Impaired glucose tolerance is not associated with lipid intolerance.

Post-prandial (pp) hypertriglyceridaemia (HTG) has an important role in the development of atherosclerosis in Type 2 diabetes. Impaired glucose tolerance (IGT) is associated with an increased risk of atherosclerosis and increased level of fasting triglycerides (TG). The aim of this study was to analyse pp HTG and the composition of TG-rich lipoproteins in carefully selected subjects with IGT in comparison to controls with normal glucose tolerance (NGT). Fifteen men with IGT and 27 men with NGT, aged 44 to 70 yr, were examined. All study participants were non-smokers and had fasting TG <4.6 mmol/l. The subjects underwent an oral glucose tolerance test (75 g glucose) and a lipid-glucose tolerance test (LGTT; 92 g fat, 126 g carbohydrate), that allowed the assessment of lipid and glucose tolerance in one test. HbA1C, plasma glucose and lipids were measured by routine methods. Lipoprotein subfraction analysis of VLDL (VLDL1: Sf60-400 and VLDL2: Sf20-60) was conducted in a fasting state, as well as 4 hr after the LGTT using a density gradient ultracentrifugation with a subsequent compositional analysis. No significant difference was found either for fasting or pp TG, or for area under curve (AUC) -TG (12.21 +/- 4.27 mmol/l x 6 hr vs 13.95 +/- 6.74 mmol/l x 6 hr; p>0.05) between the IGT and NGT. A highly significant correlation was found between the fasting TG and the AUC-TG (r=0.925; p<0.01). To avoid bias by differences in fasting plasma TG known to affect lipid tolerance we investigated 11 matched pairs for fasting TG. Also, the matched-pairs evaluation pp TG course did not differ significantly from the IGT and NGT. No significant difference for fasting or pp levels of VLDL1 and VLDL2, or for the TG content of chylomicron, VLDL1 and VLDL2 and for the percentage of TG in VLDL1 and VLDL2 was found between the IGT and NGT group. In conclusion, IGT subjects with a similar level of fasting TG do not exhibit lipid intolerance. Our data suggest that glucose intolerance should precede lipid intolerance.

Postchallenge plasma glucose and glycemic spikes are more strongly associated with atherosclerosis than fasting glucose or HbA1c level.

OBJECTIVE: To observe the relationship of fasting plasma glucose (FPG), postchallenge plasma glucose (PG) (30, 60, 90, and 120 min during an oral glucose tolerance test [OGTT], as well as maximal PG during an OGTT, postchallenge glucose spikes [PGS], and glucose under the OGTT curve), and HbA1c to intima-media thickness (IMT) as a marker of atherosclerosis. RESEARCH DESIGN AND METHODS: OGTT, ultrasound measurement of carotid IMT, and various atherosclerosis risk factors, such as family history of diabetes, obesity, and/or hyperlipoproteinemia, but without known diabetes, were analyzed in 582 individuals aged 40-70 years and at risk for type 2 diabetes. RESULTS: In univariate analysis, all examined glycemic parameters were significantly correlated to IMT. The 2-h postchallenge plasma glucose showed the strongest odds ratio (OR) of 1.88 (1.34-2.63) in relation to abnormal IMT. All PG variables, except for 30-min glucose in OGTT, showed a significant OR, whereas the OR for HbA1c and FPG was not significant. In logistic regression analysis, 2-h PG was identified as the strongest determinant of IMT from all glycemic parameters. The 2-h PG and PGS, but not FPG, were associated with a significant rise of IMT in tertiles of HbA1c. Glycemic parameters were strongly related to each other and to many atherosclerosis risk factors. In multivariate analysis including a variety of atherosclerosis risk factors, 2-h PG was a significant independent determinant of IMT. CONCLUSIONS: PG and PGS are more strongly associated with carotid IMT than FPG and HbA1c level and modify substantially the risk for atherosclerosis, estimated by HbA1c alone, in a cohort at risk for diabetes and in the early diabetes stage.

Urate attenuates oxidation of native low-density lipoprotein by hypochlorite and the subsequent lipoprotein-induced respiratory burst activities of polymorphonuclear leukocytes.

Oxidation converts native low-density lipoprotein (LDL) into a signal molecule promoting inflammatory processes during atherogenesis. The exact contribution of different antioxidants in prevention of LDL oxidation is not known. Uric acid efficiently scavenges oxidants including hypochlorite. We investigated the effect of different urate concentrations (25-500 micromol/l) on the oxidation of isolated native LDL by sodium hypochlorite (1000 micromol/l). While relative electrophoretic mobility declined continuously with increasing urate concentrations in the oxidation medium, lipid peroxidation as measured by TBARS was blunted only at high molar urate/NaOCl ratios. By decreasing oxidative modifications, urate dose-dependently (beginning with a urate/NaOCl ratio of 1:40) diminished stimulatory effects of oxidized LDL on the respiratory burst of resting polymorphonuclear leukocytes (PMNL). Protecting effects of urate against the proinflammatory action of oxidized LDL on activated cells were evident only at a molar urate/NaOCl ratio of 1:2 suggesting different sensitivities of PMNL to LDL oxidation state in dependence on their activity state.

Partial sequence identification of grapevine-leafroll-associated virus-1 and development of a highly sensitive IC-RT-PCR detection method.

Using immunocapture reverse transcription PCR (IC-RT-PCR) a specific PCR product from GLRaV-1 infected vine samples was amplified with the help of degenerate primers deduced from the conserved HSP70 region of closteroviruses. 511 basepairs of the 5'end of GLRaV-1 HSP70 gene were identified. Within this region, putative GLRaV-1 specific primers were designed and an IC-RT-PCR detection procedure was developed which is about 125 times more sensitive than the established ELISA method. No PCR product was amplified in GLRaV-2,-3 and -4 infected plants which indicates the specificity of the primers. This procedure may serve as an alternative method for GLRaV-1 detection where the sensitivity of ELISA is insufficient.

Physicochemical binding properties of the proteoglycan receptor for serum lipoproteins.

Proteoheparan sulfate can be adsorbed to a methylated silica surface in a monomolecular layer via its transmembrane hydrophobic protein core domain. Due to electrostatic repulsion, its anionic polysugar side chains are stretched out into the blood substitute solution representing a co-receptor for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. The binding process was studied by ellipsometric techniques showing that oxLDL had a deleterious effect on heparan sulfate proteoglycan binding and conformation. Ca2+ binding to and storage on the proteoheparan sulfate/LDL compound formed a 'heterotrimeric' HS-PG/LDL/Ca2+ complex of high stability, aggregability and deposit coating. On the other hand, HDL bound to heparan sulfate proteoglycan protected against LDL docking and completely suppressed calcification of the proteoglycan/lipoprotein complex.

Diminished susceptibility to in vitro oxidation of low-density lipoproteins in hypercholesterolemia: key role of alpha-tocopherol content.

Oxidizability of low-density lipoproteins (LDL) in hypercholesterolemia is of clinical relevance, but previous studies revealed diverging results. Therefore, we studied ex vivo oxidation of LDL in plasma samples of 57 hypercholesterolemic and 20 normocholesterolemic volunteers. LDL were isolated by ultracentrifugation and analyzed for lipids and alpha-tocopherol. The formation of conjugated dienes, lipoperoxides and malondialdehyde was measured at 0.1 micromol/l LDL, 3.2 micromol/l CuSO4. We found prolongation of the lagtime (53.6/65.8/71.4 min) with tertiles (< or = 3.17/3.89/14.2 mmol/l) of LDL-cholesterol (LDL-C). Regression analysis revealed that the lagtime increased with the apparent concentrations of cholesterol (P = 0.003) and alpha-tocopherol (P = 0.001) in the oxidation assay. A multiple regression model with the apparent concentrations of alpha-tocopherol, triglycerides and cholesterol explained 40% of the variation in lagtime. The close relationship between plasma concentrations of LDL-C and LDL-alpha-tocopherol (P = 0.002) indicated that LDL contained more of this antioxidant in hypercholesterolemia. This might provide an explanation for the positive relationship between lagtime and LDL-C. The latter was independent of whether LDL-C or LDL-protein was chosen for standardization of the oxidation assay. The formation of conjugated dienes (P = 0.000), lipoperoxides (P = 0.038) and malondialdehyde (P = 0.001) increased with the cholesterol level in the assay. This may be due to the increased load of LDL with cholesterol esters as a substrate for oxidation in hypercholesterolemia. Our data do not support the opinion that hypercholesterolemia is characterized by increased susceptibility of LDL to oxidation.

Increased intimal-medial thickness in newly detected type 2 diabetes: risk factors.

OBJECTIVE: To examine carotid intimal-medial thickness (IMT) and its determinants in newly detected type 2 diabetic subjects, classified according to the new criteria of the American Diabetes Association, in comparison with age- and sex-matched control subjects with normal glucose tolerance. RESEARCH DESIGN AND METHODS: This study was case-controlled, with matched pairs for 71 newly diagnosed type 2 diabetic individuals. Subjects aged 40-70 years were recruited from a risk population for diabetes seen in the Risk Factors in IGT for Atherosclerosis and Diabetes (RIAD) Study. Standard risk factors, 75-g oral glucose tolerance test with real insulin, proinsulin, and C-peptide, and ultrasound measurement of the IMT of the common carotid artery were performed. RESULTS: The diabetic subjects, both men and women, displayed carotid intimal-medial thickening, even in the subgroup with fasting plasma glucose between 7.0 and 7.8 mmol/l. HbA1c was significantly increased in the diabetic patients (6.33 vs. 5.48%). Insulin, proinsulin, and C-peptide were also significantly higher. Among the coronary risk factors, triglycerides and plasminogen activator inhibitor were significantly increased. After age and sex adjustment. IMT in the diabetic group was correlated to triglycerides and the total-to-HDL cholesterol ratio. In the total group, IMT was significantly correlated to blood pressure, 2-h glucose in oral glucose tolerance testing, triglycerides, albuminuria, and the total-to-HDL cholesterol ratio, and inversely correlated to HDL cholesterol. No independent determinant of IMT was found in the diabetic group by multivariate analysis. CONCLUSIONS: Newly detected type 2 diabetic patients exhibit a higher degree of early atherosclerosis than normal glucose-tolerant subjects matched for age and sex. Our data suggest that hyperglycemia, together with a clustering of risk factors, and in particular dyslipidemia, may cause intimal-medial thickening in the early phases of diabetes.

Relation of free and specifically bound leptin to insulin secretion in patients with impaired glucose tolerance (IGT).

Impaired glucose tolerance (IGT) is frequently associated with an increased fat mass and an altered fat distribution. The adipocyte derived hormone, leptin has been shown to interact with insulin at various levels and may be intimately involved in this process. However, only limited data concerning the interaction of insulin, glucose tolerance and leptin are available and no data exist on the potential influence of bound vs. free circulating leptin. We therefore studied free and bound leptin in 136 patients (77 males, 59 females) with IGT, in relation to plasma glucose, insulin, proinsulin and C-peptide levels as well as serum free and bound leptin concentrations during an oral glucose tolerance test (oGTT). The expected positive relation of free serum leptin levels with body mass index (BMI) was found. Free leptin concentrations were higher in women than in men. Analysis in tertiles revealed a significant relation between free leptin (16-58, 60-160, and 169-932 pmol/l) and mean fasting insulin levels (65, 93, and 100 pmol/l). This relationship remained significant in a multiple regression analysis with BMI and gender as covariates. Similar independent relationships to leptin serum levels were observed for HbA1c and plasma C peptide levels and the proinsulin/insulin ratio but not for plasma glucose and proinsulin levels. These data suggest a fine tuning of leptin by small changes in circulating insulin levels observed in impaired glucose tolerance.

Antioxidative action of the novel calcium channel antagonist mibefradil on low-density lipoproteins.

OBJECTIVE: Mibefradil is a novel calcium channel antagonist that selectively blocks T-channels. It acts to reduce hypertension, is cardioprotective and reduces ischemic episodes. Oxidative modification of low-density lipoproteins (LDL) is well known to contribute to coronary atherosclerosis and we therefore investigated to see whether mibefradil had antioxidative action on LDL. METHODS: Human LDL were isolated by ultracentrifugation. In vitro oxidation of LDL (0.1 micromol x l(-1) protein) in the presence of various concentrations of mibefradil was initiated by 3.2 micromol x l(-1) copper ions. The kinetics of formation of conjugated dienes was followed photometrically. Malondialdehyde and lipoperoxides were determined at maximum oxidation. LDL (0.3 micromol x l(-1)) were also pre-incubated with mibefradil (120 micromol x l(-1)). Excessive mibefradil was separated by column technique. The resultant LDL were oxidized using copper ions or (AAPH) 2,2'-azobis(2-amidinopropane) hydrochloride. RESULTS: The presence of mibefradil in the concentration range from 10 to 200 micromol x l(-1) had dose-dependent effects. These were protection of LDL against oxidation measured as prolongation of the lagtime up to 250%, and reduction in the formation of malondialdehyde down to 65% and of lipoperoxides to 20%. Pre-incubation of LDL with mibefradil prolonged the lagtime of Cu-mediated oxidation up to 132% and of AAPH-mediated oxidation up to 138%. CONCLUSION: In addition to the T-channel blocking and antiproliferative effects, our results provide arguments for a protective role of mibefradil (10-200 micromol x l(-1)) on LDL against in vitro oxidation. This was shown with three independent parameters (lagtime, malondialdehyde and lipoperoxides) and in different oxidation models.

Prevalence of diabetes-specific autoantibodies in patients at risk for adult onset diabetes mellitus.

To evaluate the potential of autoimmune markers in identifying patients with slowly progressive IDDM in the prediabetic state, we screened a population of 151 patients aged 37-70 years with impaired glucose tolerance (IGT) for the presence of islet cell antibodies (ICA), insulin autoantibodies (IAA), antibodies to glutamic acid decarboxylase (GADA), and antibodies to tyrosine phosphatase IA-2 (IA-2A). Autoantibodies were found in 5 (3.3%) patients with IGT suggesting the presence of an autoimmune-mediated beta cell destruction. All of them were positive for high level ICA (> 20 JDF-U) and 1 ICA positive subject had additional GADA (100 GADA-U). In contrast, none of the subjects had IA-2A or IAA. We here demonstrate a low prevalence of autoimmune diabetes among middle-aged subjects with IGT. ICA and GADA but not IA-2A or IAA may represent autoimmune markers for slowly progressive IDDM before the manifestation of the disease.

Hypochlorite-modified low-density lipoprotein stimulates human polymorphonuclear leukocytes for enhanced production of reactive oxygen metabolites, enzyme secretion, and adhesion to endothelial cells.

Hypochlorite-oxidized low-density lipoprotein ((-)OCl-LDL) has been shown to stimulate various functions of human polymorphonuclear leukocytes (PMNLs). Incubation of PMNLs with (-)OCl-LDL (produced by incubation of 0.4 mM LDL cholesterol with 1 mM NaOCl for 40 min at 37 degrees C) but not native or copper-oxidized LDL induced a substantial generation of reactive oxygen species (ROS) as measured by means of chemiluminescence with one peak at 10-12 min. Upon stimulation with (-)OCl-LDL about 70% of ROS (hydrogen peroxide and superoxide anion) were released from the cells into the extracellular environment. The (-)OCl-LDL-induced increase of the respiratory burst was dependent upon the dose, exposure time, and extent of LDL oxidation. Cytochalasin B, an inhibitor of phagocytosis, markedly diminished the LDL-induced ROS generation to nearly 40% of control values. (-)OCl-LDL enhanced the adhesion of PMNLs to human umbilical venous endothelial cells 2.5-fold as compared to native LDL and promoted the secretion of the active granule enzymes lysozyme and beta-glucuronidase. Together, the results suggest a potential role of LDL-activated PMNLs in initiating and/or maintaining the inflammatory process during the early phase of atherosclerotic lesion development. Alternatively, PMNLs may also play a protective role by phagocytosing oxidized LDL and, thus, preventing further detrimental atherogenic effects of oxidized LDL.

Glibenclamide, but not acarbose, increases leptin concentrations parallel to changes in insulin in subjects with NIDDM.

OBJECTIVE: To hypothesize if glibenclamide, which increases insulin levels, also increases leptin concentrations. RESEARCH DESIGN AND METHODS: Leptin is a hormone that regulates weight in mice. In obese humans, leptin concentrations are increased, suggesting resistance to the effects of this hormone. Although short-term infusion of insulin during the hyperinsulinemiceuglycemic clamp does not increase leptin concentration, the effect of oral antidiabetic agents on leptin concentration is unknown. Differing effects can be expected, since glibenclamide acts via stimulation of insulin secretion, whereas acarbose inhibits alpha-glucosidases of the small intestine and has no direct effect on insulin levels. We examined the effect of acarbose (n = 4), glibenclamide (n = 6), and placebo (n = 6) on insulin and leptin levels during 24-h periods before and after 16 weeks of therapy. RESULTS: We observed a significant diurnal variation in leptin concentrations. This was inversely related to insulin levels during the 24-h follow-up with usual diet. Neither the placebo nor acarbose altered leptin concentrations. However, glibenclamide increased leptin concentrations parallel to insulin levels. There were only minor changes in body weight during the l6-week follow-up: decrease in the placebo group (change -0.5 kg/m2, P = 0.07) and acarbose (change -0.7 kg/m2, P = 0.046) and increase in the glibenclamide group (change 0.8 kg/m2, P = 0.27). However, individual subjects who gained weight had increases in their leptin concentrations. The diurnal variation in leptin concentrations was preserved after glibenclamide. CONCLUSIONS: Glibenclamide increases circadian leptin and insulin concentrations, whereas acarbose does not. This observation may help to explain weight gain in subjects treated with glibenclamide and stable weight in those treated with acarbose in the long run.

Cholesterol crystals in hydroceles: sonographic detection and possible significance.

OBJECTIVE: The aim of this study of seven patients who had elective surgical repairs of a hydrocele was to try to differentiate by microscopy and chemical analysis hyperechoic hydrocele fluid from the more typical anechoic hydrocele fluid. CONCLUSION: Hyperechoic hydrocele fluid is related to the presence of cholesterol crystals. Cholesterol crystals were noted on microscopic examination in all three patients with hyperechoic hydrocele fluid. No cholesterol crystals were evident in the four patients with typical anechoic hydrocele fluid.

Effects of fluvastatin therapy on lipids, antioxidants, oxidation of low density lipoproteins and trace metals.

OBJECTIVE: Oxidation of low density lipoproteins (LDL) is thought to be an important step in the development of atherosclerosis. Trace metals are involved in oxidative processes. It was of interest to determine whether lipid-lowering therapy with fluvastatin, a potent HMGCoA reductase inhibitor, affected LDL oxidation and trace metal levels. METHODS: Twenty hypercholesterolemic patients were treated with fluvastatin (40 mg twice daily) or placebo for 8 weeks in a double-blind, randomized study. LDL composition, antioxidants and oxidizability as well as plasma zinc, copper, selenium and manganese concentrations were investigated. RESULTS: After fluvastatin treatment, total cholesterol was reduced by 24%, triglycerides fell by 26% and plasma Zn fell by 8%. Cu, Se and Mn changed insignificantly. LDL were separated by ultracentrifugation. LDL were reduced by 18%, LDL-C by 29% and LDL-TG by 19%. The concentrations of alpha-tocopherol and retinol in LDL changed insignificantly. LDL preparations were incubated with copper ions (204 mumol.1(-1) LDL-C/3.2 mumol.1(-1) Cu) and formation of conjugated dienes was monitored at 234 nm for 5 h. Treatment with fluvastatin caused a reduction of diene production by 16% and of diene production rate by 14%, effects being significantly different from placebo (P = 0.02). The change of the lagtime did not reach significance; however, it was positively correlated with the change in LDL alpha-tocopherol. In the placebo group, no significant effects were observed. CONCLUSION: Fluvastatin therapy had lipid-lowering and antioxidative effects.

Increased clearance of low density lipoprotein precursors in patients with heterozygous familial defective apolipoprotein B-100: a stable isotope approach.

In familial defective apolipoprotein B-100 (FDB) the presence of a mutant apolipoprotein (apo) B-100 (FDB3500Q/W) in LDL markedly reduces their affinity for the LDL receptor, leading to elevated LDL cholesterol levels. However, the hypercholesterolemia in most FDB patients is relatively mild when compared with, e.g., familial hypercholesterolemia (FH). In order to study mechanisms that may partly alleviate the clinical consequences of FDB, we investigated the in vivo kinetics of apoB-100-containing lipoproteins in five FDB heterozygotes (total cholesterol: 7.84 +/- 1.37 mmol/I; total apoB: 1.68 +/- 0.37 g/l; mean +/- SD) and six normolipidemic controls (4.61 +/- 0.62 mmol/l; 0.98 +/- 0.12 g/l) using a stable isotope approach. During and after a 10-12 h primed, constant infusion of either [13C6]phenylalanine or [2H3]leucine, tracer enrichment was determined in apoB-100 from ultracentrifugally isolated VLDL1 (Sf 60-400), VLDL2 (Sf 20-60), IDL (Sf 12-20), LDL1 (Sf 7-12), and LDL2 (Sf 0-7). The rates of apoB-100 production, catabolism, and transfer were estimated by model-based compartmental analysis. The overall fractional catabolic rate (FCR) of IDL apoB-100 in FDB was substantially increased (2.99 +/- 0.68 pools/day vs. 1.70 +/- 0.23 pools/day in controls, P < 0.01). The fractional rate of apoB-100 transfer from IDL to LDL in FDB was decreased (0.97 +/- 0.13 pools/day vs. 1.24 +/- 0.10 pools/day, P < 0.05). The FCR of LDL apoB-100 in FDB was decreased (0.18 +/- 0.07 pools/day vs. 0.56 +/- 0.05 pools/, P < 0.01). Finally, the input rate of LDL apoB-100 in FDB was markedly decreased (9.45 +/- 2.96 mg/kg day1 vs. 15.54 +/- 1.70 mg/kg day1, P < 0.05). Our data suggest that the relatively small increase of LDL concentrations in FDB is due to an increased clearance of LDL precursor particles via the LDL-receptor and apoE-receptors as well as a decreased conversion of IDL to LDL - two mechanisms that distinguish FDB from FH.

Impact of concentrations of glycated hemoglobin, alpha-tocopherol, copper, and manganese on oxidation of low-density lipoproteins in patients with type I diabetes, type II diabetes and control subjects.

The late organ complications in diabetic patients are associated with enhanced oxidation of low-density lipoproteins (LDL). The role of vitamin and trace metal concentrations in this process is not clear. Therefore, we compared the oxidative susceptibility and alpha-tocopherol concentration of LDL with the levels of glycated hemoglobin (HbA1c), copper and manganese. Sixty-three diabetic patients (23 female and 40 male; 53 of type II, 10 of type I) and 35 control subjects (17 female and 18 male) were investigated. The in vitro-formation of conjugated dienes in purified LDL preparations in the presence of copper was followed as absorbance at 234 nm. LDL exhibited a shorter lagtime (44.5 +/- 10.1 vs. 67.8 +/- 16.0 and 50.1 +/- 14.3 vs. 68.8 +/- 14.6 min) for type I and type II diabetic patients vs. sex and age-matched controls, P < 0.001. For all subjects together the lagtime was inversely correlated to HbA1c (r = -0.230, P = 0.023) and positively correlated to LDL alpha-tocopherol/LDL (mol/mol). This ratio was lower in diabetic patients (P < 0.01 for type II) than in control subjects. The copper and manganese plasma levels were not different between diabetic and nondiabetic groups. However, parameters of LDL oxidizability (amount and rate of oxidation) were positively correlated with both copper and manganese concentrations. We conclude that in diabetes the resistance of LDL against oxidation is diminished in relation to the quality of glucose control.

Very fast ultracentrifugation of serum lipoproteins: influence on lipoprotein separation and composition.

A very short run time and small sample volumes in the separation of lipoproteins by preparative ultracentrifugation are needed for several investigations. Recently, a very fast sequential separation method was described that needs only 100 min for one run in a centrifugal field of 625,000 x g. We studied the influence of centrifugal fields of this dimension on lipoprotein separation and lipoprotein particle integrity using a Beckman Optima TLX ultracentrifuge with a TLA-120.2 rotor. Rotor speed (120/90/60/30.10(3) rev./min) and run time (100 min/3 h/6.7 h/27 h) were selected in such a way that the product of centrifugal field and run time remained constant. The first conditions correspond to the very fast ultracentrifugation (VFU) procedure with a centrifugal field of 625,000 x g. Thirty different plasma samples covering a wide range of lipid and protein concentrations were separated in the course of two centrifugal runs at densities of 1.006 and 1.063 kg/l which yielded very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and the subnatant of low-density lipoproteins, including high-density lipoproteins (HDL) and concomitant sedimented plasma proteins. The major lipid components of the lipoproteins, triacylglycerols, free and esterified cholesterol, phospholipids and the apolipoproteins B and A-I, were estimated considering the masses of the tube contents after a slicing procedure. Measurements of lipids and proteins showed a very good recovery of better than 94% and 91%, respectively, and precision-within-series (coefficient of variation) of better than 4.2% and 6.5%, respectively. The effects of the rotor speed on the lipoprotein structure appeared to be weak. With increasing rotor speed, VLDL and LDL lipid constituents principally tended to decrease, whereas they increased in the subnatant of the LDL-run. The mean lipoprotein mass composition, considering the mass percentage of each measured particle constituent, did not show significant alterations. Total protein decreased in VLDL and in LDL and increased in the subnatant of the LDL-run. As checked by an enzyme-linked immunosorbent assay (ELISA) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein effects were due to nearly complete disappearance of contaminating plasma proteins, especially albumin as the major contamination of VLDL and LDL. The apolipoproteins (apo) B-100, A-I, E and C-I to C-III remained nearly unaffected. The main advantages of VFU were the very short run time (cumulative flotation time is 3.4 h) and the elemination of albumin without repeated runs. The procedure was suitable for the assessment of lipid and protein constituents in lipoproteins from very small plasma samples (500 microliters).

Recovery of cholesterol and triacylglycerol in very-fast ultracentrifugation of human lipoproteins in a large range of concentrations.

Very-fast ultracentrifugation using a benchtop ultracentrifuge was applied to the analysis of lipoproteins in 0.5 ml of human plasma. VLDL, IDL and LDL were flotated at densities of 1.006, 1.019 and 1.063 kg/l in runs lasting 30, 100 and 100 minutes. Chylomicrons, if present, were flotated in a separate run. HDL were isolated by precipitation of the apolipoprotein B-containing lipoproteins from total plasma using polyethylene glycol. Three series of separations were routinely performed: 1. VLDL run alone (632 samples), 2. VLDL run + LDL run (122 samples), and 3. Chylomicron separation + VLDL run + IDL run (92 samples). The concentrations of cholesterol and triacylglycerol were obtained for plasma, chylomicrons, VLDL, IDL, LDL and HDL. Plasma values ranged from 1.8 to 37.1 mmol/l cholesterol and 0.26 to 50.2 mmol/l triacylglycerol. The plasma triacylglycerol concentrations were corrected for free glycerol by 3% (for triacylglycerols < 2.5 mmol/l) and by 2% (for triacylglycerols > or = 2.5 mmol/l). The recovery rate of lipids after ultracentrifugation was determined by comparing the concentrations in lipoproteins and in plasma. It was near to 100% and decreased for samples with extremely high lipid concentrations.