Simultaneous high nutritional single cell oil and lipase production by Candida viswanathii.

BACKGROUND: Omega fatty acids are a family of polyunsaturated fats associated with several health benefits. Lipases are enzymes with potential application in several food processes such as flavor and aroma, surfactants and formulations for the dairy and bakery industries. In this study, single cell oil and lipase production by Candida viswanathii CCR8137 were evaluated simultaneously from renewable carbon sources under nitrogen limitation. METHODS: Enzyme and single cell oil were obtained in submerged cultivations supplemented with triolein, tributyrin, corn oil, sunflower oil, canola oil and olive oil. The effects of glucose on lipid accumulation, fatty acid profile, enzyme production and cell morphology were also evaluated. RESULTS: The highest lipid accumulation (44.5%, w/w) was obtained from triolein, whereas olive oil was the best inducer of lipase synthesis (26.8 U/mL). Nitrogen limiting cultivations were a key parameter for an organic source which showed higher lipid accumulation and enzyme production than the tested inorganic nitrogen source. Glucose was a poor inducer of lipase synthesis, though increased values of lipid accumulation were observed from this carbon source with a maximum of 63.1% (w/w). The fatty acid profile of lipids produced by C. viswanathii CCR8137 showed a high content of omega-9 fatty acid (C18:1 n-9). The addition of glucose to the culture media resulted in the synthesis of essential fatty acids: vaccenic, linolenic and eicosadienoic acids. CONCLUSIONS: Therefore, C. viswanathii CCR8137 strain can be considered as an oleaginous yeast able to accumulate high concentrations of intracellular lipids, which are potential additives for food industry applications as well as being able to simultaneously synthesize high yields of lipase.

Favorable effects of ezetimibe alone or in association with simvastatin on the removal from plasma of chylomicrons in coronary heart disease subjects.

OBJECTIVE: Reductions on the clearance from plasma of chylomicrons are associated with atherosclerosis. Statins improve the removal from plasma of chylomicrons in a dose dependent manner. There is controversy whether ezetimibe modifies the plasma clearance of chylomicrons. Effects of ezetimibe alone or in combination with simvastatin were compared with low and high dose of the latter, upon the kinetics of a chylomicron-like emulsion in coronary heart disease (CHD) patients. METHODS: 25 CHD patients were randomized for treatment with ezetimibe 10 mg (group 1) or simvastatin 20 mg (group 2) with progression to ezetimibe + simvastatin 10/20 mg or simvastatin 80 mg, respectively. Kinetic studies were performed at baseline and after each treatment period of 6 weeks. The fractional catabolic rates (FCR) of the emulsion labeled with (14)C-CE and (3)H-TG, that represent respectively chylomicron remnant and triglyceride removal, were calculated. Comparisons were made by ANOVA. RESULTS: The (14)CE-FCR in group 1 were 0.005 ± 0.004, 0.011 ± 0.008 and 0.018 ± 0.005 min(-1) and in group 2 were 0.004 ± 0.003, 0.011 ± 0.008 and 0.019 ± 0.007 min(-1) respectively at baseline, after 6 and 12 weeks (p < 0.05 vs. baseline, and 6 vs. 12 weeks). The (3)H-TG-FCR in group 1 were 0.017 ± 0.011, 0.024 ± 0.011 and 0.042 ± 0.013 min(-1) and in group 2 were 0.016 ± 0.009, 0.022 ± 0.009 and 0.037 ± 0.012 min(-1) at baseline, after 6 and 12 weeks (p < 0.05 vs. baseline, and 6 vs. 12 weeks). There were no differences between groups in time. CONCLUSION: Both treatments increased similarly the removal from plasma of chylomicron and remnants in CHD patients.

Screening of food grade lipases to be used in esterification and interesterification reactions of industrial interest.

Seven food grade commercially available lipases were immobilized by covalent binding on polysiloxane-polyvinyl alcohol (POS-PVA) hybrid composite and screened to mediate reactions of industrial interest. The synthesis of butyl butyrate and the interesterification of tripalmitin with triolein were chosen as model reactions. The highest esterification activity (240.63 microM/g min) was achieved by Candida rugosa lipase, while the highest interesterification yield (31%, in 72 h) was achieved by lipase from Rhizopus oryzae, with the production of about 15 mM of the triglycerides C(50) and C(52). This lipase also showed a good performance in butyl butyrate synthesis, with an esterification activity of 171.14 microM/g min. The results demonstrated the feasibility of using lipases from C. rugosa for esterification and R. oryzae lipase for both esterification and interesterification reactions.

Determination of the quantitative stereoselectivity fingerprint of lipases during hydrolysis of a prochiral triacylglycerol.

We propose a method for characterizing quantitatively the stereoselectivity of lipases during hydrolysis of triacylglycerols. Although it is of general applicability, we demonstrate it specifically for sn-1,3-regiospecific lipases. In this case the method generates a "stereoselectivity fingerprint" that consists of ratios of the specificity constants for the various reactions that produce and consume the 1,2-sn- and 2,3-sn-diacylglycerols. We use the method to determine the stereoselectivity fingerprint of several lipases during the hydrolysis of the prochiral substrate triolein. Our method opens up the possibility of correlating quantitative fingerprints with structural information, in the quest to elucidate the mechanisms underlying the stereoselectivity of lipases.

Lipid metabolism in Rhodnius prolixus (Hemiptera: Reduviidae): role of a midgut triacylglycerol-lipase.

The utilization of dietary lipids was studied in adult females of Rhodnius prolixus with the use of radiolabeled triacylglycerol (TAG). It was shown that (3)H-triolein, when added to blood meal, was hydrolyzed to free fatty acids in the posterior midgut lumen. Subsequently, free fatty acids were absorbed by posterior midgut epithelium and used in the synthesis of phospholipids, diacylglycerol (DAG) and TAG. Phospholipids, DAG and free fatty acids were then found in hemolymph, from where they were rapidly cleared, and label was found in the fat body, mainly associated with TAG. Radioactive lipids, especially TAG and phospholipids, also accumulated in the ovaries. The TAG-lipase activities of posterior midgut luminal content and tissue were characterized by incubation of these samples with (3)H-triolein in the presence of the detergent Triton X-100 and determination of the amounts of released radioactive free fatty acids. Under the conditions employed here, the release of free fatty acids was proportional to the incubation time and to the amount of sample obtained from insect midgut (enzyme source) that was added. TAG-lipase activities were affected by pH and posterior midgut tissue showed optimum activity around pH 7.0-7.5, but the luminal content had the highest activities as pH decreased. Differences in activities were observed according to calcium concentration in the medium. TAG-lipase activities were also affected by the concentration of NaCl and were activated in the presence of increasing salt concentrations. These activities were inhibited by phenylmethylsulphonyl fluoride (PMSF). On the second day after blood meal, when digestion is very intense, TAG-lipase activities were maximal and then gradually decreased.

High cholesterol intake modifies chylomicron metabolism in normolipidemic young men.

Whether the consumption of egg yolk, which has a very high cholesterol content without excess saturated fats, has deleterious effects on lipid metabolism is controversial. Absorbed dietary cholesterol enters the bloodstream as chylomicrons, but the effects of regular consumption of large amounts of cholesterol on the metabolism of this lipoprotein have not been explored even though the accumulation of chylomicron remnants is associated with coronary artery disease (CAD). We investigated the effects of high dietary cholesterol on chylomicron metabolism in normolipidemic, healthy young men. The plasma kinetics of a chylomicron-like emulsion, doubly-labeled with 14C-cholesteryl ester (14C-CE) and 3H-triolein (3H-TG) were assessed in 25 men (17-22 y old, BMI 24.1 +/- 3.4 kg/m2). One group (n = 13) consumed 174 +/- 41 mg cholesterol/d and no egg yolk. The other group (n = 12) consumed 3 whole eggs/d for a total cholesterol intake of 804 +/- 40 mg/d. The nutritional composition of diets was the same for both groups, including total lipids and saturated fat, which comprised 25 and 7%, respectively, of energy intake. Serum LDL and HDL cholesterol and apoprotein B concentrations were higher in the group consuming the high-cholesterol diet (P < 0.05), but serum triacylglycerol, apo AI, and lipoprotein (a) did not differ between the 2 groups. The fractional clearance rate (FCR) of the 14C-CE emulsion, obtained by compartmental analysis, was 52% slower in the high-cholesterol than in the low-cholesterol group (P < 0.001); the 3H-TG FCR did not differ between the groups. Finally, we concluded that high cholesterol intakes increase the residence time of chylomicron remnants, as indicated by the 14C-CE kinetics, which may have undesirable effects related to the development of CAD.

Effects of hydrochlorothiazide and propranolol treatment on chylomicron metabolism in hypertensive objects.

Modifications in chylomicron metabolism caused by antihypertensive drugs were investigated in hypertensive subjects because previous studies had indicated that diuretics and beta-blockers modify the plasma lipid concentrations through mechanisms that were not fully understood. A triglyceride-rich emulsion resembling lymph chylomicrons, labeled with (3H) triolein and (14C) cholesteryl oleate, was infused intravenously into mildly hypertensive patients after 8 weeks on placebo and subsequently on hydrochlorothiazide (n = 10) or propranolol (n = 8). The residence time of both radioactivities in plasma was utilized for the simultaneous calculation of the particle remnant removal rate and of the lipoprotein lipase activity expressed as a delipidation index = 1 - [(3H) triolein residence time/(14C) cholesteryl oleate residence time]. Treatment with hydrochlorothiazide diminished the delipidation rate value whereas propranolol mildly increased the removal rate of the remnant particle. These alterations of the chylomicron kinetics were not accompanied by changes in plasma triglycerides, glucose, and insulin concentration as measured in the fasting state. The impairment of the lipoprotein lipase activity by thiazides and the faster removal rate of the whole particle by propranolol could explain the reason why in previous clinical studies the simultaneous use of these drugs does not aggravate the hyperlipidemia known to be induced by thiazides alone.

Delayed intravascular catabolism of chylomicron-like emulsions is an independent predictor of coronary artery disease.

The atherogenic role of a delayed intravascular catabolism of chylomicrons has been suggested by univariate analysis of case-control studies. However, it is not established whether this association is caused by a direct atherogenic effect of these lipoproteins or results from the presence of concurrent and metabolically-related coronary artery disease (CAD) risk factors. In this study, the plasma kinetics of a chylomicron-like emulsion doubly labeled with 14C-cholesteryl oleate (CE) and 3H-triolein (TG) was determined in 93 subjects with or without angiographically-defined CAD. As compared with controls and even after adjustment for body mass index (BMI), LDL- and HDL-cholesterol, and the presence of traditional risk factors, CAD patients had 45% smaller fractional clearance rate (FCR) of TG, 41% smaller FCR-CE and 19% smaller dilapidation index (DI; P < 0.05). Among CAD patients, those with highest angiographic score had 66% smaller FCR-TG (P = 0.007), 50% smaller FCR-CE (P = 0.01) and 27% smaller DI (P = 0.004). In a multivariate logistic regression analysis, FCR-CE (P < 0.0001) and DI (P = 0.001) were the only independent predictors for the presence of CAD. In conclusion, we presently show that the rate of lipolysis and removal from the circulation of chylomicron-like emulsions constitutes an independent predictor of CAD and a marker of CAD severity.

Liver denervation affects hepatocyte mitochondrial fatty acid transport capacity.

The effect of liver denervation on the activity of hepatic carnitine palmitoyltransferase (CPT) system, which catalyses the transfer of long-chain fatty acids into the mitochondria, was studied in rats. Noradrenaline content in phenol-denervated liver (D) was reduced by 87%. CPT I and II activities (measured by radioassay after detergent separation of the enzymes) were decreased (p < 0.001) in D (2.6 +/- 0.1 and 0.68 +/- 0.2 nmol min(-1) mg(-1) protein, respectively) as compared with controls (4.7 +/- 0.3 and 2.5 +/- 0.2 nmol min(-1) mg(-1) protein, for CPT I and II, respectively). A less intense immunoreactive band for denervated liver CPT II was obtained after Western blotting. Concomitantly, long-chain fatty acid incorporation (p < 0.001), evaluated after administration of [14C]-oleate and total fat content (p < 0.001) were increased in D in relation to controls, while incorporation of exogenous [14C]-oleate into secreted VLDL, was decreased (p < 0.01). The effect of sympathetic denervation on CPT activity was different from that evoked by adrenodemedullation, which caused an augmentation of CPT activity (p < 0.01), when compared with the liver of intact rats. The effects of denervation and adrenodemedullation on the other parameters of lipid metabolism studied, were similar. The results strongly suggest a role of liver sympathetic innervation in the regulation of liver lipid metabolism.

Metabolic handling of different fatty acids during rehabilitation from childhood malnutrition

The gastrointestinal handling and post absorptive metabolism of [1, 1, 1-13 C] triolein (TO) and [1, 1, 13 C] tripalmitin (TP) were studied in two groups of eight severely malnourished children (5-0 months): on admission (Phase 1), during rapid-catch up growth (Phase 2) and when weight for height had reached 90 percent of the reference (Phase 3). Total excretion of 13 C label in stool (over 3 days) and breath as 13 CO 2 (over 24 hours) were analysed by isotope radio mass spectrometry. Stool 13 C excretion at admission was approximately 10 percent of the administered dose for both trials but varied markedly between subjects, was significantly reduced during rehabilitation in the TO trial (Phase 2:0.5 +or- 1.0; Phase 3: 1.3 +or- 0.9; p<0.05) and tended to decline on the P trial. ANOVA analysis of the magnitude and time course of 13 C excretion in breath (from area under the curve), excretion tended to decrease during rehabilitation in the TO trial but remained unchanged on the TP trial. These results suggest that the efficiency with which dietary triacylglycerol is handled within the gastrointestinal tract is generally impaired in severely malnourished children at admission but improves during rehabilitation.(Au)

Lipid binding capacity of spider hemocyanin.

The spider hemocyanin capacity to bind different lipid classes was evaluated by measuring some binding kinetic parameters. A very high lipoprotein (VHDL) which contains hemocyanin, was isolated from Polybetes pythagoricus hemolymph plasma and delipidated. Hemocyanin was bound separately to labelled palmitic acid, phosphatidylcholine, cholesterol, and triolein resulting in several artificial lipoprotein structures. It was possible to corroborate in vitro the lipid-hemocyanin interactions which had been previously observed and, consequently, the apolipoprotein role played by the respiratory pigment of spiders. Lipoproteins were analysed by gel filtration chromatography, and three subfractions with different hemocyanin structures were obtained. The four lipid classes were only bound to the hexameric structure (420 Kda), possibly to low polarity sites. Upon radioactivity measurements of the protein-associated lipids, maximal binding ratios (Mr), dissociation constants (Kd), and the maximal binding effectiveness at low lipid concentrations (Eo) were calculated. Lipid/protein ratios were increased proportionally to each available lipid concentration, following a hyperbolic binding model. Values of saturation, affinity, and maximal binding efficiency to hemocyanin were found to be different for each lipid class assayed. The highest lipid/protein ratio (41.5) was obtained with the free fatty acid and the lowest (7.2) with triolein. Phosphatidylcholine and cholesterol showed the highest relative affinities for hemocyanin (Kd = 63 x 10(-5) M and 74 x 10(-5) M, respectively). Phosphatidylcholine at low concentrations, similar to the physiological ones, presented the highest Eo value. Maximal lipid/protein ratios reached in vitro, were greater than those in P. pythagoricus VHDL, pointing out that hemocyanin could play the apolipoprotein role even under physiological conditions with a very high plasma lipid concentration. J. Exp. Zool. 284:368-373, 1999.

Effects of ethanol intake on lipid metabolism in the lactating rat.

Female rats receiving alcohol (20%) in drinking water during lactation (AL) were compared to pair-fed animals (PF) and normal controls (C) fed ad lib. All animals were killed on the 12th day of lactation. When compared to C rats, food intake decreased in both AL and PF groups, and this effect was followed by a lower body weight and mammary gland (MG), liver, and parametrial adipose tissue weights. Mammary glands triacylglyceride concentration (TG) was much lower in PF than in AL, although in the latter, values did not reach those of C, and had higher liver TG concentration than any of the other groups. Both PF and AL rats had lower plasma TG, glycerol, and free fatty acid concentrations and higher beta-hydroxybutyrate concentration than C rats. When compared to C rats, the rate of lipogenesis in MG was higher in both PF and AL rats, whereas in liver it was higher in PF and lower in AL rats, and in adipose tissue it was higher in PF and unchanged in AL rats. The appearance of 14C lipids 4 h after oral [14]triolein in both MG and liver was lower in AL and PF rats and only lower in adipose tissue of AL rats as compared to the c rats. Lipoprotein lipase and hormone-sensitive lipase activities were lower in MG in both PF and AL rats than in C, whereas in adipose tissue the activity of lipoprotein lipase did not differ between AL and C rats and the activity of HSL was lower in the former. These findings therefore show that in spite of reduced uptake of orally administered triglycerides due to decreased LPL activity, maternal alcohol feeding during lactation in the rat preserves the mammary gland triglyceride content thanks to enhanced lipogenetic activity. On the other hand, it causes liver triglycerides accumulation, probably as a result of the decreased rate of triglycerides released into circulation, and these changes are not caused by the reduced food intake of the animals.

'In vitro' cholesteryl ester bidirectional flow between high-density lipoproteins and triglyceride-rich emulsions: effects of particle concentration and composition, cholesteryl ester transfer activity and oleic acid.

The cholesteryl ester flow between high-density lipoproteins (HDL) and triglyceride-rich lipoproteins was investigated utilizing HDL fractions and synthetic lipid emulsion particles (EM). HDL was labeled in vitro with [1,2-3H(n)]cholesteryl ester or with [1,2,6,7-3H(n)]cholesteryl oleate, whereas EM were made with [4-14C]cholesteryl oleate (CO) or [carboxyl-14C]triolein (TO). The cholesteryl ester (CE) transfer rate between HDL and EM was spontaneous to some extent (because it occurred in saline medium), saturable, enhanced in a dose-dependent manner by the plasma fraction at D > 1.21 g/ml ascribed to its CETP activity, and greater for HDL3 than for HDL2. Unesterified fatty acids in the medium elicited two opposing effects: (1) enhanced the spontaneous cholesteryl oleate shift to EM and inhibited the reverse flow from EM to HDL3 but not to HDL2; (2) partially impaired the plasma D > 1.21 g/ml-induced bidirectional cholesteryl oleate flow. Approx. 2 mol TO from EM exchange for 1 mol CO from HDL3. Net cholesteryl ester balance was dependent on the concentration of HDL-cholesteryl ester, and independent from EM-cholesteryl oleate, possibly due to the much smaller concentration of the latter. These in vitro experiments shed light on the complex physical chemistry of transport of cholesteryl ester and triolein between HDL and TG-rich lipoproteins which occurs during the metabolism of chylomicrons in plasma.

Malignant hypertension is accompanied by marked alterations in chylomicron metabolism.

Malignant hypertension (MH) is a severe complication of untreated arterial hypertension that damages the vascular system. It is often accompanied by disturbances in lipid metabolism that could contribute to its pathophysiology. We examined chylomicron metabolism in MH patients using a triglyceride-rich emulsion known to mimic natural chylomicrons when injected into the bloodstream. The emulsion was labeled with [3H]triolein and [14C]cholesteryl oleate and injected intravenously into 15 normolipidemic MH patients aged 29 to 56 years (8 men) for comparison with 17 healthy control subjects. Consecutive plasma samples were taken at regular intervals during 1 hour for determination of the disappearance curves of the labels. The fractional clearance rate of the [3H]triolein emulsion in MH patients was twice as small as that of control subjects (0.061 +/- 0.012 and 0.141 +/- 0.074 min-1, respectively). On the other hand, [14C]cholesteryl oleate fractional clearance rate was not statistically different in MH patients and control subjects (0.032 +/- 0.004 and 0.056 +/- 0.014 min-1, respectively). These results indicate that in MH, lipolysis (measured by the fractional clearance rate of [3H]triolein) is pronounced diminished, whereas the removal of the remnant particles (measured by the fractional clearance rate of [14C]cholesteryl oleate) is not importantly affected. In conclusion, there is an alteration in the circulatory transport of dietary lipids that may be an important component in the vascular disease associated with MH.