ash, from 23 to 61 g kg1 wet matter (WM). Stocking density and graded size after harvest significantly affected their composition. Higher total lipid contents were found in large (>0.6 g) P. helleri(16–19 g kg1 WM) and those grown at the lowest density(1000 m2: 18 g kg 1 WM) than in small (≤0.6 g) ones (14 g kg1 WM) and those grown at the highest densities (4000–6000 m2: 13–16 g kg1 WM). Several fatty acids within a very broad profile (some 30 identified) reflected this pattern, yet their ARA/EPA/DHA ratios were relatively unaffected. Feeding the polychaete-assisted sand filters (PASF) with fish meal to increase worm biomass productivity significantly increased their DHA content. Other components (e.g. protein, phospholipids, cholesterol, carbohydrate, amino acids, nitrogen, minerals and bromophenols) and nutritional factors (e.g. maturity, feeding seaweed and endemic shrimp viral content) were also investigated. Results suggest that PASF-produced P. helleri have a well-balanced nutritional profile for penaeid shrimp and fish broodstock

Serum Lipids and Urinary Estrogens of Non-Pregnant Menstruating Young Women

Twelve university women students served as experimental subjects in a study of the serum lipids and urinary estrogens of healthy nonpregnant menstruating young women, who were living under their usual conditions. The subjects maintained constant weight on their ordinary diets during the entire study period. Antecubital blood and 24-hour urine specimens were collected on certain days which represented different stages of the menstrual cycle. Quantitative analyses were made on serum total cholesterol, lipid phosphorus (phospholipids), triglycerides and total lipids. Gas-liquid chromatographic analysis of the fatty acid composition of each serum lipid component was also made. Urinary estrone, 17β- estradiol and estriol were separated and quantitatively determined by chromatographic and spectrophotometric techniques. Basic data on serum lipid levels , composition of the fatty acids of cholesterol esters, phospholipids and triglycerides and urinary estrogens were obtained on these young women . Findings included the following: 1. Mean values of serum total cholesterol, phospholipids, triglycerides and total lipids were 162, 165, 105 and 544 mg per cent, respectively. The interindividual variation was greater than intraindividual variation. The values of triglycerides were more variable than those of cholesterol and phospholipids. 2. The major fatty acids in lipid fractions were palmitic, stearic, oleic and linoleic. The highest amounts of fatty acid in cholesterol esters, phospholipids and triglycerides were linoleic, 51; palmitic, 28; and oleic, 33 per cent, respectively. Inter- and intra-individual variations were high. 3. The urinary estrogen values showed that 17 B-estradiol (E 2 ), was usually present in the least and estriol (E 3 ), in the greatest amounts. The mean values of E 1 (estrone), E 2 , E 3 and Et (total) were as follows: 8. 7, 4. 8, 16. 4, and 29. 9 μg per 24-hour urine. 4. The menstrual cycle did affect the urinary excretion of estrogens which showed the lowest values during the first week and then rose to a peak which occurred on or about the time of ovulation or mid-cycle. Then it fell and rose again between the third and fourth week of the cycle. The second peak was usually lower than the first one. 5. Cyclical changes of the concentrations of serum total cholesterol, phospholipids and total lipids have been observed. These changes appeared to be influenced by the estrogenic hormonal activity of the menstrual cycle. The increased excretion of urinary estrogens with a decreased (negative correlation) concentration of serum lipids was recognized. 6. Linoleic acid in cholesterol esters, as well as palmitic acid in phospholipids were found in cyclic changes. The patterns were quite similar to those of serum lipids

Effect of High Fat Diets Containing No Cholesterol on the Properties of Rabbit Serum Lipoproteins and the Catabolism of Very Low Density Lipoproteins

154 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1980.New Zealand white rabbits were successively maintained on normal chow, a semisynthetic diet high in safflower oil (SAFF diet), and a semisynthetic diet high in hydrogenated coconut oil (COCO diet) for 9 weeks each.The -mobility of VLDL on agarose from rabbits fed the COCO diet indicates the presence of an altered lipoprotein particle which may be atherogenic. In addition, this VLDL was cholesteryl ester-enriched and triglyceride-poor. This alteration is perhaps explained by the stimulation of e novol cholesterogenesis in the liver. ALthough a similar effect has been reported after the cholesterol feeding of rabbits, the significance of the present studies is that while no dietary cholesterol was provided, an altered VLDL is produced with similar properties as those seen under conditions of both cholesterol-induced and human type III hyperlipoprotein-emias.When the SAFF diet was fed, the VLDL also became cholesteryl esterenriched and triglyceride-poor but it remained pre-beta in its mobility on agarose gels. In addition, this diet appeared to help prevent the hypercholesterolemic and hyperlipoproteinemic effects of the high saturated fat diet.Partially purified triglyceride lipase activities from postl-heparin plasma of both intact and supradiaphragmatic rabbit preparations were characterized using triglyceride emulsions as substrate. When radio-labeled VLDL from the experimentally-fed animals were used as substrate for these enzyme preparations, similar activities were observed whether preparations from intact or supradiaphragmatic rabbits were used. However, activities were lower than when normal chow-fed rabbit VLDL was assayed indicating that the VLDL from experimentally-fed animals is not as effective a substrate for the lipase enzyme. As a result, a build-up of the VLDL has been hypothesized under those dietary conditions which may be atherogenic. This finding is also consistent with the particle size of the various VLDL as estimated by surface to core ratios, the larger particles (i.e. chow-fed rabbit VLDL) being catabolized more rapidly.The preparation of VLDL remnants from VLDL of rabbits fed the various diets was also carried out. The remnants prepared from chow-fed rabbit VLDL were triglyceride-poor and cholesteryl ester-enriched. Loss of phospholipid and apo C peptides occurred while the free cholesterol content remained about the same. In addition, the mobility of these remnants on agarose was, for the most part, beta, while the VLDL originally showed pre-beta mobility. The VLDL from either SAFF-fed or COCO-fed rabbits was already appreciably cholesteryl ester-enriched and triglyceride-poor. Yet when remnants were prepared using either of these VLDL, it became further triglyceride-depleted and cholesteryl ester-enriched. Surface to core ratios of these VLDL remnants were different, indicating that the COCO diet-fed rabbit VLDL remnant was a larger particle than that in the SAFF case.Finally, the fatty acid composition of the VLDL was, in general, fund to reflect that of the diets fed. The fatty acid composition of VLDL remnants, however, showed preliminary evidence for the preferential hydrolysis of linoleic acid by the triglyceride lipase preparations when VLDL is the substrate.Ope