Measurement of lipid hydroperoxides in plasma lipoproteins by a new highly-sensitive 'single photon counting' luminometer.

The lipid hydroperoxide content of isolated, native human plasma lipoproteins, was measured, by the luminol-based chemiluminescent reaction, using a highly sensitive single photon counting instrument. The reaction was specific for lipid hydroperoxides since the signal completely disappeared after treatment with the selenoperoxidase specific for lipidic substrates. In this analytical procedure the whole kinetic of photon emission induced by lipid hydroperoxides and hemin in the presence of luminol is integrated, taking advantage of the mono-exponential fitting of the decay of photon emission. The addition of a detergent to the reaction mixture improved the precision of the measurements apparently by preventing oxidative chain reactions affecting the shape of the decay of photon emission. The sensitivity of the instrument allowed measurements on samples containing just a few picomoles of hydroperoxides, small enough to minimize the effect of antioxidants and quenchers possibly present in the sample (as in the case of lipoproteins). Thus, by using an internal calibration with a phospholipid hydroperoxide, the evaluation of the lipid hydroperoxide content in whole, native lipoproteins was possible without previous extraction and chromatographic separation. Data obtained from plasma lipoproteins isolated by different procedures suggest that lipid hydroperoxide content increases during ultracentrifugation.

Determination of lipid hydroperoxides in native low-density lipoprotein by a chemiluminescent flow-injection assay.

Lipid hydroperoxides have been implicated in the pathogenesis of atherosclerosis. This work was therefore set up to obtain a fast and specific chemiluminescent assay for measuring hydroperoxides in native low-density lipoprotein (LDL). The apparatus was a complete HPLC system including two pumps, an autosampler, a computer and a chemiluminescent detector with a T-mixing coil in the place of the column. Samples were injected from the autosampler and mixed with luminescent reagent (3 microM luminol and 1 microM microperoxidase in 0.1 M carbonate buffer (pH 10)) in the T-piece. To generate a calibration curve, linoleic acid hydroperoxide was obtained by incubating soybean lipoxygenase with linoleic acid. The calculated conjugated diene concentration was in good agreement with the nominal linoleic acid hydroperoxide concentration. The chemiluminescence was linear with the amount of linoleic acid hydroperoxide injected and the detection limit was about 3 pmol linoleic acid hydroperoxide. The chemiluminescence induced by copper-oxidized LDL was linear with concentration; the detection limit, when compared with linoleic acid hydroperoxide, was similar. The reproducibility of the linoleic acid hydroperoxide and of oxidized LDL hydroperoxide was examined in single pools. The coefficient of variation on the triplicates of each pool was about 3%. The titre of the linoleic acid hydroperoxide and oxidized LDL peroxides was quite stable for at least 10 days when stored under argon at 4 degrees C in the presence of EDTA. The mean value of the LDL hydroperoxides in 16 control subjects was 145.20 +/- 98.81 pmol/mg LDL protein. In conclusion, the microperoxidase-luminol-dependent chemiluminescence flow-injection assay is a rapid, sensitive and selective method for measuring lipid hydroperoxides in native LDL.

Kinetic analysis of lipid-hydroperoxides in plasma.

We increased the precision of chemiluminescent procedure for measuring lipid hydroperoxides in plasma or lipoproteins by (i) escaping from extraction and chromatography of lipids, (ii) using detergent dispersed lipids, and (iii) calculating the results by fitting the photon emission rate with the integrated equation, which describes the model of the series of reactions. The use of kinetics instead of the crude integration of cps increases precision because at each measurement the correct reaction pathway is tested. This was relevant for the optimization of the analytical procedure, contributing to the elimination of possible side reactions. The relationship between lipid hydroperoxide content in the sample and cps is not linear; thus, the calculation of results through internal calibration is carried out using an exponential equation. This is in agreement with the reaction mechanism and raises the point of the linear calibration previously reported in other chemiluminescent procedures. Although sensitive and precise, this procedure suffers for being time consuming, requiring approximately 30 min per sample. Moreover, since no chromatography is used, information about the hydroperoxides in different lipid classes is missing. Obviously this will be solved when a validated procedure for quantitatively extracting lipid hydroperoxides is available.

Effect of plasma on the degradation of hydroperoxides of unesterified linoleic acid and copper-peroxidized LDL.

The determination of lipid hydroperoxides in plasma and lipoproteins recently reached a clinical relevance in disorders such as atherosclerosis, where oxidative reactions have been suggested to play a fundamental pathogenetic role. The peroxide content of lipoproteins is usually measured after ultracentrifugation and extraction. During this procedure, some peroxides might decompose causing a too low recovery. To screen this possibility, the disappearance, in the presence of human plasma, of hydroperoxides of linoleic acid and Cu-oxidized low density lipoprotein (LDL) have been investigated, using both a iodometric titration and an enzymatic assay. While only in the presence of GSH plasma decomposes linoleic acid hydroperoxides quite rapidly, peroxides in Cu-oxidized LDL were stable both in presence as well as in absence of GSH. This indicated that lipid hydroperoxides are stable in plasma and that peroxides of Cu-oxidized LDL are not substrate for the glutathione-dependent peroxidase activity in plasma. The relevant decrease of the iodometric titre of LDL peroxides observed in the presence of elevated amounts of plasma was shown to be artifactual, since some compounds extracted from plasma do react with iodine generated by peroxides. Whole plasma itself, indeed, has been shown to reduce back to I- appreciable amount of free iodine.

Antioxidants inhibit the expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 induced by oxidized LDL on human umbilical vein endothelial cells.

The oxidative modification of low density lipoprotein (LDL) and the endothelial expression of adhesion molecules are key events in the pathogenesis of atherosclerosis. In this study we evaluated the effect of oxidized LDL on the expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on human umbilical vein endothelial cells (HUVECs). The hypothesis that oxidized LDL functions as a prooxidant signal was also evaluated, by studying the effect of different radical-scavenging antioxidants on expression of adhesion molecules. LDL was oxidized by using Cu2+, HUVECs or phospholipase A2 (PLA2)/ soybean lipoxygenase (SLO), the degree of oxidation being measured as thiobarbituric acid-reactive substances (TBARS) and conjugated dienes (CD). Exposure of 200 micrograms/ml of native LDL to 1 microns Cu2+, HUVECs and to PLA2/ SLO resulted in four- to fivefold higher levels of TBARS and CD than in native LDL. Cu(2+)-(1 microM), HUVEC-, and PLA2/SLO-oxidized LDL caused a dose-dependent, significant increase of ICAM-1 and VCAM-1 (p < .01). The expression of E-selectin did not change. LDL oxidized with a 2.5 and 5 microM Cu2+ did not increase ICAM-1 and VCAM-1 significantly. Both the Cu(2+)- and HUVEC-oxidized LDL, subjected to dialysis and ultrafiltration, induced ICAM-1 and VCAM-1 expression. After incubation with the ultrafiltrate, the expression of ICAM-1 and VCAM-1 was not significantly different from that obtained with native LDL. LDL pretreated with different antioxidants (vitamin E and probucol) and subjected to oxidation by Cu2+ and HUVECs induced a significantly lower expression of ICAM-1 and VCAM-1 than nonloaded LDL (p < .01). The pretreatment of HUVECs with vitamin E and probucol significantly reduced the expression of VCAM-1 on HUVECs induced by oxidized LDL (p < .01); the effect on ICAM-1 was much less evident. In conclusion, oxidized LDL can induce the expression of different adhesion molecules on HUVECs; this induction can be prevented by pretreating either the LDL or the cells with radical-scavenging antioxidant.

Predisposition to LDL oxidation in patients with and without angiographically established coronary artery disease.

Oxidative modification of low density lipoprotein (LDL) may play an important role in the mechanism of atherosclerotic damage to blood vessels. In the present study the LDL isolated from the plasmas of 73 coronary artery disease (CAD) patients, 28 valvular heart disease (VHD) patients, 59 subjects affected by type IIa hyperlipoproteinemia and 71 controls was oxidatively modified by incubation with copper ions. In 15 CAD and 15 Type IIa patients and 15 controls the LDL chemical composition and polyunsaturated fatty acid (PUFA) content were also measured. Differences in the LDL susceptibilities to lipid peroxidation were studied by measuring the changes of fluorescence intensity. The lag phase in the CAD patients was found to be significantly lower than in the VHD and controls (P < 0.001). The lag phase in the type IIa patients was significantly higher than in the CAD patients (P < 0.01), and significantly lower than the VHD and controls (P < 0.01). The LDL isolated from the type IIa patients had an increase in the relative content of free and esterified cholesterol (P < 0.05), while the CAD patients had a decrease in the relative content of free cholesterol (P < 0.05), and an increase in the relative content of protein (P < 0.05). The lowest value of the LDL cholesterol to protein ratio and LDL size, was found in the CAD patients (P < 0.05). When expressed in micrograms/mg LDL cholesterol, the concentration of the LDL PUFAs was significantly higher in the CAD group than in the others (P < 0.05). The LDL alpha-tocopherol concentration was quite similar in the different groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The calcium-channel blocker lacidipine reduces the development of atherosclerotic lesions in the apoE-deficient mouse.

BACKGROUND: Lacidipine is a widely used calcium-channel blocker, which has both long-lasting antihypertensive activity and also antioxidant properties. Previous studies have demonstrated the ability of lacidipine to reduce the development of atherosclerotic lesions in several animal models. OBJECTIVE: The present study investigated the antiatherosclerotic potential of lacidipine in the apoE-deficient mouse, an experimental model of atherosclerosis showing progressively complex and widespread lesions which closely resemble the inflammatory-fibrous plaques seen in humans. METHODS: Lacidipine was administered daily by gavage for 10 weeks at dose levels of 0 (control), 0.3, 1.0 and 3.0 mg/kg. RESULTS: Lacidipine administration reduces the extension of atherosclerotic lesions in the aorta of the apoE-deficient mouse without affecting plasma lipid levels. We also show that apoE-deficient mice have four-fold higher values of the proatherogenic peptide, endothelin, compared with the wild-type C57BL/6 mouse and that lacidipine administration reduced, in a dose-dependent manner, the concentrations of plasma endothelin. CONCLUSION: Lacidipine has anti-atherogenic effects in the apoE-deficient mouse, and reduces plasma endothelin concentrations.

Lacidipine inhibits the activation of the transcription factor NF-kappaB and the expression of adhesion molecules induced by pro-oxidant signals on endothelial cells.

OBJECTIVE: The adhesion of monocytes to endothelium, an early event in atherosclerosis is mediated by cell adhesion molecules. Signal-transduction pathways for these binding molecules include the translocation of the transcription factor NF-kappaB; moreover, intracellularly generated oxygen-derived free radicals play a major role in this process. In this study we evaluated the extent to which lacidipine, a calcium antagonist with antioxidant properties, affects the expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin on human umbilical vein endothelial cells, induced by different pro-oxidant signals such as oxidized low density lipoprotein (LDL) and tumor necrosis factor-alpha (TNF-alpha). METHODS: We incubated 5 micromol/l Cu2+-oxidized LDL and TNF-alpha (2 ng/ml) with human umbilical vein endothelial cells for 48 and 6 h, respectively. ICAM-1, VCAM-1 and E-selectin were measured by flow cytometry. NF-kappaB was evaluated by electrophoretic mobility shift assay. RESULTS: The incubation of 5 micromol/l Cu2+-oxidized LDL not only caused a dose-dependent increase in ICAM-1, VCAM-1 and E-selectin (P < 0.001), but also synergically increased their TNF-alpha-induced expression (P < 0.001). The addition of lacidipine to human umbilical vein endothelial cells significantly reduced the expression of ICAM-1, VCAM-1 and E-selectin induced by TNF-alpha alone or with oxidized LDL (P < 0.001). The reduction in adhesion molecule expression caused by lacidipine was paralleled by a significant fall in NF-kappaB translocation. CONCLUSIONS: The results suggest that lacidipine may have prevented NF-kappaB-mediated adhesion molecule expression by exerting its effects on oxygen-derived free radicals. The results support previous observations that lacidipine may have therapeutic effects in atherosclerosis.

Oxidized low-density lipoprotein increases the production of intracellular reactive oxygen species in endothelial cells: inhibitory effect of lacidipine.

OBJECTIVE: The mechanisms by which oxidized low-density lipoprotein (ox-LDL) induces the expression of adhesion molecules on endothelial cells (HUVECs) are still not clear. The signal transduction pathways for these binding molecules include the translocation of the transcription factor NF-kB and the intracellular reactive oxygen species (ROS) are said to play a key role in this process. Aim of this study was (1) to evaluate the effect of ox-LDL on intracellular production of ROS in culture of HUVECs; (2) to evaluate if the intracellular increase of ROS induced by ox-LDL is mediated by the binding to a specific endothelial receptor; (3) to ascertain if lacidipine can decrease ox-LDL-induced ROS production in HUVECs. METHODS: Five microM Cu2+ ox-LDL were incubated with HUVECs for 5 min. 2',7'-Dichlorofluorescein (DCF) as an expression of intracellular ROS production, was measured by flow cytometry. RESULTS: ox-LDL induced a significant dose-dependent increase in DCF production (P < 0.001) through the binding to a specific receptor. The preincubation of HUVECs with radical scavengers compounds and lacidipine significantly reduced (P < 0.001) the ox-LDL-induced DCF production. CONCLUSIONS: ox-LDL increased the intracellular formation of ROS through the ligation to a specific endothelial receptor. Preincubation of HUVECs with lacidipine, a calcium antagonist with antioxidant properties, significantly reduced the intracellular ROS formation induced by ox-LDL. We propose that the effect of lacidipine on adhesion molecule expression and on NF-kB activation can be explained by its effect on intracellular ROS formation.

Comparative effects of different dihydropyridines on the expression of adhesion molecules induced by TNF-alpha on endothelial cells.

OBJECTIVE: Lacidipine has already been demonstrated to reduce the expression of some adhesion molecules induced by pro-oxidant signals on endothelial cells. In order to verify if this effect is a peculiarity of this molecule, or belongs to other dihydropyridinic compounds (DHPs), the activity of lacidipine was compared with that of lercanidipine, amlodipine, nimodipine and nifedipine. DESIGN AND METHODS: The compounds were incorporated in human umbilical vein endothelial cells (HUVECs) using native low-density lipoprotein as a carrier. The drug concentrations in HUVECs were measured by mass spectrometry. Human recombinant tumour necrosis factor-alpha was then incubated with HUVECs for 7 h at 37 degrees C for adhesion molecule expression. RESULTS: The cellular amount of lacidipine, lercanidipine and amlodipine was similar, while nimodipine and nifedipine were almost undetectable or undetectable, respectively. Lacidipine, at any concentration, determined a dose-dependent significant decrease of the expression of intercellular adhesion molecule-1 (ICAM-1) ICAM-1, vascular cell adhesion molecule-1 (VCAM-1) VCAM-1 and E-selectin (P < 0.01). Lercanidipine and amlodipine determined variable decreases of adhesion molecules at the intermediate and highest concentrations. Nimodipine and nifedipine determined no effect on ICAM-1, VCAM-1 and E-selectin. The lowest IC50, i.e. the concentration determining the 50% reduction of ICAM-1, VCAM-1 and E-selectin expression was obtained with lacidipine for all the adhesion molecules considered (P < 0.01). CONCLUSIONS: It is concluded that the effect of the DHPs used in this study on adhesion molecule expression is determined first by their lipophilicity and then by their intrinsic antioxidant activity.

Predisposition to LDL oxidation during copper-catalyzed oxidative modification and its relation to alpha-tocopherol content in humans.

The predisposition to LDL oxidation during copper-catalyzed oxidative modification and its relationship with LDL alpha-tocopherol concentration was studied in 41 control subjects. The results show that the predisposition of LDL to oxidation expressed as duration of the inhibition period and rate of the propagation period varied greatly in the controls, but did not correlate with the values of LDL alpha-tocopherol. On the contrary the experiments with alpha-tocopherol incorporated in LDL demonstrate that even small increases of incorporated alpha-tocopherol, under circumstances where other variables were probably largely unaffected, increased proportionally the length of the inhibition period and reduced the rate of the propagation period. The values of LDL alpha-tocopherol achieved after the enrichment turned out to be positively correlated with the duration of the inhibition period and negatively with the rate of the propagation period. Finally the results of this study also show that there was a variability in the LDL alpha-tocopherol decay of different subjects under the same oxidative stress. In our conditions however, the time in which alpha-tocopherol contributed to the LDL protection was much shorter than the mean length of the inhibition period. The results demonstrate that the variability in the predisposition to LDL oxidation during copper-catalyzed oxidative modification is not determined only by the concentration of alpha-tocopherol in LDL and that therefore its value as a sole indicator of antioxidant status is probably inadequate.

The susceptibility of low-density lipoprotein to in vitro oxidation is increased in hypercholesterolemic patients.

It has been suggested that the oxidative modification of low-density lipoprotein (LDL) plays a major role in atherogenesis. We evaluated the oxidative resistance to copper-induced oxidative changes of LDL derived from patients affected by type IIa hyperlipoproteinemia compared with healthy subjects and faced the question of the importance of the antioxidants and polyunsaturated fatty acids (PUFAs) contained in LDL in determining its variability. LDL isolated from the plasmas of 25 subjects affected by familial hypercholesterolemia and 15 control subjects was oxidatively modified with Cu2+ in vitro, and the differences in LDL susceptibilities (lag and propagation phases) to lipid peroxidation were studied by measuring the changes in fluorescence intensity. LDL alpha-tocopherol and PUFAs were also measured. The lag phase was significantly lower and the propagation phase significantly higher in the type IIa patients than in control subjects (p < 0.01). The linoleic and arachidonic acids, expressed as percentage of total LDL fatty acids, were significantly higher in type IIa patients than in the control subjects (p < 0.01). There was a positive significant correlation between the LDL cholesterol and the linoleic and arachidonic acids as percentage of total LDL fatty acids (p < 0.01). Both linoleic and arachidonic acids turned out to be negatively correlated with the lag phase and positively with the propagation phase (p < 0.01). The concentration of LDL alpha-tocopherol was similar in the two groups. Therefore, type IIa patients have a greater susceptibility to LDL oxidation than control subjects. This may be due to a relative higher concentration of linoleic and arachidonic acids in LDL derived from patients with familial hypercholesterolemia.

Increased susceptibility of LDL to in vitro oxidation in patients on maintenance hemodialysis: effects of fish oil and vitamin E administration.

Oxidized low-density lipoproteins (LDL) play an important role in the pathogenesis of atherosclerosis. An increased sensitivity of red blood cell membranes to lipid peroxidation has been previously demonstrated in patients with chronic renal failure, suggesting that the antioxidant defence of lipoproteins might be impaired. Fish oil supplementation has been proposed in dialysis patients, but it is still unclear if the positive effects of fish oil depend only on its polyunsaturated fatty acid content or on other factors, such as the usually added antioxidants. Moreover, the increased concentration of highly peroxidable n-3 polyunsaturated fatty acids induced by fish oil in LDL particles could favour LDL oxidation and possibly the development of atherosclerosis. The present study was designed to evaluate the susceptibility of LDL to in vitro oxidation (lag phase) and the rate of lipid peroxidation (propagation phase) by fluorescence development during copper exposure in 14 hemodialysis patients. A further aim was to compare the effects on lipid metabolism and LDL oxidation of fish oil supplementation (20 ml containing vitamin E 20 IU as antioxidant) for 30 days and of vitamin E administration (50 IU) for another 30 days. The length of the lag phase and vitamin E concentration were significantly reduced (p < 0.01) in hemodialysis patients and increased significantly (p < 0.01) after administration of both fish oil and vitamin E. Fish oil supplementation also reduced plasma lipids significantly (p < 0.01) and increased the propagation phase (p < 0.01). Our results demonstrate that the susceptibility of LDL to oxidation is enhanced in hemodialysis patients, suggesting a possible relationship between excessive LDL peroxidation and accelerated atherosclerosis. The increased susceptibility of LDL to in vitro oxidation can be explained, at least partially, by a reduced LDL vitamin E concentration. Since fish oil increased the lag phase to the same extent as vitamin E supplementation, the positive effect of fish oil could be partly explained by its antioxidant content.

Fluorimetric determination and pharmacokinetic studies of fazadinium bromide in dogs.

A rapid fluorimetric method for the estimation in biological fluids of the neuromuscular-relaxant drug 1,1'-azobis-3-methyl-2-phenyl-1H-imidazo-[1,2-a]-pyridinium dibromide (fazadinium bromide, AH 8165) is described. The drug is hydrolized by alkali to give 3-methyl-2-phenyl-1H-(1,2-a)-imidazo-pyridinium bromide, which is extracted by cyclohexane and measured for fluorescence. Intravenous administration (1 mg/kg) in dogs gives initial blood levels of unchanged drug of 4.34 +/- 0.25 microgram/ml. The decay of serum concentrations is rapid. The extent of urinary excretion of unchanged drug is 15% within 6 h.

Alphaxalone distribution in rat tissues.

A method for the extraction and gaschromatographic determination of 3alpha-hydroxy-5alpha-pregnane-11,20-dione (alphaxalone, main active component of Althesin) in microgram amount in plasma and tissues is described. The slow i.v. infusion of Althesin in the rat gives uniform alphaxalone distribution in tissues, slight localization in the fat and brain levels comparable to plasma concentration.

Properties of Sepharose-bound beta-lactamase from Enterobacter cloacae.

beta-Lactamase from Enterobacter cloacae P99 was immobilized onto Sepharose by the cyanogen bromide activation method and the properties of the Sepharose-bound enzyme were compared with those of soluble and cell-bound enzyme. The immobilized beta-lactamase showed enhanced stability to storage at 4 degrees C (approximately 1 year) in respect to the free enzyme in solution (few days). The optimum pH for activity is similar for both Sepharose- and cell-bound beta-lactamase and extends over a broader pH range (pH 6-9) than the soluble enzyme (pH 8-9). Immobilization leads also to significant enhancement of thermal stability. Effective enzyme inhibition by flucloxacillin occurs with both soluble and Sepharose-bound beta-lactamase, whereas the cell-bound enzyme is much less (10(-5) times) inhibited. These results indicate that immobilized beta-lactamase could be usefully employed as a tool for investigating the properties of newly designed beta-lactamase inhibitors.

Synergistic interaction of bactericidal drugs and a factor present in serum ultrafiltrate.

Ultrafiltrate of serum of guinea-pig and man contains a factor which interacts synergistically with cephaloridine, kanamycin, and nalidixic acid against Escherichia coli. The factor on its own is not bactericidal. Gel filtration showed that the main peaks of the fractions with biological activity correspond to a MW of about 200. Chemical analysis excluded the presence of carbohydrates, proteins, ribonucleic acid at levels detectable with standard methods. Moreover the activity of the factor was not destroyed by meat nor by acid and enzymatic hydrolysis. A sequential mechanism or a reaction of the factor with drugs are considered as two possible explanations of the interaction.

A simple test for predisposition to LDL oxidation based on the fluorescence development during copper-catalyzed oxidative modification.

When human low density lipoprotein (LDL) obtained from 10 volunteers was incubated in air at 37 degrees C in the presence of various concentrations of copper, an increase in fluorescence was observed with emission maximum at 430 nm when excitation was performed at 360 nm. The fluorescence increase was inhibited by ethylenediamine tetraacetic acid and by 4-methyl-2,6-di-tert-butylphenol. The fluorescence was found to be tightly bound to the protein moiety. Furthermore, Cu2+ modification of LDL was associated with a decrease in the reactive amino groups of apolipoprotein B and in the uptake of the lipoprotein by rabbit fibroblasts. Under our conditions, the fluorescence increase showed two consecutive periods; an inhibition period during which the fluorescence increased only weakly, and a propagation period with a rapid increase in fluorescence that was linear for at least 5 h. Both periods were influenced by copper concentration. The study also shows that the extent of fluorescence generated upon LDL oxidation varied greatly in the volunteers. Thus, while the results demonstrate that the fluorescence increase may likely monitor the extent of the apoB derivatization, the calculation of the fluorescence development rate of the propagation period together with the duration of the inhibition period may constitute a quantitative measurement of the susceptibility of apoB to be derivatized.

High-density lipoprotein cholesterol concentrations and postheparin hepatic and lipoprotein lipases in obesity: relationships with plasma insulin levels.

The concentration of high-density lipoprotein (HDL) cholesterol in patients with severe obesity is generally subnormal. The exact mechanism linking obesity with reduced levels of HDL cholesterol remains unclear. In this study we evaluated the postheparin plasma lipolytic enzymes lipoprotein lipoprotein lipase (LPL) and hepatic lipase (HL) in a group of 24 obese women compared with controls and analyzed the interrelationships between insulin, postheparin lipolytic enzymes and HDL subfractions. Total HDL cholesterol was significantly lower in the obese subjects than in the controls, and the difference was mainly due to HDL2 cholesterol concentrations. Mean fasting glucose, insulin and the summated means of glucose (sigma glucose) after the oral glucose tolerance test (OGTT) were not significantly different in the two groups. The summated means of insulin (sigma IRI) after the OGTT were significantly higher in the obese women than in the controls. LPL, HL and the HL-to-LPL ratio were significantly higher in the obese women than in the controls. HL and LPL correlated positively with sigma glucose, sigma IRI and body mass index (BMI) and negatively with plasma triglycerides. Partial correlation analysis demonstrated that, when exposed to similar sigma IRI values, HL and LPL were no longer correlated with sigma glucose, plasma triglycerides and BMI. HDL2 cholesterol levels were negatively correlated with HL, posthepatic plasma lipolytic activity, sigma glucose, plasma triglycerides and BMI. HDL2 cholesterol concentrations were directly correlated with LPL. Partial correlation analysis showed that when exposed to similar HL and LPL values, HDL2 cholesterol values were no longer correlated with sigma glucose, sigma IRI, plasma triglycerides and BMI. Therefore, our results demonstrate that the low HDL2 cholesterol levels found in obese women may be related to the high levels of HL and to the high HL-to-LPL ratio which in turn could be determined by the peripheral insulin resistance.