Pro-apoptotic effects of low doses of dimethoate in rat brain.

Dimethoate (DMT), a widely used Organophosphorous insecticide, was administered for 5 weeks (sub-chronic) at low dose (15 mg/kg b.w.) to male Wistar rats with the aim to simulate potential exposure to pesticide residues in food and water. The induction of cell death programs was investigated in two brain regions, cortex (Cx) and substantia nigra (SN), after the exposure period. We found that DMT increased cytochrome C (CytC) release from mitochondria, the Bax/Bcl-2 ratio, the activity of caspase-3 and calpains, in both brain regions compared to VEH injected ones. DMT treatment induced oxidative damage of lipids with a consequent enrichment in saturated over unsaturated fatty acids. However, the activity of mitochondrial respiratory complexes was not affected by DMT treatment. The activation of the pro-apoptotic pathway can be correlated with a decrease of TH-immunoreactive neurons in SN, comparable to the reduction observed in this cell population by aging. The results of this work contribute to understand the toxic mechanism of DMT and the possible etiological role that residues of this insecticide, might play in neurodegenerative diseases.

Effects of Copper and/or Cholesterol Overload on Mitochondrial Function in a Rat Model of Incipient Neurodegeneration.

Copper (Cu) and cholesterol (Cho) are both associated with neurodegenerative illnesses in humans and animals models. We studied the effect in Wistar rats of oral supplementation with trace amounts of Cu (3 ppm) and/or Cho (2%) in drinking water for 2 months. Increased amounts of nonceruloplasmin-bound Cu were observed in plasma and brain hippocampus together with a higher concentration of ceruloplasmin in plasma, cortex, and hippocampus. Cu, Cho, and the combined treatment Cu + Cho were able to induce a higher Cho/phospholipid ratio in mitochondrial membranes with a simultaneous decrease in glutathione content. The concentration of cardiolipin decreased and that of peroxidation products, conjugated dienes and lipoperoxides, increased. Treatments including Cho produced rigidization in both the outer and inner mitochondrial membranes with a simultaneous increase in permeability. No significant increase in Cyt C leakage to the cytosol was observed except in the case of cortex from rats treated with Cu and Cho nor were there any significant changes in caspase-3 activity and the Bax/Bcl2 ratio. However, the A ß (1-42)/(1-40) ratio was higher in cortex and hippocampus. These findings suggest an incipient neurodegenerative process induced by Cu or Cho that might be potentiated by the association of the two supplements.

Role of copper and cholesterol association in the neurodegenerative process.

Age is one of the main factors involved in the development of neurological illnesses, in particular, Alzheimer, and it is widely held that the rapid aging of the world population is accompanied by a rise in the prevalence and incidence of Alzheimer disease. However, evidence from recent decades indicates that Cu and Cho overload are emerging causative factors in neurodegeneration, a hypothesis that has been partially investigated in experimental models. The link between these two variables and the onset of Alzheimer disease has opened up interesting new possibilities requiring more in-depth analysis. The aim of the present study was therefore to investigate the effect of the association of Cu + Cho (CuCho) as a possible synergistic factor in the development of an Alzheimer-like pathology in Wistar rats. We measured total- and nonceruloplasmin-bound Cu and Cho (free and sterified) contents in plasma and brain zones (cortex and hippocampus), markers of oxidative stress damage, inflammation, and programmed cell death (caspase-3 and calpain isoforms). The ratio beta-amyloid (1-42)/(1-40) was determined in plasma and brain as neurodegenerative biomarker. An evaluation of visuospatial memory (Barnes maze test) was also performed. The results demonstrate the establishment of a prooxidative and proinflammatory environment after CuCho treatment, hallmarked by increased TBARS, protein carbonyls, and nitrite plus nitrate levels in plasma and brain zones (cortex and hippocampus) with a consequent increase in the activity of calpains and no significant changes in caspase-3. A simultaneous increase in the plasma A ß 1-42/A ß 1-40 ratio was found. Furthermore, a slight but noticeable change in visuospatial memory was observed in rats treated with CuCho. We conclude that our model could reflect an initial stage of neurodegeneration in which Cu and Cho interact with one another to exacerbate neurological damage.

Pesticide-induced decrease in rat testicular steroidogenesis is differentially prevented by lipoate and tocopherol.

We have previously demonstrated that the sub-chronic administration of low doses of Toc or α-Toc, glyphosate and zineb to rats (i.p. 1/250 LD50, three times a week for 5 weeks) provoked severe oxidative stress (OS) in testicles. These effects were also reflected in plasma. Lipoic acid (LA) and α-tocopherol are considered as antioxidants due to their ability to neutralize reactive oxygenated species (ROS) and reset endogenous antioxidant levels. To investigate the possible protective effect on reproductive function, LA and Toc (i.p. 25, 50 and 100mg/kg) were administered simultaneously with the pesticide mixture (PM) for 5 weeks. Both drugs prevented OS and the damage to proteins and lipids caused by PM in a dose-dependent manner. The PM-induced increase levels of prostaglandins E2 and F2α was completely restored by LA but not by Toc. Similarly, only LA was able to restore the inhibition of testosterone production, the decrease of 3ß- and 17ß-hydroxysteroid dehydrogenases activities, and the elevation of gonatropins (FSH and LH) levels produced by PM. Furthermore, LA was more efficient than Toc in normalizing the histological alterations produced by PM administration, suggesting that pesticides act though other mechanisms that generate oxidative stress. In our experimental model LA displayed a higher protective role against pesticide-induced damage than that observed by Toc administration. Our results suggest that LA administration is a promising therapeutic strategy for coping with disorders suspected to be caused by OS generators - such as pesticides - in male reproductive system.

Dietary lipid-induced changes in enzymes of hepatic lipid metabolism.

OBJECTIVE: To investigate the effect of different dietary oils on the main hepatic enzymes involved in metabolism and their impact on oxidative stress status. METHODS: Twenty-four male Wistar rats were fed for 60 d on the same basal diet plus different lipid sources from commercial oils: soybean (S), olive (O), coconut (C), and grape seed (G). After sacrifice, the liver lipid fatty acid composition, enzymatic and non-enzymatic components of the antioxidant defense system, and the activity of enzymes involved in lipid metabolism were determined. The concentration of Ca(2+) in plasma and liver homogenates was also measured. RESULTS: The diets produced significant changes in the total and polar lipid fatty acid compositions and alterations in key enzyme activities involved in lipid metabolism. The S and G groups showed significantly increased oxidative stress biomarkers. The enzymatic and non-enzymatic components of the antioxidant defense system were increased in the O and C groups. The highest levels of nitrite plus nitrate were observed in the S and G groups compared with the O and C groups in plasma and in liver homogenates. These were directly correlated with the Ca(2+) concentration. The most beneficial effects were obtained with olive oil. However, it is necessary to study in more detail appropriate mixtures of olive and soybean oils to provide an adequate balance between ω-3 and ω-6 fatty acids. CONCLUSION: Different dietary oils modify the lipid composition of the plasma and liver, local and systemic antioxidant statuses, and the activity of the key enzymes of lipid metabolism. The interrelation between Ca(2+) and nitrite plus nitrate could be the causal factor underlying the observed changes.

Hypothyroidism modifies lipid composition of polymorphonuclear leukocytes.

Thyroid hormones are important regulators of lipid metabolism. Polymorphonuclear leukocytes (PMN) are essential components of innate immune response. Our goal was to determine whether hypothyroidism affects lipid metabolism in PMN cells. Wistar rats were made hypothyroid by administrating 0.1 g/L 6-propyl-2-thiouracil (PTU) in drinking water during 30 days. Triacylglycerides (TG), cholesterol and phospholipids were determined in PMN and serum by conventional methods. The mRNA expression of LDL receptor (LDL-R), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR), sterol regulatory element binding protein 2 (SREBP-2), and diacylglycerol acyltransferase 2 (DGAT-2) were quantified by Real-Time PCR. Cellular neutral lipids were identified by Nile red staining. We found hypothyroidism decreases serum TG whereas it increases them in PMN. This result agrees with those observed in Nile red preparations, however DAGT-2 expression was not modified. Cholesterol synthesizing enzyme HMGCoAR mRNA and protein was reduced in PMN of hypothyroid rats. As expected, cholesterol content decreased in the cells although it increased in serum. Hypothyroidism also reduced relative contents of palmitic, stearic, and arachidonic acids, whereas increased the myristic, linoleic acids, and the unsaturation index in PMN. Thus, hypothyroidism modifies PMN lipid composition. These findings would emphasize the importance of new research to elucidate lipid-induced alterations in specific function(s) of PMN.

Clinical parameters and biomarkers of oxidative stress in agricultural workers who applied copper-based pesticides.

Copper based-pesticides are widely used in agricultural practice throughout the world. We studied the (i) concentration of Cu and proteins involved in Cu homeostasis, (ii) plasma redox status, and (iii) biomarkers of exposure in Cu-based pesticide applicators in order to compare them with clinical biochemical tests. Thirty-one professional applicators and 32 control subjects were recruited. Oxidative stress biomarkers, ceruloplasmin (CRP), metallothioneins (MTs), copper, hematological parameters, and biochemical markers for pancreatic, hepatic and renal function were measured in plasma. Copper was increased in the exposed group compared to the control group concomitantly with TBARS, protein carbonyls, and nitrate+nitrite levels. In the exposed group, α-tocopherol and the FRAP assay were lower and LDH, transaminases, GGT, ALP, urea, creatinine, CRP and MTs were higher than in the control group. The relative leukocyte subclasses were also different between the two groups. Clinical chemistry tests did not surpass the upper reference limit. Our results suggest that the incorporation of oxidative stress biomarkers to biochemical/clinical tests should be considered for validation and included in the human health surveillance protocols.

Carnosine and neocuproine as neutralizing agents for copper overload-induced damages in cultured human cells.

Copper is dangerous when it is present in excess, mainly because it can participate in the Fenton reaction, which produces radical species. As a consequence of copper pollution, people are involuntarily exposed to a copper overload under sub-clinical and sub-symptomatological conditions, which may be very difficult to detect. Thus, we investigated (i) the possible use of the chelator molecules carnosine and neocuproine to prevent the Cu overload-induced damage on cellular lipids and proteins, as tested in human cell culture systems, and (ii) the differential response of these two chelating agents in relation to their protective action, and the type of copper ion involved in the process, by using two types of human cultured cells (HepG2 and A-549). Cu treatment clearly enhanced (p<0.01) the formation of protein carbonyls, thiobarbituric acid-reactive substances (TBARS) and the concentration of nitrate plus nitrites, with a concomitant decrease in cell survival, as estimated by the trypan dye exclusion test and lactate dehydrogenase leakage. Simultaneous treatment with Cu and carnosine or neocuproine indicated that carnosine is more efficient than neocuproine in protecting both types of cells from the effect of cupric ions on both the cell-associated damages and the decrease in the cellular viability. This observation was supported by the fact that carnosine is not only a complexing agent for Cu(II), but also an effective antioxidant that can dismutate superoxide radicals, scavenge hydroxyl radicals and neutralize TBARS formation. Carnosine should be investigated in more detail in order to establish its putative utility as an agent to prevent copper-associated damages in biological systems.

Early alterations in vascular contractility associated to changes in fatty acid composition and oxidative stress markers in perivascular adipose tissue.

AIM: To test the early effect of fructose-induced changes in fatty acid composition and oxidative stress markers in perivascular adipose tissue (PVAT) upon vascular contractility. METHODS: Adult male Wistar rats were fed a commercial diet without (CD) or with 10% fructose (FRD) in the drinking water for 3 weeks. We measured plasma metabolic parameters, lipid composition and oxidative stress markers in aortic PVAT. Vascular contractility was measured in aortic rings sequentially, stimulated with serotonin (5-HT) and high K+-induced depolarization using intact and thereafter PVAT-deprived rings. RESULTS: Comparable body weights were recorded in both groups. FRD rats had increased plasma triglyceride and fructosamine levels. Their PVAT had an increased saturated to mono- or poly-unsaturated fatty acid ratio, a significant decrease in total superoxide dismutase and glutathione peroxidase activities and in the total content of glutathione. Conversely, lipid peroxidation (TBARS), nitric oxide content, and gluthathione reductase activity were significantly higher, indicating an increase in oxidative stress. In aortic rings, removal of PVAT increased serotonin-induced contractions, but the effect was significantly lower in rings from FRD rats. This effect was no longer observed when the two contractions were performed in PVAT-deprived rings. PVAT did not affect the contractions triggered by high K+-induced depolarization either in CD or FRD rats. CONCLUSIONS: FRD induces multiple metabolic and endocrine systemic alterations which also alter PVAT and the vascular relaxant properties of this tissue. The changes in PVAT would affect its paracrine modulation of vascular function.

Alterations in copper homeostasis and oxidative stress biomarkers in women using the intrauterine device TCu380A.

Copper ions participate in the Häber-Weiss reaction to produce ROS, which can be toxic when in excess. The purpose of this study was to measure the copper concentration (Cu) in the plasma of women using Cu-IUDs and determine (i) the effect of Cu on oxidative stress biomarkers, (ii) the levels of copper transport proteins in the plasma and (iii) the status of some liver damage markers in relation to the length of the intrauterine device use. Thirty-nine controls and 35 T380-IUD users were recruited. Various oxidative stress biomarkers, ceruloplasmin (CRP), metallothioneins (MTs), Cu and enzyme activities involved in liver function were measured in the plasma. The Cu concentration was higher in women with IUDs, concomitantly with time-dependent increases in the main oxidative stress biomarkers (TBARS, protein carbonyls, glutathione and nitrates+nitrites), hepatic enzymes (LDH and transaminases), MTs and CRP. We concluded that the use of Cu-IUDs for more than 2 consecutive years should be avoided in order to prevent oxidative damage.

Alteration and recovery of the antioxidant system induced by sub-chronic exposure to microcystin-LR in mice: its relation to liver lipid composition.

The effects of MC-LR on antioxidant system in liver and kidney and its effects on hepatic lipid composition after prolonged exposure to sublethal doses of microcystins (MCs) were studied in mice. Mice were treated i.p. with 25 microg of MC-LR/kg body weight or saline solution every 2 days for a month (inflictive stage), then progression or recovery was studied for 1 and 2 months of wash-out. During the inflictive stage, MC-LR-induced oxidative damage and significant changes in liver lipids of treated mice were compared with control mice. A clear dependence of the enzyme defense system was demonstrated with reduced glutathione and alpha-tocopherol availabilities and a concomitant elevation in NOx production. Sub-chronic MC-LR toxicosis produced alterations in lipid components that included a decreased EFA/non-EFA, SFA/PUFA, and n-3/n-6 ratios all of which exhibited a pattern of slow recovery during the recovery periods.

Microtubule depolymerization modifies the incorporation of fatty acids into glycerolipids.

BACKGROUND: The influence of cytoskeletal integrity on fatty-acid (FA) metabolism is an almost unexplored field of biochemical research. This study therefore investigated the influence of cytoskeletal integrity on the incorporation of palmitate and eicosa-8,11,14-trienoate into glycerolipids of Hep G2 human hepatoma cells. MATERIAL/METHODS: Attached cultures and suspended cells were exposed to colchicine (COL, 10 microM) or dihydrocytochalasin B (DHCB, 20 microM) and supplemented with [14C]FAs bound to delipidated BSA or [14C]glycerol during 0-300 min of incubation. Various key enzymes of lipid metabolism were also determined after COL or DHCB treatment. RESULTS: Incorporation of both FAs into phospholipids (PLs) was strongly reduced by COL treatment especially in the PE and PC subfractions at short incubation times and in PS and SM for 300 min. COL also produced increased incorporation of both FAs into neutral lipids (NL), especially in TG and its precursors (MG and DG). DHCB increased the labeling into lyso-PL and reduced incorporation into PE and SM. However, this drug did not modify the [14C]NL to [14C]PL ratio. DG-acyltransferase and phosphatidate phosphohydrolase were stimulated by COL treatment. Phospholipase A2 activity was reduced significantly by COL and stimulated by DHCB treatment. CONCLUSIONS: It was demonstrated that the microtubule and microfilament network is involved in the incorporation of FAs and in its channeling to neutral lipids and phospholipids. These effects had differential characteristics depending on the type of FA involved and may have potential significance in the understanding of physiological and/or pathological processes.

Alpha-tocopherol protects against oxidative damage to lipids of the rod outer segments of the equine retina.

Oxidative stress is a possible risk factor for eye diseases. Lipid peroxidation is one of the major events induced by oxidative stress and is particularly active in polyunsaturated fatty acid (PUFA)-rich biomembranes. This work evaluated endogenous lipid antioxidants, in vitro non-enzymatic lipid peroxidation of rod outer segment membranes (ROS), the fatty acid composition during oxidative damage of total lipids from equine retina and ROS, and the protective action of alpha-tocopherol (alpha-Toc). The major lipid soluble antioxidant was alpha-Toc followed by retinoids and carotenoids. The retina contained a high percentage of PUFAs, mainly docosahexaenoic acid (22:6n-3) and arachidonic acid (20:4n-6). Lipid peroxidation of the equine ROS, induced by Fe(2+)-ascorbate, was monitored using chemiluminescence (CL) with or without pre-treatment with alpha-Toc. With alpha-Toc pre-treatment, CL values were significantly decreased. The most abundant fatty acid was 22:6n-3. After 3h incubation, 95% of total PUFAs were destroyed by peroxidation, whereas in alpha-Toc pre-treated ROS the percentage was significantly decreased. The results show that the retina has an endogenous lipid soluble antioxidant system. ROS were highly sensitive to oxidative damage, since their fatty acid composition was markedly modified during the lipid peroxidation process. The protective role of alpha-Toc as an antioxidant was evident and it could be used in the treatment of equine ocular diseases in which free radicals are involved.

Dietary lipids modify redox homeostasis and steroidogenic status in rat testis.

OBJECTIVE: The present study explored the effect of dietary oils on lipid composition, antioxidant status, and the activity of the main steroidogenic enzymes in the testis. METHODS: Forty Wistar rats were randomly assigned to one of four groups (n = 10) fed for 60 d on the same basal diet plus different lipid sources as commercial oils: soybean, olive, coconut, or grapeseed. After sacrifice, testicular lipids and fatty acid composition, free radical biomarkers, antioxidant levels, hormones, and steroidogenic enzymes were determined. RESULTS: The lipid composition of diets produced significant changes in neutral/phospholipids, free/esterified cholesterol, and plasmalogen proportion. Fatty acid patterns of these lipids were also strongly modified, influencing the double bond index. We also found a close correlation between the type of diet and the generation of free radicals. The oxidative stress in testes was higher with the grapeseed oil-supplemented diet and decreased with the other diets in this order: soybean oil > olive oil > coconut oil. Animals fed with the olive oil and coconut oil diets showed the highest testicular levels of antioxidants in addition to significantly high levels of testosterone and 3beta- or 17beta-hydroxysteroid dehydrogenase enzymes. CONCLUSION: Different oils in the diets strongly modified the homeostasis of the testicular antioxidant defense system and, in consequence, affected steroidogenic function, showing a clear correlation with the damage induced. According to our results, an appropriate mixture of olive and soybean oils could be a healthy recommendation.

A reliable biomarker derived from plasmalogens to evaluate malignancy and metastatic capacity of human cancers.

Antigen tumor markers employed in monitoring therapeutical approaches are limited by their specificity (Sp) and sensitivity (Se). The aim of this study was to investigate the suitability of a lipid tumor marker derived from ether-linked phospholipids and to compare it with others usually assayed in clinical practice. Complex lipids from normal and pathological breast, lung, and prostate tissue were isolated and analyzed by TLC and c-GLC methods. Results were compared as pooled samples, or by means of the averaged percent changes with respect to the composition observed in the normal tissue of the same patient. Sp, Se, negative-predictive (NPV) and positive- predictive values (PPV) were established for conventional markers and for the proposed lipid-derived marker. Results demonstrated that the content of monoenoic fatty acyl chains was significantly increased in total lipids, phosphatidylethanolamine, and especially in ethanolamine-containing ether lipids of neoplastic tissues with respect to their corresponding normal ones. Major changes were observed in the plasmalogen sub-fraction where the ratio monoenoic/saturated fatty acids can distinguish with high Se normal tissues from either benign or neoplastic tissues from breast, lung, or prostate lesions. Analyses of fatty acyl chains from ethanolamine-containing plasmalogens provided a reliable tumor marker that correlated with high Se and linearity with metastases spreading. This fact may be useful in prognosis of the most frequently observed human cancers.

Oxidative stress biomarkers and hormonal profile in human patients undergoing varicocelectomy.

The aetiology of varicocele is multifactorial although hormonal imbalance and oxidative stress play a key role in the progression of illness. No conclusive evidence has been presented previously, describing the changes in these two factors and the evolution of patients after varicocelectomy. Semen characteristics and hormonal profile were analysed in 36 infertile men with unilateral left varicocele and 33 age-paired controls (proved to be fertile men), after careful inclusion/exclusion selection criteria. Liposoluble and hydrosoluble antioxidants, oligoelements and enzyme activities of the antioxidant defence system were also determined in plasma and erythrocyte from antecubital and spermatic veins, and in spermatozoa. Data were compared between groups at different times before and after varicocelectomy. Decreased levels of liposoluble and hydrosoluble antioxidants and increased activities of the antioxidant defence system enzymes were observed in patients compared with controls. Varicocelectomy normalized this condition at different post-surgical times. Levels of Zn and Se in seminal plasma, protein carbonyls and fragmented DNA remained elevated up to 1 month after surgery. Luteinizing and follicle stimulating hormone concentrations exhibited a biphasic behaviour while testosterone was diminished in patients but normalized soon after varicocelectomy. The results clearly demonstrate the link between the antioxidant defence system, hormonal status and semen characteristics along the post-varicocelectomy period. We suggest that oxidative biomarkers may be appropriate in controlling the evolution of post-varicocelectomy patients, and antioxidant supplementation may improve the clinical condition of infertile men with varicocele.

Lipid metabolism in rats is modified by nitric oxide availability through a Ca++-dependent mechanism.

We studied lipid metabolism and the antioxidant defense system in plasma and liver of rats fed diets supplemented with L(omega)-nitro-L-arginine methyl ester (L-NAME), isosorbide dinitrate (DIS), L-arginine (Arg), or the associations of these drugs. Liver hydroperoxide and thiobarbituric-acid-reactive substance (TBARS) levels were decreased by Arg and increased by L-NAME or DIS treatments. Oxidized glutathione and conjugated dienes were increased by DIS. Nitrate + nitrite levels and serum calcium ([Ca(++)]) were incremented by Arg or DIS and reduced by L-NAME. Superoxide dismutase and catalase activities decreased under Arg treatment, while L-NAME or DIS caused stimulation. Liver high-density lipoprotein (HDL) cholesterol was increased by DIS or NAME (alone or associated with Arg). Free fatty acids and neutral and polar lipids were increased by Arg, L: -NAME, and DIS. However, predominating phospholipid synthesis increased the neutral/polar ratio. Decreased levels of nitric oxide (NO) (low [Ca(++)]) was directly associated with increased fatty acid synthetase, decreased phospholipase A(2), carnitine-palmitoyl transferase, and fatty acid desaturase activities. Raised NO (high [Ca(++)]) inversely correlated with increased phospholipase-A(2) and acyl-coenzyme A (CoA) synthetase and decreased fatty acid synthetase and beta-oxidation rate. Arg or DIS produced changes that were partially reverted by association with L-NAME. Based on these observations, prolonged therapeutical approaches using drugs that modify NO availability should be carefully considered.

Microtubular integrity differentially modifies the saturated and unsaturated fatty acid metabolism in cultured Hep G2 human hepatoma cells.

The influence of cytoskeleton integrity on the metabolism of saturated and unsaturated FA was studied in surface cultures and cell suspensions of human Hep G2 hepatoma cells. We found that colchicine (COL), nocodazol, and vinblastin produced a significant inhibition in the incorporation of labeled saturated FA, whereas incorporation of the unsaturated FA remained unaltered. These microtubule-disrupting drugs also diminished Delta9-, Delta5-, and Delta6-desaturase capacities. The effects produced by COL were dose (0-50 microM) and time (0-300 min) dependent, and were antagonized by stabilizing agents (phalloidin and DMSO). Dihydrocytochalasin B (20 microM) was tested as a microfilament-disrupting drug and produced no changes in either the incorporation of [14C] FA or the desaturase conversion of the substrates. We hypothesized that the interactions between cytoskeleton and membrane proteins such as FA desaturases may explain the functional organization, facilitating both substrate channeling and regulation of unsaturated FA biosynthesis.

Neutral and polar lipid metabolism in liver microsomes of growing rats fed a calcium-deficient diet.

We studied the incorporation of (14)C-labeled fatty acids and glycerol into different classes of glycerolipids in an in vitro system containing liver microsomes from growing Wistar rats fed a calcium-deficient (CaD; 0.5 g/kg) diet for a 60-day period. Desaturase activities and incorporation of the elongation-desaturation metabolites into specific neutral and polar glycerolipids were also studied and correlated with the activities of various enzymes involved in complex lipid metabolism (acyl-CoA synthase, acyl-CoA hydrolase, DAG-acyltransferase, DAG-kinase, lysophospatidate-acyl-CoA transferase, phosphatidate-phosphohydrolase and phospholipase A(2)). Low calcium condition led to a significant increase in the incorporation (relative amounts and specific activities) of both labeled fatty acids and glycerol with a preferential increase of labeling in neutral lipids rather than in phospholipids. Acyl-CoA synthetase, diacylglycerol acyltransferase and diacylglycerol-3-P acyltransferase activities were increased in low calcium microsomes while diacylglycerol kinase, phospholipase A(2) and palmitoyl-, stearoyl-, linoleyl-, alpha-linolenyl, and eicosatrienoyl-desaturases were decreased. The modifications observed in the interlipid and lipid/protein relationships, enzyme activities, and pattern of incorporation of labeled precursors into each glycerolipid class, suggest that decreased intake of calcium should be considered as a harmful risk factor for the development of cardiovascular diseases.

Correlation between fatty acyl composition in neutral and polar lipids and enzyme activities from various tissues of calcium-deficient rats.

In this study we investigated the changes induced by feeding rats a calcium-deficient diet (0.5 g Ca/kg diet) during 65 d after weaning. Phospholipase A2, acyl-Co synthetase and FA delta9-, delta6-, and delta5-desaturase activities were also determined. Calcium deficiency evoked a general alteration in the quality and proportion of the FA chains acylated to neutral and polar lipids from liver, lungs, spleen, brain, kidneys, fat, articular cartilage, erythrocyte ghosts, and plasmas, characterized by an increment of saturated FA and a significant depletion of polyunsaturated acids derived from linoleate and alpha-linolenate. Several interlipid and lipid/protein relationships were also modified in microsomes from calcium-deprived rats, with a concomitant reduction in the rotational mobility of the probe diphenylhexatriene. Phospholipase A2 and acyl-CoA synthetase activities were also decreased and increased, respectively, in some tissues from calcium-deficient rats, whereas delta9-, delta6- and delta5-desaturases were significantly depressed. We conclude that changes in tissue fatty acyl composition evoked by calcium deprivation are due to alterations in the acylation/deacylation cycles via inhibition of the phospholipase A2. These changes were reflected in the physicochemical properties of the membranes, which in turn inhibits desaturase activities. A possible failure in the transcriptional rate for desaturase-mRNA was also discussed.