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).

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.

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.

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.

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.

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.

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.

Effects of p-chlorophenoxyisobutyric acid (CPIB) on the human liver.

Serial liver biopsies were carried out in 67 patients with HLP and/or fatty liver before, during short- and long-term therapy with CPIB and after termination of therapy. Results (1) Decrease of liver glycogen from 4.17% to 2.69% (wet weight, P less than 0.02). (2) Insignificant changes of liver triglyceride content. (3) Significant decrease of manganese, while the concentrations of zinc and copper in the liver biopsy specimens remained unchanged. (4) No signs of liver intoxication or cancerogeneous effects of light-microscopic pictures. (5) Significant increases in numbers of mitochondria and cristae as well as a hypertrophy of endoplasmic reticulum with longer lasting therapy. (6) Striking focal proliferation of cristae mitochondriales in 3 cases on longterm treatment. (7) Regression of the mitochondrial alterations after termination of the CPIB therapy. Our findings suggest that an increased number of mitochondria and of their inner membranes in the liver cells induced by CPIB could play an important role in the hypolipidemic action of the drug.

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.

Sonography of intrascrotal adenomatoid tumor.

A case is reported of an adenomatoid tumor of the epididymis. A discussion of the appropriate differential of mass of the epididymis as well as a review of adenomatoid tumors per se and their occurrence in the scrotum are presented.

Very-fast ultracentrifugation of human plasma lipoproteins: influence of the centrifugal field on lipoprotein composition.

A short run-time in separation of lipoproteins by preparative ultracentrifugation is desirable for several reasons. Recently, a method was described that needs only 100 min for one run in a centrifugal field of 625,000 x g. It is assumed that lipoprotein separation depends on the rotor speed but this has not been systematically studied in centrifugal fields of this order. We performed such a study. Rotor speeds of 120, 90, 60 and 30 x 10(3) rev./min and run-times of 100 min, 3 h, 6.7 h and 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. The Optima tabletop ultracentrifuge, the rotor TLA-120.2 and thick wall open tubes for 1 ml were used. Thirteen different plasma samples covering a wide range of cholesterol and triglyceride concentrations were separated into VLDL, LDL and HDL in the course of two centrifugal runs at densities of 1.006 and 1.063. The constituents of the lipoproteins were calculated considering the mass of the tube contents after slicing. Recoveries of cholesterol, triglycerides and protein were 97%, 98% and 90%, respectively. The influence of the rotor speed on the apparent composition of the lipoproteins was small. With increasing rotor speed, VLDL-cholesterol became higher (by 14%, P < 0.001), VLDL-triglyceride became lower (by 6%, P < 0.012), LDL-cholesterol became lower (by 9%, P < 0.000). The effects on LDL-triglyceride and on HDL-cholesterol and HDL-triglyceride, did not reach statistical significance. Protein in VLDL and in LDL decreased and increased in 'HDL' (the subnatant of the LDL run). As checked by SDS-PAGE the protein effects were due to complete disappearance of albumin from VLDL and LDL while the apolipoproteins B-100, E and C-I to C-III remained unaffected. It is concluded that the main advantages of VFU are the short run-time and the disappearance of albumin from VLDL and LDL. The other compositional changes need to be further investigated.

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.

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.

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.

Elimination and metabolism of a fat emulsion containing medium chain triglycerides (lipofundin MCT 10%).

Medium chain triglycerides (MCT) are supposed to be advantageous on account of rapid energy supply in parental nutrition. However, data on the elimination rate of MCT-containing emulsions during an intravenous fat-tolerance test (IVFTT) are scarce. We performed this test (0.1 g lipid/kg body weight) in 18 young healthy volunteers (nine females and nine males) using Lipofundin MCT 10% (50% MCT; egg phospholipids as emulsifier). Our results indicate that both elimination and metabolization of the emulsion are fast: a prompt decrease of light-scattering index and of triglyceride concentrations in serum, an immediate appearance of post-load fatty acids and of beta-hydroxybutyrate were observed. This was in good agreement with the findings obtained during 6-hr infusions in the same probands. Fractional elimination rates k2 obtained from light-scattering indices are 7.29 +/- 2.73%/min in males and 11.59 +/- 3.38%/min in females, indicating a higher removal capacity in women. In the same subjects, the corresponding k2 values for Lipofundin S 10% (containing only long chain triglycerides) were higher, reflecting an elimination rate that is faster due to the use of soya bean phospholipids as emulsifier. In comparison, k2 values based on the course of the triglyceride concentrations are generally lower.

Elimination kinetics of Lipofundin MCT: bolus injection and infusion compared.

Fifteen healthy young probands (nine males, six females) underwent an intravenous fat tolerance test (IVFTT) and, on the following day, a fat infusion lasting 6 hr. The emulsion tested was Lipofundin MCT 10%. One half of its triglyceride mass contains medium chain fatty acids. The IVFTT was started by injection of 0.1 g lipid per kg body weight into the fasting proband. Lipid elimination was estimated by measurement of light-scattering intensity of serum samples collected during a 60-min period. Individual fraction elimination rate constants covered a considerable range (K2 = 8.84 +/- 3.45%/min). The infusion test was performed at a rate of 0.1 g lipid per kg body weight and hr and lasted 6 hr. Serum triglyceride concentrations were determined enzymatically. They increased from 0.941 +/- 0.285 mmol/liter at the fasting state to a plateau level of 1.753 +/- 0.306 mmol/liter during infusion, and returned to initial levels 1 to 2 hr after the infusion was terminated. Individual triglyceride increments during infusion were significantly correlated with half-life periods of lipid elimination during IVFTT (r = 0.792, p less than 0.001). This relationship was derived using a model of the stationary state during infusion. We conclude that elimination kinetics of exogenous fat given either as bolus or infusion are ruled by the same fractional elimination rate constant K2. The IVFTT provides an estimate of the stationary triglyceride increment during a lipid infusion lasting several hr.

Elimination of lipofundin S during the intravenous fat tolerance test in patients with low, medium, and high fasting triglyceride concentrations.

The intravenous fat tolerance test with Lipofundin S (0.5 ml of 20% emulsion/kg body weight) was performed in 22 male nondiabetic patients. According to their fasting triglycerides (TG), the patients were arranged into three groups: low (less than 2.8 mmol/liter), medium (2.8-5.7 mmol/liter), and high (greater than 5.7 mmol/liter) concentrations. Fractional elimination rates of injected Lipofundin S decreased from 11.08 in low TG to 4.57%/min in high TG; they were positively correlated with fasting levels of high-density lipoprotein cholesterol but negatively with those of TG. The same pattern of correlations was observed with fractional catabolic rates of endogenous TG as measured after injection of tritium-labeled glycerol. The intravenous Lipofundin S load effected transient TG and free fatty acid elevations which were delayed in high TG. The elimination mechanisms of injected Lipofundin S and of endogenous TG are compared.

Computer program for iterative evaluation of fractional turnover rates by exponential regression: application to the turnover of VLDL-triglycerides in blood.

A microcomputer program ('EXREG') in GW BASIC was developed for fitting experimental data of A(T) to the equation A = A0 + A1.exp(-K.T) by exponential regression. In an iterative procedure K-values are tested to yield the maximum coefficient of determination. The accuracy of K is further improved in consecutive steps. The program is used to obtain the fractional turnover rate K of radiolabelled very low density lipoprotein (VLDL)-triglycerides in patients suffering from disturbances of lipid metabolism. A is the radioactivity of VLDL-triglycerides which is measured after isolation from blood. Seventeen consecutive blood samples are taken within a period of 24 h after injection of radioactive glycerol. T is the time after injection. The production rate of VLDL-triglycerides is calculated using K, the VLDL-triglyceride concentration, the body weight and height.