Polar lipid and fatty acid distribution in carotenolipoprotein complexes extracted from sea buckthorn fruits.

Sea buckthorn (Hippophae rhamnoides L., fam. Elaeagnaceae) fruits are rich in pigments and lipoproteins located in membranes and the fleshy mesocarp. In spite of many reports concerning the neutral lipids in the mesocarp, no data about the polar lipids and their fatty acid composition are available even though they play important structural and physiological roles in cell membranes and may offer interesting applications as emulsifiers and nutrients in cosmetic preparations. Carotenolipoprotein complexes are located particularly in fruit membranes where polar lipids may function as bridge compounds between the polar (protein) and non-polar (carotenoid) moieties. The fatty acid compositions of total and individual polar lipids separated from carotenolipoprotein complexes were determined by HPTLC and GC. The polar lipids included 61% phospholipids and 39% galactolipids, which contained mainly 16:0, 16:1 (9c), 18:1 (9c), 18:1 (11c) and 18:2 (9c, 12c) fatty acids. Almost all polar lipids showed high ratios of 16:0/16:1 (11c) and 18:1 (9c)/18:1 (11c), and higher quantities of 18 carbon unsaturated fatty acids than of the saturated analogue. Galactolipids proved to be richest in 18:1 (9c) and 18:3 (9c, 12c, 15c) fatty acids, while phospholipids contained higher concentrations of 16:0 and 18:1 (9c).

Carotenoid incorporation into natural membranes from artificial carriers: liposomes and beta-cyclodextrins.

Liposomes and beta-cyclodextrin (beta-CD) have been used as carriers for the incorporation of three dietary carotenoids (beta-carotene (BC), lutein (LUT) and canthaxanthin (CTX)) into plasma, mitochondrial, microsomal and nuclear membrane fractions from pig liver cells or the retinal epithelial cell line D407. The uptake dynamics of the carotenoids from the carriers to the organelle membranes and their incorporation yield (IY) was followed by incubations at pH 7.4 for up to 3 h. The mean IYs saturated between 0.1 and 0.9 after 10-30 min of incubation, depending on membrane characteristics (cholesterol to phospholipid ratio) and carotenoid specificity. Mitochondrial membranes (more fluid) favour the incorporation of BC (non-polar), while plasma membranes (more rigid) facilitate the incorporation of lutein, the most polar carotenoid. A high susceptibility of BC to degradation in the microsomal suspension was observed by parallel incubations with/without 2,6-di-t-buthyl-p-cresol (BHT) as antioxidant additive. The beta-CD carrier showed to be more effective for the incorporation of lutein while BC was incorporated equally into natural membranes either from liposomes or from cyclodextrins. The presence of cytosol in the incubation mixture had no significant effects on the carotenoid incorporations.

Influence of glutethimide on rat brain mononucleotides by sub-chronic codeine treatment.

It was investigated the in vivo effect of glutethimide on the intracellular neuroadaptation characteristic for m-opioid receptor tolerance induced by chronic codeine treatment and reflected by increased levels of adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA). AC activity was appreciated by cyclic-AMP (cAMP) formation, the levels of adenine and guanine nucleotides in brain extracts being assayed using a high performance liquid chromatographic method. The concomitant chronic administration of codeine and glutethimide resulted in a pronounced and long-lasting energetic depletion of the neurons, consistent with the high risk of overdose, and increase of cAMP's stable metabolite, 5'-AMP. This increase is persistent even after withdrawal and suggests an interference with the adenylyl cyclase system involved in the development of tolerance of opioid receptor and in relapse and provides a possible explanation of addiction and fast increase of doses observed in humans abusing this combination.

Competitive carotenoid and cholesterol incorporation into liposomes: effects on membrane phase transition, fluidity, polarity and anisotropy.

Pure 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) or mixed DPPC:1,2-dipalmitoyl phosphatidyletanolamine (DPPE):1,2-dipalmitoyl diphosphatidylserine (DPPS) (17:5:3) liposomes were incorporated with 5 mol% dietary carotenoids (beta-carotene, lutein and zeaxanthin) or with cholesterol (16 and 48 mol%) in the absence or presence of 15 mol% carotenoids, respectively. The carotenoid incorporation yields ranged from 0.42 in pure to 0.72 in mixed phospholipid liposomes. They decreased significantly, from 3 to 14%, in the corresponding cholesterol-doped liposomes, respectively. Highest incorporation yields were achieved by zeaxanthin and lutein in phospholipid liposomes while in cholesterol-containing liposomes, lutein was highest incorporated. The effects on membrane structure and dynamics were determined by differential scanning calorimetry, steady-state fluorescence and anisotropy measurements. Polar carotenoids and cholesterol cause similar, dose-dependent effects: ordering and rigidification revealed by broadening of the transition peak, and increase of anisotropy. Membrane hydrophobicity is determined by cholesterol content and carotenoid polarity. In cholesterol-doped liposomes, beta-carotene is less incorporated than in cholesterol-free liposomes. Our observations suggest effects of carotenoids, even at much lower effective concentrations than cholesterol (8 to 80-fold), on membrane structure and dynamics. Although they are minor constituents of animal membranes, carotenoids may act as modulators of membrane phase transition, fluidity, polarity and permeability, and therefore, can influence the membrane physiology and pathology.

Carotenoid incorporation into microsomes: yields, stability and membrane dynamics.

The carotenoids beta-carotene (BC), lycopene (LYC), lutein (LUT), zeaxanthin (ZEA), canthaxanthin (CTX) and astaxanthin (ASTA) have been incorporated into pig liver microsomes. Effective incorporation concentrations in the range of about 1-6 nmol/mg microsomal protein were obtained. A stability test at room temperature revealed that after 3 h BC and LYC had decayed totally whereas, gradually, CTX (46%), LUT (21%), ASTA (17%) and ZEA (5%) decayed. Biophysical parameters of the microsomal membrane were changed hardly by the incorporation of carotenoids. A small rigidification may occur. Membrane anisotropy seems to offer only a small tolerance for incorporation of carotenoids and seems to limit the achievable incorporation concentrations of the carotenoids into microsomes. Microsomes instead of liposomes should be preferred as a membrane model to study mutual effects of carotenoids and membrane dynamics.

Effects of long-term administration of lithium and hydrochlorothiazide in rats.

The biochemical and histological changes following 60 days administration of daily doses equivalent to 1/20 LD(50) of lithium lactate and hydrochlorothiazide, as such and in association, were studied in male Wistar rats. No mortality or overt signs of toxicity were observed during the experiment and the serum activities of transaminases, alkaline phosphatase and cholinesterase were not significantly modified compared to controls. The histopathological examination of all the investigated organs: kidney, liver, brain and spleen, revealed significant lesions which were time-dependant and more pronounced in the association group. Although the changes were mostly inflammatory and conqestive, it was proved that the concomitant administration of lithium and hydrochlorothiazid is potentially dangerous, increasing lithium's nephrotoxicity and the thiazide diuretic's hepatotoxicity.

Carotenoid composition of Rosa canina fruits determined by thin-layer chromatography and high-performance liquid chromatography.

The carotenoid composition of fruits of Rosa canina (Rosaceae) was determined comparatively by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) in total extracts and in three different fractions derived from previous separation of the total fruit extract on alumina columns. Both chromatographic analyses revealed as major carotenoids: beta-carotene, lycopene, beta-chryptoxanthin, rubixanthin, zeaxanthin and lutein. The distribution of these compounds was reproducible by TLC and by HPLC. The I-III fractions eluted successively from alumina columns by increasing the polarity of the solvents were analysed also by TLC and HPLC. In all situations, carotenoids were better separated and identified by gradient HPLC systems than by isocratic HPLC or TLC.

Genotoxicity of Some Metal-Based Antineoplastics, Evaluated by SOS Chromotest and Cytogenetic Analysis.

The paper reports the screening results of two metal-based antineoplastic drugs with mutagenic potential, such as Romcis (trademark of Cisplatinum, produced in Romania) and diphenylantimony(III) diisopropyldithiophosphate (PADTF). Their effects were compared with those induced by Cyclophosphamide. Two mutagenicity tests, the SOS Chromotest and cytogenetic analysis were applied. The tests were carried out with or without metabolic activation (addition of S(9)-mix), either in E. coli PQ 37 cultures, using four doses (0.3, 3, 30 and 300 pmol compound/assay) for the SOS Chromotest or in leukocyte cultures using 0.3 mM from each compound, for cytogenetics. The dose- response relationships and SOSIP values revealed an indirect mutagenic potential for Cyclophosphamide, amplified by S(9) mix in bacterial cultures and an antiproliferative, clastogenic effect on lymphocytes. For Romcis and diphenylantimony(III) diisopropyldithiophosphate, a significant positive response by SOS Chromotest was recorded, which correlated with increased frequencies of chromosomal aberrations.

"In vivo" effects of lithium sulphate on the turnover of rat tissue nucleotides.

The effects of lithium sulphate following acute administration to Wistar rats - in therapeutic and toxic doses - were investigated, using as markers the levels and turnover of brain, kidney and liver nucleotides. Adenine- and guanine-dependent nucleotides were assayed concomitantly, using a high- performance liquid chromatographic method. Following acute administration, lithium's effects on 3',5'- cAMP levels were more significant at hepatic and cerebral level, in a dose-dependant manner. The effect on 3',5'-cGMP had tissue specificity and was not dose-dependant. It might be possible that 3',5'-cGMP is a better indicator of lithium toxicity than 3',5'-cAMP.

In vitro yeast (Saccharomyces cerevisiae) presqualene and squalene synthesis related to substrate and cofactor availability.

Squalene synthase catalyses the synthesis of squalene from trans-farnesyl diphosphate in 2 separate steps requiring NAD(P)H. The kinetics of this enzyme in different fractions extracted from a wild-type Saccharomyces cerevisiae were studied. Although this protein is known to be a membrane-bound enzyme, we have found a cytosolic squalene synthase activity besides the microsomal enzyme. A spectrophotometric enzyme assay, not involving isotopic labelling, was established. The relative synthesis of presqualene and squalene was evaluated by using different substrate and cofactor concentrations during the incubation. The involvement of a single catalytic site promoting the 2 reactions of squalene synthesis is suggested.

Histopathologic investigations of acute and subchronic toxicities of some organotin compounds in chickens.

The acute and subchronic oral toxicities of 4 organotin derivatives were investigated in chickens. The LD50 values oscillated between 200 and 400 mg/kg bw and revealed a moderate or weak toxicity. Subchronic dosing with 2 doses from each compound at 2 and 10 mg/kg bw induced dose-dependent lesions seen at necropsy and histologic examinations. Involution of the thymus and bursa Fabricius, fatty degeneration of the liver, and nephrotic syndromes were the most evident effects. The butyltin derivatives induced more drastic lesions than did the phenyltins.

Antitumor Organometallics. IV. The Mutagenic Potential of Some Diphenylantimony(III) Dithiophosphorus Derivatives.

Two diphenylantimony(III) derivatives of dithiophosphorus ligands, i.e. Ph(2)SbS(2)PPh(2) and Ph(2)SbS(2)P(OPr-i)(2), which were previously found to exhibit antitumor properties, have been now investigated for potential mutagenic effects in healthy and Ehrlich ascites tumor-bearing mice. Two short-term tests, i.e. the micronucleus test and the cytogenetic analysis, were used as end-points for mutagenicity. The results are consistent with a mutagenic potential for both organoantimony(III) compounds tested, the effect being higher for the phosphorodithioato derivative.

Antitumor organometallics. I. Activity of some diphenyltin(IV) and diphenylantimony(III) derivatives on in vitro and in vivo Ehrlich ascites tumor.

Diphenyltin(IV) and diphenylantimony(III) derivatives of dithiophosphorus ligands, i.e. Ph2Sn(S2PPh2)2 (1), Ph2Sn[S2P(OPr)2]2 (2), Ph2SbS2PPh2 (3) and Ph2SbS2P(OPri)2 (4), have been tested in vitro and in vivo against Ehrlich ascites tumor. All four compounds were almost equally effective in vitro, exhibiting inhibitory effects on cell proliferation, viability and protein synthesis, and exacerbated respiration and Ca-ATPase activity. In mice bearing Ehrlich ascites tumor cells, all four compounds inhibited the tumor growth, the organometallic phosphorodithioates being more active than phosphinodithioate analogues, and the organoantimony derivatives more active than organotins. Compound 4 (5 mg/kg/day, i.p., on days 1,3 and 5) produced an increase in life span of 83% and a cure rate of 30% in mice bearing this tumor.

Antitumor organometallics. II. Inhibitory effects of two diphenyl-antimony (III) dithiophosphorus derivatives on in vitro and in vivo Ehrlich ascites tumor.

(Diphenylphosphinodithioato) diphenylantimony (III), Ph2SbS2PPh2 (1) and (diisopropylphosphorodithioato) diphenylantimony(III), Ph2SbS2P(OPri)2 (2) were tested in vivo in male AKR mice and in vitro against Ehrlich ascites tumor cells. Both compounds exhibited dose-dependent inhibitory effects on in vivo tumor growth and on in vitro cell proliferation, cell viability, respiration and protein synthesis. The activities of some enzymes involved in energetic metabolism (Ca-ATPase, LDH, G6Pase) were exacerbated in vitro. The inhibitory effects could be related to the imbalance between ATP-producing and ATP-consuming processes in Ehrlich ascites tumor cells and also to their cell-cycle specificities.

The first organoantimony(III) compounds possessing antitumor properties: diphenylantimony(III) derivatives of dithiophosphorus ligands.

Two organoantimony (III) derivatives, i.e., diphenylantimony diphenyl-dithiophosphinate, Ph2SbS2PPh2, and diphenylantimony diizopropyldithiophosphate, Ph2SbS2P(OPri)2, were shown to exhibit antitumor properties. Both compounds produced strong tumor inhibition effects in mice bearing Ehrlich ascites tumor. Moreover, the dithiophosphate (15 mg/kg, i.p., in days 1, 3 and 5) produced a cure rate of 30%.

Metabolic disturbances induced by organotins through subchronic treatment in chickens. First communication: growth dynamics, carbohydrate and protein metabolism.

Four organotin derivatives (tributyltin acid, dibutyltin chloride, triphenyltinchloride, and triphenyltin-bis (diethyl) dithiophosphate) were tested through subchronic treatment on 19 days old chickens. Growth dynamics as well as carbohydrate and protein metabolism were investigated, in order to evaluate their effects on metabolic activity. Biochemical liver and blood constituents revealed growth retardation and general depression of metabolic activity in experimental groups. Some correlations were found to exist chemical structure, dose use for compound, and effects. Butyltins were more toxic than phenylderivatives and their effects were proportionale to the dose.

Metabolic disturbances induced by organotins through subchronic treatment in chickens. Second communication: mineral metabolism.

4 organotin derivatives (tributyltin acid, dibutyltin chloride, triphenyltin chloride, and triphenyltinbis (diethyl) dithiophosphate) were tested through subchronic treatment in 19 days old chickens. Their effects upon mineral metabolism were studied through biochemical investigations in kidney, bone, and blood. Bone mineral disturbances (depletion of Ca/P ratio) induced Ca elimination in blood, immobilised as calcium urates in kidney, and also increased values for alkaline phosphatase, following tin accumulation in bone. Butyl and phenyltin chloride were more toxic than dithiophosphate, and the intensity of effects was proportionate to the dose.

Changes of the respiratory coefficient in cultures of rat liver cells.

The increase of oxygen consumption in liver cell cultures in medium 199 with addition of serum or of regenerative liver extract pleads for the existence of a factor stimulating energy-dependent processes (mitoses, protein synthesis), which is active both in vivo and in vitro. This stimulating factor is of liver origin, but with systemic offer. The concentration of the stimulating factor, which is the highest 3 days after operation, decreases as the normal liver cell population is re-established. The stimulating factor shows a limited specificity for tissues with epithelial structure and in vivo increased mitotic index the leukocytes being reactive only to very high concentrations of this factor. An optimal concentration of the stimulating factor exists which induces the more marked increase of oxygen consumption.