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Patients with familial hypercholesterolemia (FH) are at an increased risk of premature cardiovascular disease (CVD). The benefits of statin therapy are not well known since no placebo controlled studies have been performed in these patients. The aim of this study was to determine the CVD event and mortality risk in statin-treated patients with FH. A total of 345 FH patients were followed prospectively for 8 years. Mortality from CVD was compared to that of the general population. The absolute risk of CVD in patients without a previous history of CVD was 3% per year for men and 1.6% for women. Mortality from CVD in patients without a previous history was 1.4-fold (95% CI = 0.6-3.3) increased and ischaemic heart disease (IHD) mortality was 2.6-fold (95% CI = 1.1-6.3) higher compared to the general population. This mortality risk was highest in patients aged 40-59 years. Female FH patients had no increased CVD or IHD mortality risk. Over a period of 8 years the event risk of patients with a history of CVD was almost 30% per year under age 40 years and 15% in patients aged 60 years and over. When compared to the general population, mortality from other causes than CVD was lower for patients with FH, the relative risks not reaching statistical significance. The relative risk of mortality from all causes was 1.5 (P < 0.05) for men and 1.0 for women. In conclusion, male patients with FH, treated from middle-age with statins remain at an increased risk of developing CVD.

Significant familial aggregation was observed for plasma levels of lathosterol (an indicator of whole-body cholesterol synthesis) and plant sterols campesterol and beta-sitosterol (indicators of cholesterol absorption) in 160 Dutch families consisting of adolescent mono- and dizygotic twin pairs and their parents. For lathosterol a moderate genetic heritability in parents and offspring (29%) was found. In addition, shared environment also contributed significantly (37%) to variation in plasma lathosterol concentrations in twin siblings. However, a model with different genetic heritabilities in the two generations (10% in parents and 68% in offspring) fitted the data almost as well. For plasma plant sterol concentrations high heritabilities were found. For campesterol heritability was 80% and for beta-sitosterol it was 73%, without evidence for differences in heritability between sexes or generations. No influence of common environmental influences shared by family members was seen for either campesterol or beta-sitosterol. Taken together, these results confirm and expand the hypothesis that individual differences in plasma levels of noncholesterol sterols are moderately (lathosterol) to highly (plant sterols) heritable.

Type III hyperlipoproteinemia (HLP) is mainly found in homozygous carriers of apolipoprotein E2 (apoE2, Arg158-->Cys). Only a small percentage (< 5%) of these apoE2 homozygotes develops hyperlipidemia, indicating that additional environmental and genetic factors contribute to the expression of type III HLP. In the present study, first, the prevalence of type III HLP among apoE2 homozygotes was estimated in a Dutch population sample of 8888 participants. Second, 68 normocholesterolemic and 162 hypercholesterolemic apoE2 homozygotes (type III HLP patients) were collected to investigate additional factors influencing type III HLP expression. In the Dutch population sample, apoE2 homozygosity occurred with a frequency of 0.6% (57 of 8888 individuals). Among the 57 E2/2 subjects, 10 type III HLP patients were identified (prevalence 18%). Comparison of normocholesterolemic E2/2 subjects and type III HLP patients showed that the latter had a significantly increased body mass index (25.6 +/- 4.0 versus 26.9 +/- 3.8 kg/m(2), respectively; P=0.03) and prevalence of hyperinsulinemia (26% versus 63%, respectively; P<0.001). Multiple linear regression analysis demonstrated that most of the variability in type III HLP expression can be explained by fasting insulin levels (partial correlation coefficient approximately 0.50, P<0.001). In contrast to men, apoE2 homozygous women aged > or = 50 years had significantly higher plasma lipid levels than their counterparts aged < 50 years. These data demonstrate that the expression of type III HLP in E2/2 subjects is elicited to a large extent by hyperinsulinemia. In addition, in female apoE2 homozygotes, the expression increases with age; this increase is most likely due to the loss of estrogen production.

Objective: To investigate the short-term effect of treatment with tibolone on plasma lipid and lipoprotein levels in postmenopausal women with type III hyperlipoproteinemia (HLP)., Design and Intervention: Patients were randomized to receive, in a double-blind cross-over fashion, a fixed dose of tibolone, 2.5 mg once daily or placebo for 8 weeks. The two treatment periods were separated by a wash-out period of 6 weeks. At each visit body weight and blood pressure were determined. Before and after each treatment period, fasting venous blood samples were obtained from the patients for biochemical measurements., Setting: The Leiden University Medical Center., Subjects: Postmenopausal women with type III HLP (aged < or = 65 years) were recruited from the Lipid Clinics of the Leiden University Medical Center, the Amsterdam Medical Center, the Utrecht Medical Center and the University Hospital Rotterdam. Five out of 25 women with type III HLP were eligible to be included in the study. Four of the five included patients completed the study according to the protocol. One patient was excluded from blinded therapy because total cholesterol levels increased above 20 mmol L(-1)., Main Outcome Measures: A significant reduction of plasma triglyceride, total cholesterol, VLDL cholesterol and VLDL triglyceride levels., Results: Plasma triglyceride and total cholesterol levels decreased from 6.82 +/- 3.58 to 2.45 +/- 1.36 mmol L(-1) and from 13.53 +/- 3.64 to 6.61 +/- 2.03 mmol L(-1), respectively (both P < 0.05). The body mass index remained unchanged. The glycated haemoglobin percentage decreased significantly from 5.8 to 5.3%. Treatment with tibolone resulted in a profound reduction in plasma apolipoprotein E, VLDL cholesterol and VLDL triglyceride levels (mean reductions of 66, 77 and 70%, respectively, P < 0.05)., Conclusions: Tibolone is a valuable adjuvant to current therapy in postmenopausal women with type III HLP.

Objective: In hypertriglyceridemic patients, hypertension occurs frequently and may be associated with hyperinsulinemia and elevated plasma levels of free fatty acids (FFA). Besides the lipid-lowering effects, fibrates have been shown to reduce blood pressure in hypertensive patients. The present study was undertaken to investigate the effects of bezafibrate on hemodynamics in relation to insulin, FFA, sympathetic activity, renal sodium absorption, cyclic-GMP (cGMP) and endothelin-1 in hypertriglyceridemic patients., Subjects and Methods: Hypertriglyceridemic patients (17) were randomized to receive in a double-blind placebo-controlled study bezafibrate or placebo for 6 weeks. At the end of both treatment periods, blood pressure and heart rate were measured automatically. Plasma insulin, FFA, aldosterone, catecholamines, cGMP, endothelin-1 levels and 24 h urine catecholamines and sodium excretion were assessed., Results: Bezafibrate therapy decreased serum triglycerides (-65%, P < 0.001) and hemodynamic parameters: heart rate decreased from 69 to 66/min (P = 0.009), systolic blood pressure from 137 to 132 mmHg (P = 0.01), diastolic blood pressure from 81 to 79 mmHg (P = 0.07) and mean blood pressure from 102 to 99 mmHg (P = 0.06). Bezafibrate therapy reduced FFA and insulin (-55 and -57% respectively, both P < 0.001), while sympathetic activity and renal sodium absorption were not affected. cGMP increased (+17%, P = 0.008), whereas endothelin-1 levels tended to decrease upon bezafibrate therapy (-10%, P = 0.077), Conclusion: Bezafibrate reduces heart rate, blood pressure, insulin and FFA in hypertriglyceridemic patients. The hemodynamic effects cannot be attributed to changes in sympathetic activity or renal sodium absorption. Instead, based on the increase in plasma cGMP levels, the bezafibrate-induced hemodynamic effects are most likely to be caused by bezafibrate-induced improvement of endothelial function.

Hypertriglyceridemia, a risk factor for cardiovascular disease, has been associated with hypercoagulability, but whether platelet activation is implicated is unknown. This study was designed to compare the in vivo platelet activation status between patients with severe hypertriglyceridemia and age- and sex-matched control subjects, and to evaluate the effects of triglyceride-lowering therapy. Sixteen patients with primary hypertriglyceridemia were included in a double-blind, placebo-controlled cross-over trial with 400 mg bezafibrate once daily. Platelet activation was analysed by double label flow cytometry, using monoclonal antibodies against GP53, P-selectin, and platelet-bound fibrinogen. Surface expression of the lysosomal membrane protein GP53 was significantly higher in the hypertriglyceridemic patients at baseline as compared to the group of age- and sex-matched controls (16.3+/-4.8% vs. 8.9+/-3.4%, respectively, P<0.001). No differences in the expression of P-selectin and fibrinogen binding were observed. In response to bezafibrate therapy, the expression of GP53 in the patient group decreased from 16.3+/-4.8% to 13.1+/-4.1% (P=0.018). The expression of P-selectin and fibrinogen binding was not affected by bezafibrate therapy. In conclusion, patients with hypertriglyceridemia have an increased in vivo platelet activation status, which can be improved by bezafibrate therapy.

Searching for familial hypercholesterolaemia (FH) in children is useful only if efficacious treatment is to be administered shortly and if there are relatives with ischaemic heart disease at very early ages. In all other cases, the (psychological) drawbacks probably outweigh the doubtful benefit of early intervention. The search for the major homozygous form of FH begins with cholesterol assay in both parents, in the absence of FH in either of them, the above-named restrictions apply.

Oestrogen replacement therapy is associated with a decreased risk of cardiovascular disease in postmenopausal women. Patients with non-insulin-dependent diabetes mellitus (NIDDM) have an increased cardiovascular risk. However, oestrogen replacement therapy is only reluctantly prescribed for patients with NIDDM. In a double blind randomized placebo controlled trial we assessed the effect of oral 17 beta-estradiol during 6 weeks in 40 postmenopausal women with NIDDM. Glycated haemoglobin (HbA1c), insulin sensitivity, suppressibility of hepatic glucose production, lipoprotein profile and parameters of fibrinolysis were determined. The oestrogen treated group demonstrated a significant decrease of HbA1c and in the normotriglyceridaemic group a significantly increased suppression of hepatic glucose production by insulin. Whole body glucose uptake and concentrations of non-esterified fatty acids did not change. LDL-cholesterol- and apolipoprotein B levels decreased, and HDL-cholesterol, its subfraction HDL2-cholesterol and apolipotrotein A1 increased. The plasma triglyceride level remained similar in both groups. Both the concentration of plasminogen activator inhibitor-1 antigen and its active subfraction decreased. Tissue type plasminogen activator activity increased significantly only in the normotriglyceridaemic group. Oestrogen replacement therapy improves insulin sensitivity in liver, glycaemic control, lipoprotein profile and fibrinolysis in postmenopausal women with NIDDM. For a definite answer as to whether oestrogens can be more liberally used in NIDDM patients, long term studies including the effect of progestogens are necessary.

Lifestyle including eating habits, physical training, smoking, drinking alcoholic beverages etc. can to a certain extent maintain or spoil our health. The physiological mechanisms of haemostasis and of lipoprotein metabolism play a role in acute cardiovascular diseases but also in a great number of chronic diseases in which vascular pathology is prominent. Imparied fibrinolysis and increased lipid levels are often incriminated in vascular disease. Lifestyle can modify fibrinolysis as well as lipid levels. Physical training, moderate eating habits, no smoking, moderate alcohol intake will be a beneficial influence on both fibrinolysis and lipid levels. The possibility that long-term pharmacological intervention may adversely affect fibrinolysis and lipid levels should always be considered.

In type II diabetes mellitus the altered hormonal state after menopause may represent an additional cardiovascular risk factor. Estrogen replacement therapy (ERT) is associated with a decreased cardiovascular risk, at least in nondiabetic postmenopausal women. We studied the effect of ERT on plasma lipids and lipoproteins and on LDL oxidation in 40 postmenopausal women with type II diabetes but with minimal vascular complications in a randomized placebo-controlled trial. Twenty patients were treated orally with 2 mg/d micronized 17 beta-estradiol and 20 patients with placebo for 6 weeks. Plasma total cholesterol (-6%, P = .04), LDL cholesterol (-16%, P = .0001), and apoB (-11%, P = .001) levels decreased and HDL cholesterol (20%, P = .0001) and apoA-I (14%, P = .0001) levels increased after ERT compared with placebo. Glycated hemoglobin (HbA1c) decreased significantly after ERT (-3%, P = .03), the cholesterol content of the LDL particles decreased (-5%, P = .006), triglyceride content increased (16%, P = .01), and LDL particle size did not change significantly. ERT had no effect on parameters of LDL oxidation. We conclude that plasma levels of HDL cholesterol, apoA-I, LDL cholesterol, apoB, and glycated hemoglobin are improved in postmenopausal women with type II diabetes mellitus after treatment with 17 beta-estradiol, indicative of a better metabolic control, and that ERT has no effect on LDL oxidizability.

Apolipoprotein (apo) E2 and high insulin levels are associated with the severity of hypertriglyceridemia in patients with combined hyperlipidemia. To study how these determinants affect very low-density lipoprotein (VLDL) in combined hyperlipidemic patients, we characterized VLDL particles in 106 unrelated patients with combined hyperlipidemia. The study was performed after 9 weeks of standardized dietary intake and after an overnight fast. Patients heterozygous for apoE2 had significantly higher mean levels of VLDL cholesterol by 0.71 mmol/l (95% CI, 0.30 to 1.12 mmol/l, P < 0.005) and VLDL triglycerides by 0.88 mmol/l, (95% CI, 0.30 to 1.47 mmol/l, P < 0.005) compared to patients without apoE2. The VLDL triglyceride content per particle and the calculated diameter of the VLDL particles were similar in both groups, which indicate a higher number of circulating VLDL particles in heterozygous apoE2 carriers. Patients with high fasting insulin levels (> or = 80 pmol/l) had a higher mean serum VLDL triglyceride level by 0.56 mmol/l (95% CI, 0.04 to 1.07 mmol/l, P < 0.05). The calculated VLDL diameter was larger by 3.7 nm (95% CI, 1.2 to 6.2 nm, P < 0.005) and the particles contained more triglycerides by 2.7 weight percent (95% CI, 0.3 to 5.1 weight percent, P < 0.05). These insulin-dependent changes in VLDL particles were only present in the absence of apoE2. In conclusion, patients heterozygous for apoE2 have higher numbers of circulating VLDL particles, whereas patients with high fasting insulin levels have larger, triglyceride enriched VLDL particles.

In a sample of Dutch families consisting of parents aged 35-65 years and their twin offspring aged 14-21 years, a significant difference between generations was observed in phenotypic variances and in genetic heritabilities for plasma levels of total cholesterol, triglycerides, high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol, and apolipoproteins (apo) A1, A2, B, and E. For all traits parents were more variable than their offspring. This increase in phenotypic variance was best explained by a genetic model in which individual specific environmental variance increased with increasing age. Genetic variance was the same across generations for nearly all traits except triglycerides and apoE, for which a decrease in genetic variance was observed. This model led to large intergenerational differences in genetic heritabilities. Heritabilities for children were between 65 and 87%, while heritabilities for their parents were between 10 and 50%. No evidence was found for effects of a shared family environment.

Objective: To test the hypothesis that the progestogen medrogestone has no effect on changes in lipoprotein metabolism evoked by continuous estrogen replacement therapy, paying special attention to high-density lipoproteins (HDL)., Design: Open multicenter randomized comparative trial., Patients: Postmenopausal hysterectomized women aged 49 to 64 years., Intervention: Continuous oral treatment with 0.625 mg daily of conjugated estrogens (CE) alone (n = 55) or CE plus 5 mg of the progestogen medrogestone orally during the last 12 days of each 28-day cycle (n = 59)., Main Outcome Measures: At baseline and at cycles 3, 6, and 13 we measured the plasma levels of apolipoprotein (Apo) A1, cholesterol in total HDL and in its subfractions HDL2 and HDL3, using density gradient ultracentrifugation., Results: High-density lipoprotein cholesterol increased from baseline at all assessments in both treatment groups, being significantly greater in the CE group (+15% at cycle 13) than in the CE and medrogestone group (+8%). However, HDL2-cholesterol increased in both treatment groups, but with no significant difference between the two groups. High-density lipoprotein 3 cholesterol increased only in the CE group (+7% at cycle 13); there was no significant change in HDL3-cholesterol in the CE and medrogestone group. Low-density lipoprotein (LDL) cholesterol decreased from baseline at all assessments in both treatment groups (-6% and -9%, respectively, at cycle 13). The change in very low-density (VLDL) lipoprotein cholesterol was not significant in either of the two groups. Medrogestone had no significant effects on the estrogen-induced increases in apo A-1 and triglycerides nor on the decreases in ApoB and LDL-cholesterol. Neither hormone significantly affected VLDL-cholesterol or Lp(a) levels., Conclusion: Medrogestone did not eliminate the increase in plasma HDL levels evoked by CE.

To assess the presence and composition of very-low-density lipoprotein (VLDL) in various types of hyperlipoproteinemia, a method of density gradient ultracentrifugation has been developed. After 2 hours of density gradient ultracentrifugation, human serum VLDL is separated into two distinct VLDL cholesterol peaks (VLDL1 and VLDL2). The two VLDL subfractions were detected in the serum samples from all subjects in the study, including subjects with normolipidemia (n = 10), familial dysbetalipoproteinemia (n = 12), and type IIa (n = 8), type IIb (n = 12), and type IV/V (n = 10) hyperlipoproteinemia. The cholesterol profiles obtained by the density gradient ultracentrifugation technique resembled the band patterns after electrophoresis of identical serum samples on 2% to 16% nondenaturing polyacrylamide gradient gel: VLDL1 represents relatively large VLDL particles (diameter of about 67 nm) and VLDL2 represents relatively small VLDL particles (diameter of about 38 nm). Recentrifugation of isolated VLDL1 and isolated VLDL2 did not result in any change in their density distribution. In all groups studied, the fluidity of VLDL1 was significantly higher than that of VLDL2, in accordance with the finding that VLDL1 particles were relatively rich in triglycerides and VLDL2 particles were relatively rich in cholesteryl esters. These results indicate that the two VLDL subfractions isolated represent distinct VLDL subclasses. The density gradient ultracentrifugation technique presented in this study allows the rapid isolation and characterization of VLDL subfractions from the serum samples of normolipidemic individuals and patients with hyperlipoproteinemia.

Plasma levels of histidine-rich glycoprotein (HRG) were investigated in three groups of women receiving a different dose of estrogens. First, the effect of low-dose estrogen was studied in a group of 83 postmenopausal women who were treated with 0.625 mg conjugated estrogens (CE). No significant change from baseline levels was found at the end of cycle 3 and cycle 13. Secondly, in 15 mothers and 23 daughters using oral contraceptives (OC) containing 30-50 micrograms ethinyl estradiol (EE) daily the mean HRG level was 14% and 24% lower than in a group of 144 mothers and 134 daughters not taking oral contraceptives, respectively (p < 0.05). Finally, in 11 excessively tall prepuberal girls who received 300 micrograms EE daily to reduce their final height the mean plasma HRG levels were decreased by 68% (p < 0.005). The effect of progestogens administered during low-dose and high-dose estrogen therapy appeared to be minor. The results from these three studies indicate that estrogens reduce plasma HRG levels in a dose-dependent way.

There is accumulating evidence that oxidative modification of LDL is an important step in the process of atherogenesis and that antioxidants may protect LDL from oxidation. We and others have previously shown that ingestion of pharmacological doses of the antioxidant D,L-alpha-tocopherol (vitamin E), far above the recommended daily intake (ie, 12 to 15 IU/d for adults), increases the oxidation resistance of LDL. In this study, we ascertained the minimal supplementary dose of vitamin E necessary to protect LDL against oxidation in vitro. Twenty healthy volunteers (10 men and 10 women, aged 21 to 31 years) ingested consecutively 25, 50, 100, 200, 400, and 800 IU/d, D,L-alpha-tocopherol acetate during six 2-week periods. No changes were observed in LDL triglyceride content, fatty acid composition of LDL, or LDL size during the intervention. Concentrations of alpha-tocopherol in plasma and LDL were both 1.2 times the baseline values after the first period (25 IU/d) and 2.6 and 2.2 times, respectively, after the last period (800 IU/d). There was a linear increase in LDL alpha-tocopherol levels up to an intake of 800 IU/d (r = .79, P < .0001) and a good correlation between alpha-tocopherol in plasma and LDL (r = .66, P < .0001). Simultaneously, the resistance of LDL to oxidation was elevated dose-dependently (+28% after the last period) and differed significantly from the baseline resistance time even after ingestion of only 25 IU/d.(ABSTRACT TRUNCATED AT 250 WORDS)

Using a density-gradient ultracentrifugation technique, we analyzed in detail the plasma lipoprotein profiles of 18 patients with familial dysbetalipoproteinemia (FD) who had apolipoprotein (apo) E2(Arg158-->Cys) homozygosity (the E2-158 variant, n = 6), apoE3-Leiden heterozygosity (the E3-Leiden variant, n = 6), or apoE2(Lys146-->Gln) heterozygosity (the E2-146 variant, n = 6), with average plasma cholesterol concentrations of 8.99 +/- 1.34 mmol/L, 9.29 +/- 1.55 mmol/L, and 8.46 +/- 1.10 mmol/L, respectively. No significant differences in sex, age, body mass index, dietary habits, and standard laboratory tests between the three groups were observed. The lipoprotein profiles of all FD patients were characterized by higher concentrations of very-low-density lipoprotein (VLDL) 1, VLDL2, and intermediate-density lipoprotein (IDL) and a higher cholesteryl ester content of VLDL1 and VLDL2 than in 6 normolipidemic control subjects with an average plasma cholesterol concentration of 5.90 +/- 0.53 mmol/L. Major differences between the plasma lipoprotein profiles of patients with the E2-158 variant, the E3-Leiden variant, and the E2-146 variant and the normolipidemic control subjects were in IDL cholesterol concentration (1.70 +/- 0.26, 1.50 +/- 0.26, 1.05 +/- 0.36, and 0.47 +/- 0.14 mmol/L, respectively), LDL cholesterol concentration (1.83 +/- 0.50, 3.09 +/- 0.32, 3.79 +/- 0.76, and 3.77 +/- 0.56 mmol/L, respectively), and the molar ratio of IDL cholesterol to LDL cholesterol (0.98 +/- 0.28, 0.48 +/- 0.04, 0.28 +/- 0.09, and 0.12 +/- 0.03, respectively). After 10 weeks of simvastatin treatment the concentrations of plasma cholesterol, VLDL2 cholesterol, IDL cholesterol, and LDL cholesterol in 3 patients with the E2-158 variant fell significantly, by 46%, 56%, 53%, and 48%, respectively; they also fell in 3 patients with the E3-Leiden variant, by 48%, 54%, 57%, and 52%, respectively, and in 3 patients with the E2-146 variant, by 38%, 55%, 46%, and 35%, respectively. Simvastatin therapy lowered plasma activity of cholesteryl ester transfer protein but had no significant effect on plasma activity of lecithin:cholesterol acyltransferase. It is concluded that patients with FD due to various apoE variants have different lipoprotein profiles, mainly with regard to IDL and LDL levels, although they have a number of similar features of dysbetalipoproteinemia. Simvastatin therapy effectively reduced the plasma concentrations of total cholesterol, VLDL2 cholesterol, IDL cholesterol, and LDL cholesterol in the three groups of patients studied. It is proposed that apoE-dependent defects of the conversion of IDL to LDL may be an important mechanism in the pathophysiology of FD.

Lipoprotein metabolism is involved in atherogenesis. Female sex-hormones have substantial effects on both lipoprotein metabolism and the vessel wall. Cholesterol, one of the major lipids in lipoproteins, is both the substrate for, and the target of, the steroidal sex hormones.

We compared plasma lipoprotein profiles of 15 individuals with normocholesterolemic (plasma cholesterol 4.81 +/- 0.90 mmol/L) familial dysbetalipoproteinemia (NFD) and 15 patients with hypercholesterolemic (plasma cholesterol 10.61 +/- 2.32 mmol/L) familial dysbetalipoproteinemia (HFD), matched for age and sex. All subjects were homozygous for apoE2(Arg158-->Cys). Compared with 15 normolipidemic controls (plasma cholesterol 5.47 +/- 0.92 mmol/L), subjects with NFD and HFD had greater cholesterol concentrations of large very-low-density lipoprotein (VLDL1), small VLDL (VLDL2), and intermediate-density lipoprotein, each of which was correlated to their plasma total cholesterol concentration. VLDL1 and VLDL2 subfractions were enriched in cholesteryl ester, and plasma cholesteryl ester transfer protein activities were increased in both NFD and HFD; however, absolute changes were larger in HFD than in NFD. Concentrations of low-density lipoprotein cholesterol were lower in HFD (1.89 +/- 0.48 mmol/L) and NFD (1.56 +/- 0.36 mmol/L) than in normolipidemic controls (3.35 +/- 0.73 mmol/L). We conclude that all subjects homozygous for apoE2(Arg158-->Cys) show features of dysbetalipoproteinemia.

In this report, we describe the characterization of a mutation in the low density lipoprotein (LDL) receptor gene of a true homozygous familial hypercholesterolemic (FH) patient. The combined use of denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis revealed a unique A to G transition in the penultimate 3'-nucleotide of intron 16 of the LDL receptor gene, which disrupts the acceptor splice site. cDNA sequence analysis indicated that a cryptic splice site was activated in intron 16, upstream from the original splice site, leading to the inclusion of 62 nucleotides and a reading frame-shift. The resulting new translation product contains a stretch of 154 amino acids at the carboxy-terminal that have no resemblance to the normal receptor protein. To elucidate the biological effects of the mutation, the structural and functional properties of the mutated LDL receptor protein were studied. Immunoprecipitation of the newly synthesized LDL receptors showed that an aberrant precursor form of the LDL receptor protein was synthesized, about 10 kDa larger than normal, which is not further processed to the mature form. Some 50% of the normal LDL binding activity was found on the cell surface of the patient's fibroblasts, whereas internalization and degradation of LDL were abolished.

Assessment of the apolipoprotein E (apoE) phenotype by isoelectric focusing of both hyperlipidemic and normolipidemic individuals identified five new variants. All mutations were confined to the downstream part of the APOE gene by using denaturing gradient gel electrophoresis (DGGE). Sequence analysis revealed five new mutations causing unique amino acid substitutions in the carboxyl-terminal part of the protein containing the putative lipid-binding domain. Three hyperlipoproteinemic probands were carriers of the APOE*2(Val236-->Glu) allele, the APOE*3(Cys112-->Arg; Arg251-->Gly) allele, or the APOE*1(Arg158-->Cys; Leu252-->Glu) allele. DGGE of the region encoding the receptor-binding domain was useful for haplotyping the mutations at codons 112 and 158. Family studies failed to demonstrate cosegregation between the new mutations and severe hyperlipoproteinemia, although a number of carriers for the APOE*3(Cys112-->Arg; Arg251-->Gly) allele and the APOE*1(Arg158-->Cys; Leu252-->Glu) allele expressed hypertriglyceridemia and/or hypercholesterolemia. Two other mutant alleles, APOE*4-(Cys112-->Arg; Arg274-->His) and APOE*4+(Ser296-->Arg), were found in normolipidemic probands. The lack of cosegregation of these new mutations with severe hyperlipoproteinemia suggests that these mutations do not exert a dominant effect on the functioning of apoE.

We tested the influence of the apolipoprotein E (apoE) polymorphism on the intrapair differences in the levels of plasma cholesterol, plasma triglycerides, low density lipoprotein-cholesterol, apoB and apoE in monozygotic (MZ) twins, and estimated whether or not there was a interaction between the apoE polymorphism and environmental factors. In 65 MZ twin pairs, the intrapair differences in the measured lipoprotein parameters were similar in the different apoE phenotype classes. This indicates that the effect of the apoE polymorphism is not influenced by environmental variability between the MZ pair members and accordingly identifies the APOE gene as a "level" gene.

We measured plasma levels of lipoprotein(a) (Lp(a)) in a sample of 152 Dutch adolescent mono- and dizygotic twin pairs and their parents. The distribution of Lp(a) levels was skewed, with the highest frequencies at low levels and was similar for adult men and women and their children. The relationship of Lp(a) concentrations with other lipoprotein and apolipoprotein risk factors for coronary heart disease and with lathosterol, an indicator of whole-body cholesterol synthesis, was studied dependent on sex and generation. In mothers and children there was a small positive correlation between Lp(a) levels and plasma cholesterol and apolipoprotein (apo) B. In mothers and daughters there also was a correlation between Lp(a) and LDL cholesterol levels. No correlation was found between Lp(a) levels and plasma lathosterol, suggesting that there is no relationship between Lp(a) levels and cholesterol synthesis. Associations among family members, i.e. between monozygotic and dizygotic twins and between parents and offspring were used to study familial transmission of Lp(a) levels. Results showed that almost all of the variance in Lp(a) concentrations was accounted for by genetic heritability. A small, but significant, sex difference in heritability was observed, but heritabilities were the same in parents and offspring. Heritability estimates were 93% for females and 98% for males. No evidence was found for assortative mating or for the influence of a shared family environment. These results indicate that nearly all variance in Lp(a) concentrations that is not accounted for by the apo(a) size polymorphism, is also under genetic control.

We found that the distribution of the lipophilic chemotherapeutic agent o,p'-DDD (mitotane) among serum (lipo)proteins was altered in hypertriglyceridemia, with relatively more o,p'-DDD accumulating in the chylomicron and very-low-density lipoprotein (VLDL) fraction. Intralipid, an artificial chylomicron emulsion, or isolated VLDL could extract o,p'-DDD from the other serum (lipo)proteins. There was an inverse relationship between the relative amount of o,p'-DDD found in the fraction exhibiting a density of less than 1.006 g/ml (chylomicrons plus VLDL) and the relative amount observed in the LDL or HDL fractions of serum. Our results indicate that hypertriglyceridemia may impede the entry of o,p'-DDD into the brain or the adrenals. For therapeutic monitoring of o,p'-DDD levels in severe hypertriglyceridemia, we recommend that the chylomicron and VLDL fraction first be removed from the serum by ultracentrifugation.

After 15 weeks of simvastatin therapy (20 mg/day), low density lipoprotein particle size in sera of 16 patients with type IIb hyperlipoproteinemia increased significantly from 233 +/- 5.0 A to 237 +/- 7.0 A (P less than 0.05), analyzed by 2-16% polyacrylamide gradient gel electrophoresis. Under simvastatin therapy the concentrations of total cholesterol, total triglyceride, very low density lipoprotein cholesterol and triglyceride, low density lipoprotein cholesterol and apolipoprotein B in serum fell significantly by 30%, 30%, 43%, 28%, 36% and 26%, respectively, and the concentration of high density lipoprotein cholesterol rose significantly by 14%. The changes of low density lipoprotein particle size induced by simvastatin therapy were correlated best with the changes of very low density lipoprotein triglyceride concentration (r2 = 0.438, P less than 0.01). Our results suggest that simvastatin therapy, additionally to a reduction of the serum cholesterol concentration, increases low density lipoprotein particle size which may contribute to reduction of the risk of coronary heart disease in patients with type IIb hyperlipoproteinemia.

In this study, the relation of plasma levels of lathosterol (an indicator of whole body cholesterol synthesis) and plant sterols (indicator of cholesterol absorption) with age, sex, weight, height, plasma lipids, and lipoproteins, and with apolipoprotein (apo) E phenotype, was investigated in a group of 160 nuclear families consisting of twins living with their parents. Lathosterol was higher in fathers than in mothers, but not different between boys and girls. In each of these four groups, there was a strong correlation with plasma and low-density lipoprotein (LDL)-cholesterol and -triglyceride, as well as with body weight, but not with height or high-density lipoprotein (HDL)-cholesterol. In adults, lathosterol was inversely correlated with plant sterols. Lathosterol was higher in children with E4/3 phenotype than in those with E3/3 or E3/2; in adults, lathosterol did not differ among the various E phenotypes. The plasma levels of the two plant sterols, campesterol and beta-sitosterol, were highly correlated with each other, and also with plasma or LDL-cholesterol, in each of the four groups. Plant sterols were higher in adults or children with E4/3 phenotype as compared with those with other phenotypes. In multivariate analysis (performed separately for two groups of adults and children) plasma cholesterol, plasma plant sterols, plasma triglycerides, and weight were found to make significant contributions to the variation of lathosterol in all groups, and E phenotype and sex only in one group, while age did not contribute in any group.(ABSTRACT TRUNCATED AT 250 WORDS)

We measured the serum lathosterol level, a reflection of the rate of whole body cholesterol synthesis, in 15 patients with manifest type III hyperlipoproteinemia (HLP), in 20 subjects with apolipoprotein (apo) E2/2 phenotype, but without type III HLP, in 21 patients with type IIA and 10 patients with type IIB HLP. A group of 100 subjects with apo E3/3 phenotype served as reference. Using ANCOVA, lathosterol was adjusted for serum cholesterol and triglyceride concentrations, since these parameters were found to independently correlate with lathosterol. The adjusted means (+/- SEM), in mumol/L, in these groups were 12.9 +/- 1.1, 8.2 +/- 1.1, 4.8 +/- 0.9, 9.8 +/- 1.4, and 7.8 +/- 0.4, respectively. Type III HLP patients had significantly higher lathosterol levels than all other groups except type IIB HLP. In addition, lathosterol was significantly lower in type IIA patients than in all other groups. The serum levels of plant sterols, used as a reflection of cholesterol absorption, did not differ among the various groups after adjustment for serum cholesterol. These findings suggest that an overproduction of cholesterol is one factor discriminating E2/2 homozygotes with type III HLP from those without the disease.

DNA samples from 53 unrelated Dutch patients with familial hypercholesterolemia (FH) were screened for rearrangements in the gene for the LDL receptor (LDLR) by Southern analysis. Four different mutations have been detected by hybridisation of BglII digested genomic DNA with an exon 10-14 containing cDNA probe. The mutations are defined by a 7 kb insertion near exon 11, a partial gene duplication encompassing exons 9-12, a 4 kb deletion of exons 7 and 8 and an 0.4 kb deletion comprising the 5'-part of exon 16. These four different rearrangements in the LDLR gene account for 17% of the mutations in the Dutch FH population sample. Interestingly, the 4 kb deletion was detected in 5 unrelated FH patients (9.5%) and appeared to be identical to the deletion previously described (Russell, D.W. et al., Arteriosclerosis, 9 (Suppl. I) (1989) I-8; Russell, D.W. et al., Cold Spring Harbor Symp. Quant. Biol., 51 (1987) 401). in an FH patient of Dutch origin. This suggests that the 4 kb deletion is a common mutation in the Dutch FH population.

In a group of 120 patients with heterozygous familial hypercholesterolemia (FH) the influence of the apolipoprotein E (apoE) polymorphism on pre-treatment plasma lipid levels and on the response to treatment with simvastatin was studied. The apoE phenotype distribution did not differ significantly between the FH group and a sample group of the Dutch population. Differences in pre-treatment lipid levels were not related to the apoE polymorphism in this FH population. After 12 weeks use of a daily dose of 40 mg simvastatin, the plasma total cholesterol, low density lipoprotein (LDL)-cholesterol and plasma triglyceride levels were reduced on average by 33%, 38% and 19%, respectively. At the same time high density lipoprotein (HDL)-cholesterol concentration increased on average by 7%. In the combined FH patient group (males and females) a considerable interindividual variation in response to simvastatin was observed, but was not related to the apoE polymorphism. However, considering males and females separately, we found that female FH patients with the apoE3E3 phenotype responded better on simvastatin treatment with respect to LDL-cholesterol than male FH patients with the apoE3E3 phenotype.

In a randomized comparative study, changes in lipoprotein metabolism during the use of two low-dose oral contraceptives with similar doses of ethinyl estradiol but with different progestogenically active compounds were evaluated for their effective estrogen/androgen balance. Sixty-eight healthy women who did not take hormonally active drugs or were pregnant the previous 3 months took either 75 micrograms of gestodene + 30 micrograms of ethinyl estradiol or 150 micrograms of desogestrel + 30 micrograms ethinyl estradiol during 12 cycles. During the first three cycles serum levels of the following parameters increased: triglycerides, cholesterol in high-density lipoprotein, and apolipoproteins A1, A2, and B. Additional increase was observed in apolipoprotein B only after three and six cycles. The induced changes were not significantly different in the two groups, and the levels generally remained within normal limits. The changes seen with both pills reflect a mild estrogenic dominance. On the basis of current knowledge, moderately altered lipoprotein metabolism is not expected to impose an extra risk of atherosclerosis.

The power of serum lathosterol concentration as an indicator of whole-body cholesterol synthesis was investigated in 47 human volunteers consuming two diets differing in fatty acid composition. The cholesterol balance (fecal excretion of neutral and acid steroids minus cholesterol intake) was strongly correlated with the serum level of total (free plus esterified) lathosterol and also with the ratio of serum lathosterol over serum cholesterol, both on a diet rich in polyunsaturated fatty acids (r = 0.74 for the ratio) and one containing mainly saturated fatty acids (r = 0.70 for the ratio). In a subgroup for which the serum levels of free lanosterol and other free methylsterols were also quantitated, the correlations of these levels (expressed relative to serum free cholesterol) with the cholesterol balance were lower than that found for total lathosterol (expressed relative to serum total cholesterol). A further corroboration was obtained by measuring the lathosterol/cholesterol ratio in 20 patients with familial hypercholesterolemia before and during treatment with the hydroxymethylglutaryl coenzyme A reductase inhibitor Mk-733. The ratio was lowered by 47% during drug treatment, suggesting a significant decrease of the cholesterol balance in these patients. We conclude, from the various indicators proposed to monitor whole-body cholesterol synthesis, that the lathosterol/cholesterol ratio in serum appears preferable with respect to indicative power and ease of quantitation.

We studied the effects on plasma levels of coagulation and fibrinolysis factors of two currently used "sub-50" oral contraceptive preparations (OCs), one containing 750 micrograms lynestrenol and 37.5 micrograms ethinyl estradiol (LYN-EE) and the other containing 150 micrograms levonorgestrel and 30 micrograms ethinyl estradiol (LNG-EE), in groups of about 25 women aged 21 +/- 2 years. After 3 months, plasminogen levels increased in the two experimental groups (LYN-EE and LNG-EE), by 40% and 32%, respectively. This change was positively correlated with changes in ceruloplasmin levels, indicating that an estrogenic effect might be involved. Histidine-rich glycoprotein concentration decreased by 26% and 16%, respectively. Tissue-type plasminogen activator (t-PA) activity increased by 260% and 167%; t-PA antigen decreased by 12% and 18%, and t-PA inhibitor activity decreased by 31% and 32%, respectively. In the coagulation system, in both groups factor XII increased by 47% and 34%, respectively. The main inhibitor of factor XII, C1-inactivator, decreased slightly, but this was significant only in the LNG-EE group. The von Willebrand factor antigen fell by 8% and 9%, whereas factor VIII activity did not change. Antithrombin III antigen decreased by 14% in both groups. Factor IX activity increased by 15% and 21%. The difference in hormonal effects of both preparations was reflected by the increases in sex hormone binding globulin (by 130% and 21%) and ceruloplasmin (by 98% and 51%), indicating that LYN-EE had a more estrogenic potency than LNG-EE. In a control group of 25 matched subjects, who were observed simultaneously, we found no significant changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Simvastatin (MK-733), a new inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, was administered to 38 patients with heterozygous familial hypercholesterolaemia for 24 weeks. A dose of 40 mg per day produced a mean reduction in low density lipoprotein cholesterol of 43-45% and in triglycerides of 21-31%. Mean high density lipoprotein cholesterol increased significantly by 10-13%. There were no major differences in response whether the drug was taken in one or two doses. MK-733 was tolerated well. Adverse effects were infrequent and limited to slight increases of alanine aminotransferase, creatine phosphokinase and bilirubin. This drug appears to be a potent inhibitor of cholesterol synthesis and has produced the largest therapeutic response as monotherapy in patients with familial hypercholesterolaemia.

The effect of 3-hydroxy-3-methylglutaric acid (HMG) on serum cholesterol levels was investigated in Watanabe heritable hyperlipidaemic (WHHL) rabbits. Oral administration of HMG resulted in a reduction of serum cholesterol by 39%. Bile acid drainage, by means of either cholestyramine medication or partial ileal bypass (PIB) surgery, also led to significant reductions in circulating cholesterol, by 35 and 59% respectively. Intraperitoneal injection of HMG after PIB surgery further reduced serum cholesterol by 35%. Fibroblasts from the WHHL rabbits did not show high-affinity binding, uptake or degradation of 125I-labelled low density lipoprotein (LDL). The working mechanism of these lipid-lowering lowering therapies in WHHL rabbits is discussed in relation to recent literature. The significant reductions in circulating cholesterol induced by HMG warrant further investigation into the use of this compound in the management of familial hypercholesterolaemia.

Impairment of fibrinolysis is supposed to contribute to CVD. In 38 hyperlipoproteinemic patients, known to be at risk for early CVD, fibrinolytic activity was measured before and after stimulation with DDAVP. A negative correlation was found between serum triglyceride levels and fibrinolytic activity, both before and after DDAVP. A subnormal activity was invariably found when serum triglyceride concentration was above 8 mmol/L. The defect can be attributed to low levels of extrinsic plasminogen activator. High cholesterol levels were not associated with impairment of fibrinolysis. Fibrinolytic activity and response to DDAVP were lowest in those patients with hypertriglyceridemia who also had a tendency to develop hyperchylomicronemia. (type V/IV). The low fibrinolytic activity in this type of hyperlipoproteinemia cannot be explained by obesity. Factor VIII was higher than normal in most patients with hyperlipoproteinemia; the level increased after stimulation with DDAVP in every patient. This imbalance between coagulation and fibrinolysis might increase the risk of CVD.

Few studies have presented a thorough analysis of young adults with symptoms of arterial occlusive disease. To learn more about the possible risk factors of vascular disease playing a role in these young patients, we have reviewed all patients of 45 years of age and younger with symptoms of arterial occlusive disease who had been referred to our department between 1978 and 1987. Thirty-seven patients (28 males and 9 females) were included in the study. The mean age at which the first symptoms occurred was 34 years. Most patients presented with chronic arterial obliterations of the lower extremities (31/37, 84%). In addition, 4 patients showed signs of ischaemic heart disease. A strongly positive family history of arteriosclerosis was obtained from 13 patients (35%). Hypertension was present in 7 patients (19%), diabetes in three (8%) and nicotine abuse was found in 27 patients (73%). Fifty-four percent of the patients (20/37) had undergone vascular reconstructive surgery, 19% (7/37) underwent transluminal dilatation, and 3 had had subsequent treatment of newly developed lesions. For this study, all patients were recalled to the outpatient clinic. A complete case history was taken followed by a physical examination and ECG. Laboratory examinations were performed to analyse parameters of: (a) coagulation; (b) fibrinolysis; (c) fat- and (d) methionine metabolism. Clear-cut laboratory abnormalities were found in 33 patients (33/37, 89%). Coagulation parameters were abnormal in 11 patients (30%) (protein S deficiency: 3 pts). Fibrinolysis was impaired in 15 patients (40%).(ABSTRACT TRUNCATED AT 250 WORDS)

The apolipoprotein E3-Leiden variant has been shown to be associated with familial dysbetalipoproteinemia (FD) in a dominant manner (Havekes et al., Hum Genet 1986;73:157-163). Applying the polymerase chain reaction technique, we have cloned and sequenced relevant parts of both APOE alleles of the original proband. In exon 4 of the E*3-Leiden allele a partial gene duplication encompassing 21 nucleotides was found, leading to a tandem repeat of the codons 120-126 or 121-127. Using an E3-Leiden mutation specific oligonucleotide probe, the same mutation was found in two additional independently ascertained FD patients with an E3E3 phenotype based on isoelectric focusing. The E*3-Leiden mutation will be useful in the elucidation of the etiology of dominantly inherited forms of FD.

Tetranectin is a tetrameric protein that binds to kringle 4 of plasminogen. Increase of electrophoretic mobility of the otherwise slowly migrating tetranectin in the presence of ethylenediaminetetraacetate was used to develop a reproducible electroimmunoassay to quantify plasma levels. Plasma levels in normals were found within narrow limits of 100 +/- 16 (SD)%, (100% = 0.15 mumol/l). There was no difference between males and females, smokers and non-smokers, and there were no significant changes with age from 20 to 49 years. Patients with severe liver cirrhosis showed a large variation in plasma tetranectin levels but no systematic or average reduction, in contrast to strong reductions in plasma levels of other proteins. Patients treated with L-asparaginase showed a gradual reduction in time in plasma levels of various proteins, though tetranectin showed no significant reduction. It is concluded that tetranectin can be assayed reproducibly in plasma and has a well regulated plasma level. This level is not sensitive to conditions with reductions in synthesis of many proteins, such as during cirrhosis of the liver and during L-asparaginase therapy. The reductions in plasma levels during the use of oral contraceptives and pregnancy indicate involvement of sex steroids in the metabolism of tetranectin.

The influence of hydrochlorothiazide (HCT) treatment on the plasma levels of triglycerides, total cholesterol and high density lipoprotein cholesterol (HDL-cholesterol) was studied in 10 patients with essential hypertension. After a placebo period of 4 weeks, 50 mg HCT twice daily was given for a period of 9 months, followed by a second placebo period of 4 weeks. Triglycerides, total cholesterol and HDL-cholesterol were determined at the end of both placebo periods and after 1, 3, 6 and 9 months of HCT. For the whole group, there were no significant changes in triglycerides or HDL-cholesterol, whereas total cholesterol significantly increased during HCT. In 6 patients, plasma triglycerides were higher during HCT as compared to both placebo periods. In only 4 patients did HDL-cholesterol increase during HCT. Changes in triglycerides, total cholesterol and HDL-cholesterol were not related and no correlation was found with changes in blood pressure, body weight or serum potassium. In conclusion, this study confirms a possible adverse effect of diuretic treatment on plasma lipids, which should be considered when determining therapeutic regimens for hypertension.

Heterozygous familial hypercholesterolaemia (HtFH) is associated with an increased risk of coronary artery disease. Prevention is possible by increasing the number of functioning receptors of low density lipoproteins (LDLs) in the liver. This is partly achieved by treatment with bile acid sequestrants, such as cholestyramine, but the effect is limited because of a concomitant increase in cholesterol synthesis. It was the purpose of this study to determine whether the increase in cholesterol synthesis could be influenced by treatment with cyclandelate, since it is known that cyclandelate inhibits cholesterol synthesis in rats. Ten patients received cyclandelate (3.2 g daily in 2 doses) or placebo in a double-blind cross-over study, with each treatment period of 3 months' duration. During these periods, treatment with cholestyramine (16 g daily) was continued. No evidence was found of inhibition of cholesterol synthesis by cyclandelate, as indicated by the serum concentration of the cholesterol precursor, lanosterol, which remained unchanged. Neither the serum concentration of LDL, nor those of high density lipoprotein (HDL) cholesterol, apolipoprotein B, A-I or A-II, were affected. Thus, it can be concluded that treatment with cyclandelate was not effective in lowering serum cholesterol concentrations in patients with familial hypercholesterolaemia who received concomitant cholestyramine therapy.