Endothelial dysfunction in men with small LDL particles.

BACKGROUND: It is unknown whether LDL particle size is, independent of other lipids and lipoproteins, associated with endothelial dysfunction in vivo. METHODS AND RESULTS: We determined in vivo endothelial function in 34 healthy men by measuring forearm blood flow responses to intrabrachial artery infusions of acetylcholine (ACh, an endothelium-dependent vasodilator) and sodium nitroprusside (an endothelium-independent vasodilator). LDL peak particle size was measured with gradient gel electrophoresis. Men with small LDL particles (LDL diameter 25. 5 nm, n=24, blood flow 6.9+/-3.6 versus 11.4+/-5.1 mL/dL. min, P=0. 006). The groups had comparable LDL cholesterol concentrations (3. 9+/-0.6 versus 3.7+/-1.0 mmol/L, men with small versus large LDL particles), blood pressure, glucose concentrations, and body mass indexes. LDL size (r=0.45, P=0.01) but not HDL cholesterol (r=0.31, P=0.09) or triglycerides (r=-0.19, P=0.30) was significantly correlated with endothelium-dependent vasodilation. Serum triglyceride concentrations and LDL size were inversely correlated (r=-0.44, P=0.01). In multivariate regression analysis, LDL size was the only significant determinant of the ACh-induced increase in blood flow. Sodium nitroprusside-stimulated endothelium-independent vasodilation was similar in both groups. CONCLUSIONS: Small LDL particles are associated with impaired in vivo endothelial function independent of HDL and LDL cholesterol and triglyceride concentrations. LDL size may therefore mediate adverse effects of hypertriglyceridemia on vascular function.

In vivo low density lipoprotein oxidation relates to coronary reactivity in young men.

OBJECTIVES: This study was undertaken to examine the relation of in vivo low density lipoprotein (LDL) oxidation and other lipid risk factors to coronary reactivity in normal subjects. BACKGROUND: Experimental studies have shown that oxidized LDL (ox-LDL) particles are injurious to the vascular wall by impairing its normal vasodilator function. METHODS: We used noninvasive positron emission tomographic (PET) imaging with intravenous dipyridamole to measure coronary flow reserve, a marker of coronary endothelial and smooth muscle function, in 30 healthy men (mean [+/-SD] age 34.4 +/- 3.2 years). As a marker of in vivo LDL oxidation, the autoantibody titer against ox-LDL was measured by the enzyme-linked immunosorbent assay method. RESULTS: Plasma levels of autoantibody titer against ox-LDL were inversely associated with coronary flow reserve (r = -0.42, p = 0.023). High LDL cholesterol levels (above median > 3.0 mmol/liter) were associated with a low coronary flow reserve only in subjects expressing simultaneously high levels of ox-LDL titer (above median). Subjects with simultaneously high levels of LDL cholesterol and ox-LDL titer had lower coronary flow reserve values than subjects in other groups (3.89 vs. > 5.0 in other groups, p = 0.066). CONCLUSIONS: These data provide evidence for the role of ox-LDL in affecting the coronary reactivity in vivo and support the concept that oxidative modification of LDL particles provides a mechanism by which high LDL concentrations exhibit injurious effects on the coronary vascular bed.

Effects of gemfibrozil on low-density lipoprotein particle size, density distribution, and composition in patients with type II diabetes.

OBJECTIVE: To study the effects of gemfibrozil treatment on LDL particle size, density distribution, and composition in NIDDM patients. RESEARCH DESIGN AND METHODS: We performed LDL analyses on 16 NIDDM patients with stable glycemic control. They were randomly allocated to receive either gemfibrozil (n = 8) or a placebo (n = 8) for 3 mo in a double-blind study. The LDL particle size distribution and the particle diameter of the major LDL peak were measured with nondenaturing polyacrylamide gradient gel electrophoresis. The density distribution and composition of LDL were determined with the density gradient ultracentrifugation method. RESULTS: In the gemfibrozil group the mean serum TG concentration decreased by 38%, HDL cholesterol concentration increased by 10%, and LDL cholesterol concentration by 17% (P < 0.05). During gemfibrozil therapy the mean particle diameter of the major LDL peak increased from 244 to 251 A (P < 0.05), whereas in the placebo group the mean LDL particle diameter remained unchanged. We found an inverse correlation between the changes of serum TG and the particle diameters of the major LDL peak (r = 0.85, P < 0.01). Gemfibrozil produced a shift in the LDL density distribution toward lower density. The mean peak density decreased from 1.0371 to 1.0345 g/ml because of a significant rise in the light LDL concentration from 141.0 to 183.2 mg/dl (P < 0.05), whereas the concentration of dense LDL had a tendency to decrease. In the placebo group the LDL density distribution did not change. Gemfibrozil increased the CE-to-TG ratio in LDL core lipids by 27% (P < 0.05); otherwise, the LDL composition was only slightly affected. CONCLUSIONS: The results indicate gemfibrozil-induced changes in LDL properties in NIDDM patients are similar to those previously reported in nondiabetic individuals and are related to changes in serum TG level.

Impaired endothelium-dependent vasodilation in type 2 diabetes. Relation to LDL size, oxidized LDL, and antioxidants.

OBJECTIVE: To search for determinants of endothelial dysfunction in type 2 diabetes. RESEARCH DESIGN AND METHODS: We performed a comprehensive analysis of cardiovascular risk markers and measured blood flow responses to endothelium-dependent (acetylcholine [ACh] and NG-monomethyl-L-arginine) and -independent (sodium nitroprusside [SNP]) vasoactive agents in 30 nonsmoking men with type 2 diabetes (age 51 +/- 1 years, BMI 27.8 +/- 0.4 kg/m2, HbA1c 7.4 +/- 0.3%) and 12 matched normal control men. RESULTS: ACh-induced vasodilation was 37% lower in type 2 diabetic (6.1 +/- 0.5) than in normal subjects (9.7 +/- 1.5 ml.dl-1.min-1, P < 0.01), while flows during SNP were similar (9.1 +/- 0.6 vs. 9.9 +/- 1.3 ml.dl-1.min-1, NS). The ratio of endothelium-dependent vs. -independent flow (ACh:SNP ratio) was 31% lower in type 2 diabetic (0.70 +/- 0.05) than in normal subjects (1.10 +/- 0.18, P < 0.01). Total (2.2 +/- 0.4 vs. 1.3 +/- 0.2 mmol/l, P < 0.05), VLDL, and intermediate-density lipoprotein triglycerides were significantly higher, and the mean LDL particle diameter was significantly smaller in type 2 diabetic than in normal subjects. The lag times for LDL oxidation by Cu2+ in vitro were similar in patients with type 2 diabetes (183 +/- 7) and in normal subjects (183 +/- 9 min, NS). Measured and calculated (sum of concentration of individual antioxidants in serum) total peroxyl radical-trapping capacities (TRAPs) were comparable between the groups. In the patients with type 2 diabetes, LDL size was significantly correlated with endothelium-dependent vasodilation (r = 0.43, P < 0.05), serum triglycerides (r = -0.75, P < 0.001), and the lag time for LDL oxidation in vitro (r = 0.38, P < 0.05). HbA1c was inversely correlated with the lag time for LDL oxidation in vitro (r = -0.41, P < 0.05) and TRAP. CONCLUSIONS: In summary, patients with type 2 diabetes exhibited impaired endothelium-dependent vasodilation in vivo, elevated serum triglycerides, decreased LDL size, and normal antioxidant capacity. Of these parameters, LDL size was significantly correlated with endothelial function.

Effects of postmenopausal estrogen/progestin replacement therapy on LDL particles; comparison of transdermal and oral treatment regimens.

The aim of the study was to compare the effects of continuous oral estrogen/progestin therapy to the effects of transdermal estrogen therapy combined with cyclic oral progestin on the properties of LDL particles. Eighty postmenopausal women were randomly allocated to receive either oral (continuous 17-beta-estradiol 2 mg and norethisterone acetate 1 mg per day, E2/NETA) or transdermal therapy (patches delivering continuous 17-beta-estradiol, E2, 0.05 mg/day with sequential oral medroxyprogesterone acetate, MPA, 10 mg/day for 12 days/cycle). The groups had similar mean values and ranges of age, BMI and postmenopausal status. The blood samples were taken at baseline, and twice at 1 year before and after MPA administration. LDL particle size distribution was determined by gradient gel electrophoresis and LDL was isolated by sequential ultracentrifugation for compositional analyses. Concentrations of total LDL mass, LDL cholesterol and LDL protein decreased in the oral treatment group (p < 0.01, p < 0.001 and p < 0.01, respectively), whereas they remained unchanged during the transdermal therapy. Particle size of the major LDL peak remained unchanged during both transdermal and oral therapies. HDL cholesterol concentration decreased significantly in both treatment groups (p < 0.001 for both). Serum triglyceride and HDL cholesterol concentrations were the strongest determinants of LDL particle size ( r = -0.50 and r = 0.54, respectively, p < 0.001 for both). The cholesteryl esters and free cholesterol content of the LDL particles decreased in the oral treatment group (p < 0.05). Phospholipid content of LDL increased in both groups receiving either oral or transdermal therapy (p < 0.01 for both). In conclusion, oral administration of 17-beta-estradiol and norethisterone acetate caused a decrease in LDL mass by decreasing the number and cholesterol content of LDL particles. The concomitant decrease of HDL cholesterol by progestins may partly negate this beneficial effect of LDL lowering.

LDL particle size in mildly hypertriglyceridemic subjects: no relation to insulin resistance or diabetes.

We examined 18 Type 2 diabetic and 19 non-diabetic subjects in order to determine the association between insulin resistance and LDL particle size distribution in mildly hypertriglyceridemic and hyperinsulinemic subjects with and without Type 2 diabetes. Insulin sensitivity of the patients was characterized by their insulin-stimulated glucose uptake rate determined by euglycemic clamp technique. LDL particle size distribution was determined by nondenaturing polyacrylamide gradient gel electrophoresis. Type 2 diabetic and non-diabetic subjects had closely similar serum lipid and lipoprotein concentrations as well as the mean particle diameters of the major LDL peak (246 +/- 6 A and 244 +/- 6 A, respectively). To evaluate the effect of insulin resistance on LDL particle size the participants were categorized into two subgroups using the median of their insulin-stimulated glucose uptake rate (14.67 mumol/kg/min) as a cut-off point. Neither lipid and lipoprotein concentrations nor the LDL particle size distributions differed between the more insulin resistant group (nine diabetic and nine non-diabetic subjects) and less insulin resistant group (nine diabetic and ten non-diabetic subjects). LDL particle size was not associated with the insulin-stimulated glucose uptake rate or with the mean 24-h concentration of serum insulin. Mean 24-h concentration of serum triglycerides was the strongest discriminator for LDL particle size (r = -0.44, P < 0.01). In conclusion, neither Type 2 diabetes nor insulin resistance seem to have any direct effect on LDL particle size in mildly hypertriglyceridemic subjects. The fact that LDL particle size was associated with serum triglycerides indicates that the effect of diabetes and insulin resistance on LDL particle size could be explained by the effects of insulin resistance and/or hyperinsulinism on VLDL metabolism.

Effect of gemfibrozil on high density lipoprotein subspecies in non-insulin dependent diabetes mellitus. Relations to lipolytic enzymes and to the cholesteryl ester transfer protein activity.

Twenty patients (18 men, 2 women) with non-insulin dependent diabetes mellitus (NIDDM) were randomized to receive either gemfibrozil 1200 mg daily or placebo for 3 months in a double-blind study. The effect of gemfibrozil on plasma HDL subfraction distribution was studied with sequential and density gradient ultracentrifugation and in gradient gel electrophoresis. The concentrations of apo A-I, apo A-II, Lp A-I and Lp A-I:A-II particles were measured. Postheparin plasma lipoprotein lipase (LPL) and hepatic lipase (HL) activities and plasma cholesteryl ester transfer protein (CETP) activities were also determined. Gemfibrozil increased the concentration of HDL cholesterol (P < 0.01), which was due to the rise of HDL3 cholesterol (+16%), while in the placebo group these values remained unchanged. Gemfibrozil increased the concentrations of apo A-I(+12.6%, NS), apo A-II (+28.2%, P < 0.01) and Lp A-I:A-II particles (+21.6%, P < 0.06) but there were no changes in the placebo group. Neither gemfibrozil nor placebo had any effect on the concentration of Lp A-I particles. As determined by density-gradient ultracentrifugation, gemfibrozil increased the concentration of cholesterol in the most dense HDL fractions (mean density 1.193 g/ml, +22%, P < 0.05 and mean density 1.158 g/ml, +19.3%, P < 0.05). In gradient gel electrophoresis, the gemfibrozil-induced elevations of the cholesterol and protein were most pronounced in the HDL3a (8.8-8.2 nm) region. Gemfibrozil increased LPL and HL activities by 14.7% (P < 0.05) and by 18.8% (P < 0.01), respectively, while in the placebo group LPL and HL activities remained unchanged. Plasma CETP activity was also increased during gemfibrozil treatment while in the placebo group it remained unchanged. We conclude that gemfibrozil causes multiple changes in plasma HDL metabolism. The gemfibrozil-induced elevation of HDL3 and dense HDL subpopulations may reflect the concerted action of LPL, HL and CETP on plasma HDL metabolism.

Heterozygous hepatic lipase deficiency, due to two missense mutations R186H and L334F, in the HL gene.

Hepatic lipase (HL) is an endothelial enzyme involved in the metabolism of intermediate density lipoproteins (IDL) and high density lipoproteins (HDL) in plasma. In a Finnish pedigree consisting of 18 members belonging to three generations two missense mutations RI86H and L334F in exons 5 and 7 of the HL gene co-segregated with low post-heparin HL activity. Haplotype analysis of the HL gene in family members revealed a high degree of genetic variation and demonstrated that the two missense mutations reside on the same chromosome. In vitro site-directed mutagenesis and expression of the cDNA constructs in COS-1 cells revealed that the R186H mutation leads to a protein that is not secreted while the L334F mutation results in the production of a HL protein that is secreted but has only about 30% of wild type HL activity. Carriers of the mutated HL gene exhibited clearly reduced HL activity and mass in post-heparin plasma. Probably due to their heterozygous carrier status they had only moderate elevation of total triglycerides, IDL, and LDL-triglycerides. The LDL-particles were enriched in triglycerides and depleted of cholesterol. Also their HDL2- and HDL3-particles were enriched in triglycerides.

Responses of HDL subclasses, Lp(A-I) and Lp(A-I:A-II) levels and lipolytic enzyme activities to continuous oral estrogen-progestin and transdermal estrogen with cyclic progestin regimens in postmenopausal women.

Seventy postmenopausal women took part in the study. Subjects received either continuous oral 17 beta-estradiol 2 mg/day combined with norethisterone acetate 1 mg/day (E2/NETA, Kliogest) or transdermal treatment consisting of 28 day cycles with patches delivering 17 beta-estradiol 50 micrograms/day (Estraderm) combined with cyclic medroxyprogesterone acetate 10 mg/day (E2/MPA, Provera), on days 17-28. At baseline the serum lipid and lipoprotein concentrations, composition and concentrations of high density lipoprotein (HDL) subclasses, lipoprotein (Lp)(AI) and Lp(A-I:A-II) levels were comparable in the two groups. In the E2/NETA group, after 12 months hormone replacement therapy (HRT), the HDL2 cholesterol concentration decreased by 17% (P < 0.01) and the HDL3 cholesterol remained unchanged. The concentrations of HDL2b, HDL2a and HDL3a were reduced by 30, 26 and 15%, respectively, P < 0.001, and the cholesterol:triglyceride ratio decreased significantly in all HDL subclasses. Apolipoprotein (apo) A-I concentration decreased by 5% (P < 0.05), but apo A-II, Lp(A-I) and Lp(A-I:A-II) concentrations remained unchanged. In the E2/MPA group the HDL2 and HDL3 cholesterol levels were both reduced by 6% (P < 0.05) and the HDL3a, HDL3b and HDL3c concentrations decreased by 14, 12 and 17% during the E2/MPA phase compared with baseline (P < 0.01). No major changes in the composition of HDL subclasses occurred in the E2 MPA group during treatment. The apo A-I and Lp(A-I) levels were not changed, but apo A-II and Lp(A-I:A-II) concentrations decreased by 8 and 5%, P < 0.001 and P < 0.05, respectively. At 12 months the postheparin plasma hepatic lipase (HL) activity decreased only in the E2/NETA group (by 12%, P < 0.05). The cholesteryl ester transfer protein (CETP) activity was not affected by either HRT regimen. The results of our study show that the 2 HRT regimens have multiple effects on HDL particles and HRT induced changes in HDL are not associated with changes in activities of lipolytic enzymes or CETP.

Intense physical training decreases circulating antioxidants and endothelium-dependent vasodilatation in vivo.

Physical training increases free radical production and consumes antioxidants. It has previously been shown that acute exercise markedly increases the susceptibility of LDL to oxidation but whether such changes are observed during physical training is unknown. We measured circulating antioxidants, lipids and lipoproteins, and blood flow responses to intrabrachial infusions of endothelium-dependent (acetylcholine, ACh, L-N-monomethyl-arginine, L-NMMA) and -independent (sodium nitroprusside, SNP) vasoactive agents, before and after 3 months of running in 9 fit male subjects. Maximal aerobic power increased from 53 +/- 1 to 58 +/- 2 ml/kg min (P < 0.02). All circulating antioxidants (uric acid, SH-groups, alpha-tocopherol, beta-carotene, retinol) except ascorbate decreased significantly during training. Endothelium-dependent vasodilatation in forearm vessels decreased by 32-35% (P < 0.05), as determined from blood flow responses to both a low (10.8 +/- 2.1 vs. 7.3 +/- 1.5 ml/dl min, 0 vs. 3 months) and a high (14.8 +/- 2.6 vs. 9.6 +/- 1.8) ACh dose. The % endothelium-dependent blood flow (% decrease in basal flow by L-NMMA), decreased through training from 37 +/- 3 to 22 +/- 7% (P < 0.05). Blood flow responses to SNP remained unchanged. The decrease in uric acid was significantly correlated with the change in the % decrease in blood flow by L-NMMA (r = 0.74, P < 0.05). The lag time for the susceptibility of plasma LDL to oxidation in vitro, LDL size and the concentration of LDL cholestetol remained unchanged. We conclude that relatively intense aerobic training decreases circulating antioxidant concentrations and impairs endothelial function in forearm vessels.

Phenotype expression in familial combined hyperlipidemia.

Familial combined hyperlipidaemia (FCHL) is one of the most common hereditary disorders predisposing to early coronary death. The affected family members have elevations of serum total cholesterol, triglycerides or both. Despite intensive research efforts the genetic and metabolic defects underlying this complex disorder are still unknown. To dissect the metabolism and genetics of FCHL the phenotype of an individual must be precisely defined. We assessed the influence of different diagnostic criteria on the phenotype definition and studied factors affecting the phenotype expression in 16 large Finnish families (n = 255) with FCHL. The fractile cut-points used to define abnormal lipid values had a profound influence on the diagnosis of FCHL. If the 90th percentile cut-point was used, approximately 45% of the family members were affected, in concord with the presumed dominant mode of transmission for FCHL. If the 95th percentile was used only 22% of study subjects were affected. To characterize the metabolic differences or similarities between the different lipid phenotypes, we determined very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) particles separated by ultracentrifugation. In linkage analysis no single ultracentrifugation variable could discriminate reliably affected family members from non-affected family members. Our data emphasizes the need for re-evaluation of FCHL diagnostic criteria. Preferably, the diagnosis should be based on a single, reliable metabolic marker.

Determinants of the severity and extent of coronary artery disease in patients with type-2 diabetes and in nondiabetic subjects.

BACKGROUND: Factors predicting the anatomic distribution and the severity and extent of coronary atherosclerosis in patients with clinically manifest coronary artery disease (CAD) for type-2 diabetic patients could be different than those for nondiabetic patients. OBJECTIVE: To study the determinants of severity and extent of CAD in consecutive patients with type 2 diabetes mellitus, compared with those for matched nondiabetic patients, undergoing clinically indicated coronary angiography. METHODS: Coronary angiograms of 48 men and seven women with type-2 diabetes and an equal number of nondiabetic subjects were analyzed quantitatively. Scores reflecting severity and extent of CAD were compared with potential risk factors using univariate correlation analyses and multivariate regression models. RESULTS: For the diabetics, a global coronary atheroma burden index was independently and directly related to age (P = 0.022) and to level of intermediate-density lipoprotein cholesterol (P = 0.055), and inversely to level of particles of a subtype of high-density lipoprotein (P = 0.022). Several angiographic indexes were related to the duration of diabetes and control of glycemia. For the nondiabetic group, global atheroma burden was independently related to age (P = 0.028), a history of hypertension (P = 0.028), and concentration of low-density lipoprotein (P = 0.013), and inversely to level of apolipoprotein A-I (P = 0.008). The duration of coronary disease and a history of smoking were also predictive of severe coronary atherosclerosis among nondiabetic patients. CONCLUSIONS: Classical risk factors are strong predictors of the severity and extent of coronary atherosclerosis in nondiabetic patients, but the most important determinants for type-2 diabetic patients are levels of triglyceride-rich lipoproteins and apolipoprotein A-I-containing particles of high-density lipoprotein, and factors directly related to diabetes.

Insulin glargine or NPH combined with metformin in type 2 diabetes: the LANMET study.

AIMS/HYPOTHESIS: In type 2 diabetic patients we compared 9 months of combination therapy with insulin glargine and metformin with 9 months of NPH insulin combined with metformin. The primary focus was changes in HbA(1c); secondary focus was diurnal glucose profiles and symptomatic hypoglycaemia. METHODS: In this investigator-initiated open, parallel-group clinical trial involving seven centres, 110 insulin-naive type 2 diabetic patients with poor glycaemic control (HbA(1c) >or=8.0%) on oral hypoglycaemic agents (90% using sulfonylurea plus metformin) were randomised to receive bedtime insulin glargine with metformin (G+MET) or bedtime NPH with metformin (NPH+MET) for 36 weeks. The patients were taught how to self-adjust their insulin dose and use a modem to send the results of home glucose monitoring to treatment centres. The goal was to achieve a fasting plasma glucose (FPG) of 4.0 to 5.5 mmol/l in both groups. RESULTS: During the last 12 weeks, FPGs averaged 5.75+/-0.02 and 5.96+/-0.03 mmol/l (p<0.001) and insulin doses were 68+/-5 and 70+/-6 IU/day (0.69+/-0.05 and 0.66+/-0.04 IU kg(-1) day(-1), NS) in the G+MET and NPH+MET groups, respectively. At 36 weeks, mean HbA(1c) was 7.14+/-0.12 and 7.16+/-0.14%, respectively (NS). Symptomatic, but not confirmed symptomatic, hypoglycaemia was significantly lower during the first 12 weeks in the G+MET group (4.1+/-0.8 episodes/patient-year) than in the NPH+MET group (9.0+/-2.3 episodes/patient-year, p<0.05), but not significantly different thereafter. Glucose levels before dinner were higher in the NPH+MET group (10.1+/-0.3 mmol/l) than in the G+MET group (8.6+/-0.3 mmol/l, p=0.002) throughout the 36-week study. With regard to baseline characteristics such as initial glycaemia or C-peptide, there was no difference between patients who achieved good glycaemic control (HbA(1c) <7.0%) and those who did not. Differences were seen in the following: between study centres, weight gain during the run-in period and insulin therapy, and FPG during the last 12 weeks (5.7+/-0.2 vs 6.7+/-0.3 mmol/l for patients reaching vs those not reaching target, p<0.01). CONCLUSIONS/INTERPRETATION: Good glycaemic control can be achieved with both G+MET and NPH+MET. Use of G+MET reduces symptomatic hypoglycaemia during the first 12 weeks and dinner time hyperglycaemia compared with NPH+MET.

New insights into lipid metabolism in non-insulin-dependent diabetes mellitus.

Perturbations of lipid metabolism are common in diabetes. Therefore, an understanding of the underlying mechanism of lipid metabolism and in particular the role of insulin is a critical issue. The review aims to provide evidence that hypertriglyceridaemia is central to many features of diabetic dyslipidaemia.

Regulation of low-density lipoprotein particle size distribution in NIDDM and coronary disease: importance of serum triglycerides.

An increase of low-density lipoprotein triglycerides (LDL-Tg) was found to be an independent coronary artery disease (CAD) risk factor for non-insulin-dependent diabetic (NIDDM) patients in a recent prospective study. We examined the composition and size of LDL particles in 50 NIDDM men with angiographically verified CAD (NIDDM+ CAD+) and in 50 NIDDM men without CAD (NIDDM(+)-CAD-) as compared to 50 non-diabetic men with CAD (NIDDM - CAD +) and 31 non-diabetic men without CAD (NIDDM-CAD-). The groups had similar ranges of age and BMI. LDL particle size was determined by gradient gel electrophoresis, and LDL was isolated by sequential ultracentrifugation for compositional analyses. Serum Tg was increased in NIDDM patients as compared to non-diabetic subjects (p < 0.05), and in patients with CAD as compared to subjects without the disease (p < 0.05). LDL cholesterol was lower in NIDDM patients than in non-diabetic subjects (p < 0.001). Mean diameter of LDL particles was less than 255 A, but closely comparable in all groups. The presence of NIDDM was associated with increases of Tg and protein but lowering of free cholesterol in LDL (p < 0.005 for all). In multivariate regression analyses neither NIDDM nor CAD were associated with LDL particle size, but serum Tg was the major determinant of LDL size in both NIDDM and non-diabetic subjects (p < 0.001). When the patients were divided into quartiles according to fasting serum Tg levels, the LDL particle size and free cholesterol content decreased, but Tg and protein contents of LDL particles increased from the lowest to the highest Tg quartile (analysis of variance p < 0.001 for all). When the subjects were categorized into two groups according to the median of VLDL-Tg (1.10 mmol/l) LDL size was associated with VLDL-Tg in the high but not in the low VLDL-Tg group. We conclude that in NIDDM patients with or without CAD serum Tg is the major determinant of the properties of LDL particles. The clinical implication is that in NIDDM serum Tg should be as low as possible to prevent atherogenic changes in LDL.

Abnormalities of low density lipoproteins in normolipidemic type II diabetic and nondiabetic patients with coronary artery disease.

The characteristics of low density lipoproteins (LDL) of ten non-insulin-dependent diabetic (NIDDM) and ten nondiabetic patients with coronary artery disease (CAD) were investigated and compared to LDL of ten NIDDM patients without CAD and ten healthy persons. All subjects had LDL cholesterol below 160 mg/dl and serum triglycerides below 200 mg/dl. The mean LDL particle size and particle distribution profiles were analyzed by using nondenaturing polyacrylamide gradient gel electrophoresis. The LDL composition and hydrated density distribution were investigated by using density gradient ultracentrifugation. Both NIDDM and nondiabetic CAD patients tended to have larger LDL particles than NIDDM patients without CAD and healthy subjects. The increase of LDL particle size of CAD patients was due to marked enrichment of triglycerides (TG) in their LDL. The percentage content of TG in LDL of NIDDM patients with CAD was 14.5% and in LDL of nondiabetic CAD patients 13.4% compared with 7.9% in LDL of NIDDM patients without CAD and 7.2% in normal-LDL (P less than 0.05 or less between either CAD group and NIDDM without CAD or normals). The LDL TG/apolipoprotein (apo) B weight ratio was significantly higher in both CAD groups compared with LDL of the two groups without CAD (0.70 and 0.68 vs. 0.38 and 0.34, respectively, P less than 0.05, P less than 0.05 and P less than 0.01, P less than 0.01). The LDL total lipid to apoB weight ratio was similar in all four groups. Consistent with this, the hydrated density distributions of LDL in the four groups were similar, the average peak densities being 1.0346 g/ml, 1.0331 g/ml, 1.0331 g/ml, and 1.0331 g/ml, respectively. The findings of this study demonstrate that normolipidemic patients with CAD may have marked abnormalities in th eir LDL composition and these anomalies are present in both diabetic and nondiabetic patients.

Changes of lipolytic enzymes cluster with insulin resistance syndrome. Botnia Study Group.

The activities of hepatic and lipoprotein lipase and the levels of lipo- and apoproteins were compared in two groups of normoglycaemic men representing the highest (n = 18) and lowest (n = 15) fasting insulin quintiles of first degree male relatives of non-insulin-dependent diabetic patients. The high insulin group representing insulin-resistant individuals had significantly lower post-heparin plasma lipoprotein lipase activity than the low insulin group (14.2 +/- 4.0 vs 20 +/- 5.8 mumol NEFA.ml-1.h-1, p < 0.001); hepatic lipase activity did not differ between the two groups (24.2 +/- 11 vs 18.0 +/- 5.3 mumol NEFA.ml-1.h-1, NS). The lipoprotein lipase/hepatic lipase ratio in the high insulin group was decreased by 66% as compared to the low insulin group (0.75 +/- 0.57 vs 1.25 +/- 0.65, p < 0.01). In the high insulin group both total and VLDL triglycerides were higher than in the low insulin group (1.61 +/- 0.57 vs 0.86 +/- 0.26 mmol/l, p < 0.001 and 1.00 +/- 0.47 vs 0.36 +/- 0.16 mmol/l, p < 0.001, respectively) whereas HDL cholesterol and HDL2 cholesterol were lower (1.20 +/- 0.30 vs 1.43 +/- 0.22 mmol/l, p < 0.05 and 0.49 +/- 0.21 vs 0.71 +/- 0.17 mmol/l, p < 0.05, respectively). Total cholesterol, LDL cholesterol or HDL3 cholesterol did not differ between the two groups. The mean particle size of LDL was smaller in the high insulin group than in the low insulin group (258 +/- 7 vs 265 +/- 6 A, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The Asn-291-->Ser and Ser-477-->Stop mutations of the lipoprotein lipase gene and their significance for lipid metabolism in patients with hypertriglyceridaemia.

We examined 99 Finnish patients whose serum fasting triglycerides (TG) had exceeded 6.0 mmol L-1 with special interest to their lipid, lipoprotein and post-heparin plasma lipase activities. The control group consisted of 75 healthy individuals. We also determined the frequency of the Asn-291-->Ser and Ser-447-->Stop mutations both in hypertriglyceridaemic (HTG) subjects and in control subjects. A total of 51 of the original 99 hypertriglyceridaemic patients still had TG > 6.0 mmol L-1 when measured a second time. They are referred to as persistently hypertriglyceridaemic subjects (pHTG). The remaining 48 subjects had TG < 6.0 mmol L-1 in the second measurement and are referred to as sporadically hypertriglyceridaemic subjects (sHTG). The allelic frequencies of the Ser-447-->Stop mutation in the total HTG and sHTG groups were similar to the frequencies present in the control group, but lower in pHTG patients compared with the control group (0.049 vs. 0.153, chi(2) = 6.63, P < 0.05). The Asn-291-->Ser mutation was more frequent in HTG group than in the control group (0.0606 vs. 0.013, chi(2) = 4.86, P < 0.05). This difference was due to the higher frequency of the minor allele of Asn-291-->Ser in the cohort with persistent hypertriglyceridaemia compared with the control group (0.088 vs. 0.013, chi(2) = 8.00, P < 0.01). The highest frequency (0.114) of the minor allele of Asn-291-->Ser was found in type 2 diabetic patients with persistent hypertriglyceridaemia. The carrier status of Asn-291-->Ser or Ser-447-->Stop did not predict either post-heparin plasma lipoprotein lipase (LPL) activities or lipid and lipoprotein levels in any of the groups studied. Our data suggest that overproduction of very low-density lipoproteins (VLDL) is a more important cause of hypertriglyceridaemia in the Finns than is the LPL deficiency.

ApoA-IHelsinki (Lys107-->0) associated with reduced HDL cholesterol and LpA-I:A-II deficiency.

A Finnish kindred with premature coronary heart disease and decreased HDL cholesterol levels was identified as having an apoA-I variant, apoA-I (Lys107-->0), caused by a 3-bp deletion of nucleotides 1396 through 1398 in exon 4 of the apoA-I gene. These subjects (n = 10) were heterozygous for this mutation. The mean serum HDL cholesterol concentration (26.7 +/- 9.7 mg/dL) of affected family members was 36%, lower than that of unaffected family members (P < .05). Mean serum apoA-I and apoA-II concentrations in heterozygotes were reduced by 18% and 22%, respectively, compared with normal family members (P < .05). In heterozygotes the mean concentration of lipoprotein containing both apoA-I and apoA-II (LpA-I:A-II) was 31% lower than in those with normal apoA-I (P < .001), while the mean level of lipoproteins containing apoA-I without apoA-II was similar in the two groups. HDL density-gradient ultracentrifugation showed a lack of HDL2 and small dense HDL3 in heterozygotes compared with unaffected family members. The HDL particle size distribution, as analyzed by nondenaturing gradient gel electrophoresis of heterozygotes, revealed one major peak at 8.0 to 9.7 nm, a minor peak at 7.8 to 8.5 nm, and an absence of HDL2b and HDL2a peaks. These latter peaks were observed in unaffected family members. Serum levels of LDL cholesterol, triglycerides, VLDL, IDL, and LDL subclasses were similar in the two groups. However, in heterozygotes the cholesterol-to-triglyceride ratios in VLDL2, LDL1, LDL3, HDL2b, HDL2a, and HDL3a were 8% to 54% lower than in unaffected family members (P < .05). Cholesteryl ester transfer protein activity in heterozygotes was reduced by 25% compared with unaffected family members (P < .05), while the plasma lecithin:cholesterol acyltransferase (LCAT) activity did not differ between heterozygotes and unaffected family members. The ability of isolated variant apoA-I to serve as a cofactor for LCAT in vitro did not differ from that of normal apoA-I. Our data are consistent with the concept that a low HDL cholesterol level in subjects heterozygous for the apoA-IHelsinki mutation (Lys107-->0) having normal LCAT activity is a consequence of decreased concentration of LpA-I:A-II particles and of a smaller size and reduced cholesterol content of HDL particles.

Development and validation of dry reagent time-resolved fluoroimmunoassays for zeranol and alpha-zearalenol to assist in distinguishing zeranol abuse from Fusarium spp. toxin contamination in bovine urine.

Zeranol, an oestrogenic growth promoter in food animals, is banned within the European Union (EU). However, commercially available immunoassay kits for zeranol cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the validation of a specificity enhanced, rapid dry reagent time-resolved fluoroimmunoassay (TR-FIA) for zeranol (recovery 99%, limit of detection 1.3 ng ml(-1)) demonstrating that up to 150 ng ml(-1) of Fusarium spp. toxins in urine do not lead to false-positive results. This assay will assist EU Member States to implement Council Directive 96/23/EC, which requires states to monitor for potential abuses of zeranol. A similar TR-FIA for the Fusarium spp. toxin alpha-zearalenol, using the same sample extract, is also described (recovery 68%, limit of detection 5.6 ng ml(-1)). Only the addition of diluted sample extract is required to perform these dry-reagent TRFIAs, the results being available within 1h of extract application. The EU-funded project 'Natural Zeranol' (FAIR5-CT97-3443) will use these fluoroimmunoassays to screen bovine urine in four Member States to gather data on the seasonality of Fusarium spp. toxin contamination of urine and the incidence of zeranol screening test positives.