Paola Alessandrini

In Vivo Formation of 8-Epi-Prostaglandin F2α Is Increased in Hypercholesterolemia

Abstract F2-isoprostanes are bioactive prostaglandin (PG) -like compounds that are produced from arachidonic acid through a nonenzymatic process of lipid peroxidation catalyzed by oxygen free-radicals. 8-Epi-PGF2α may amplify the platelet response to agonists, circulates in plasma, and is excreted in urine. We examined the hypothesis that the formation of 8-epi-PGF2α is altered in patients with hypercholesterolemia and contributes to platelet activation in this setting. Urine samples were obtained from 40 hypercholesterolemic patients and 40 age- and sex-matched control subjects for measurement of immunoreactive 8-epi-PGF2α. Urinary excretion of 11-dehydro-thromboxane (TX) B2, a major metabolite of TXA2, was measured as an in vivo index of platelet activation. Low-dose aspirin, indobufen, and vitamin E were used to investigate the mechanism of formation and effects of 8-epi-PGF2α on platelet activation. Urinary 8-epi-PGF2α was significantly ( P =.0001) higher in hypercholesterolemic patients than in control subjects: 473±305 versus 205±95 pg/mg creatinine. Its rate of excretion was inversely related to the vitamin E content of LDL and showed a positive correlation with urinary 11-dehydro-TXB2. Urinary 8-epi-PGF2α was unchanged after 2-week dosing with aspirin and indobufen despite complete suppression of TX metabolite excretion. Vitamin E supplementation was associated with dose-dependent reductions in both urinary 8-epi-PGF2α and 11-dehydro-TXB2 by 34% to 36% and 47% to 58% at 100 and 600 mg daily, respectively. We conclude that the in vivo formation of the F2-isoprostane 8-epi-PGF2α is enhanced in the vast majority of patients with hypercholesterolemia. This provides an aspirin-insensitive mechanism possibly linking lipid peroxidation to amplification of platelet activation in the setting of hypercholesterolemia. Dose-dependent suppression of enhanced 8-epi-PGF2α formation by vitamin E supplementation may contribute to the beneficial effects of antioxidant treatment.

Increased Levels of Soluble P-Selectin in Hypercholesterolemic Patients

BACKGROUND Hypercholesterolemia is considered a major risk factor for the development of atherosclerosis. Enhanced lipid peroxidation and persistent platelet activation can be observed in vivo in hypercholesterolemic patients and may have pathophysiological implications in the occurrence of cardiovascular events. P-selectin may play an important role in the pathogenesis of multicellular events, including atherosclerosis. We studied the impact of hypercholesterolemia and oxidative stress on plasma levels of P-selectin. METHODS AND RESULTS Plasma levels of P-selectin were measured by means of an enzyme immunoassay in 20 hypercholesterolemic patients with no clinical evidence of cardiovascular disease and in 20 sex- and age-matched normocholesterolemic subjects. Hypercholesterolemic patients had higher levels of P-selectin compared with that of control subjects (98+/-61 versus 56+/-14 ng/mL; P=.001). They also displayed increased von Willebrand Factor (vWF) levels (176+/-22 versus 119+/-12%; P=.0001). A direct correlation was observed between P-selectin and LDL cholesterol levels (p=.453). Administration of vitamin E (600 mg/d for 2 weeks) to hypercholesterolemic patients significantly reduced plasma P-selectin (40%), and an inverse correlation was observed between vitamin E and P-selectin plasma levels (p=-.446). CONCLUSIONS Hypercholesterolemia is associated with elevated plasmatic P-selectin. Altered oxidative processes leading to endothelial dysfunction and persistent platelet activation may contribute to increased soluble P-selectin levels. P-selectin may be proposed as a marker of endothelial dysfunction in hypercholesterolemic patients.

The molecular basis of lecithin:cholesterol acyltransferase deficiency syndromes: a comprehensive study of molecular and biochemical findings in 13 unrelated Italian families.

Objective—To better understand the role of lecithin:cholesterol acyltransferase (LCAT) in lipoprotein metabolism through the genetic and biochemical characterization of families carrying mutations in the LCAT gene. Methods and Results—Thirteen families carrying 17 different mutations in the LCAT gene were identified by Lipid Clinics and Departments of Nephrology throughout Italy. DNA analysis of 82 family members identified 15 carriers of 2 mutant LCAT alleles, 11 with familial LCAT deficiency (FLD) and 4 with fish-eye disease (FED). Forty-four individuals carried 1 mutant LCAT allele, and 23 had a normal genotype. Plasma unesterified cholesterol, unesterified/total cholesterol ratio, triglycerides, very-low-density lipoprotein cholesterol, and pre-&bgr; high-density lipoprotein (LDL) were elevated, and high-density lipoprotein (HDL) cholesterol, apolipoprotein A-I, apolipoprotein A-II, apolipoprotein B, LpA-I, LpA-I:A-II, cholesterol esterification rate, LCAT activity and concentration, and LDL and HDL3 particle size were reduced in a gene–dose-dependent manner in carriers of mutant LCAT alleles. No differences were found in the lipid/lipoprotein profile of FLD and FED cases, except for higher plasma unesterified cholesterol and unesterified/total cholesterol ratio in the former. Conclusion—In a large series of subjects carrying mutations in the LCAT gene, the inheritance of a mutated LCAT genotype causes a gene–dose-dependent alteration in the plasma lipid/lipoprotein profile, which is remarkably similar between subjects classified as FLD or FED.

Effects of Vitamin E Supplementation on F2-Isoprostane and Thromboxane Biosynthesis in Healthy Cigarette Smokers

BACKGROUND Increased formation of 8-iso-prostaglandin (PG) F(2alpha) and thromboxane (TX) A(2), potent agonists of platelet and vascular thromboxane (TH)/PGH(2) receptors, has been detected in cigarette smokers. We performed a randomized, double-blind, placebo-controlled study of the effects of vitamin E (300, 600, and 1200 mg/d, each dose for 3 consecutive weeks) on 8-iso-PGF(2alpha) and TXA(2) biosynthesis in 46 moderate cigarette smokers. METHODS AND RESULTS Urinary immunoreactive 8-iso-PGF(2alpha) and 11-dehydro-TXB(2), plasma vitamin E, and serum TXB(2) were measured by previously validated techniques. Baseline urinary 8-iso-PGF(2alpha) and 11-dehydro-TXB(2) excretion averaged 241+/-78 and 430+/-293 pg/mg creatinine, respectively. Urinary 8-iso-PGF(2alpha) was significantly correlated with 11-dehydro-TXB(2) (r=0.360, n=138, P<0.0001). Baseline plasma vitamin E levels averaged 20.6+/-4.9 micromol/L and were inversely correlated with urinary 11-dehydro-TXB(2) (r=-0.304, P=0.039) but not with 8-iso-PGF(2alpha) (r=-0.227, P=0.129). Vitamin E supplementation caused a dose-dependent increase in its plasma levels that reached a plateau at 600 mg (42.3+/-11.2 micromol/L, P<0. 001). This was not associated with any statistically significant change in urinary 8-iso-PGF(2alpha) or 11-dehydro-TXB(2) excretion. CONCLUSIONS Supplementation with pharmacological doses of vitamin E has no detectable effects on lipid peroxidation and thromboxane biosynthesis in vivo in healthy subjects with a mild degree of oxidant stress. These findings are consistent with the hypothesis that the basal rate of lipid peroxidation is a major determinant of the response to vitamin E supplementation and have implications for the use of vitamin E in healthy subjects as well as for the design and interpretation of clinical trials of antioxidant intervention.

Functional Lecithin: Cholesterol Acyltransferase Is Not Required for Efficient Atheroprotection in Humans

Background— Mutations in the LCAT gene cause lecithin:cholesterol acyltransferase (LCAT) deficiency, a very rare metabolic disorder with 2 hypoalphalipoproteinemia syndromes: classic familial LCAT deficiency (Online Mendelian Inheritance in Man No. 245900), characterized by complete lack of enzyme activity, and fish-eye disease (Online Mendelian Inheritance in Man No. 136120), with a partially defective enzyme. Theoretically, hypoalphalipoproteinemia cases with LCAT deficiency should be at increased cardiovascular risk because of high-density lipoprotein deficiency and defective reverse cholesterol transport. Methods and Results— The extent of preclinical atherosclerosis was assessed in 40 carriers of LCAT gene mutations from 13 Italian families and 80 healthy controls by measuring carotid intima-media thickness (IMT). The average and maximum IMT values in the carriers were 0.07 and 0.21 mm smaller than in controls (P=0.0003 and P=0.0027), respectively. Moreover, the inheritance of a mutated LCAT genotype had a remarkable gene-dose–dependent effect in reducing carotid IMT (P=0.0003 for average IMT; P=0.001 for maximum IMT). Finally, no significant difference in carotid IMT was found between carriers of LCAT gene mutations that cause total or partial LCAT deficiency (ie, familial LCAT deficiency or fish-eye disease). Conclusions— Genetically determined low LCAT activity in Italian families is not associated with enhanced preclinical atherosclerosis despite low high-density lipoprotein cholesterol levels. This finding challenges the notion that LCAT is required for effective atheroprotection and suggests that elevating LCAT expression or activity is not a promising therapeutic strategy to reduce cardiovascular risk.

Is glycation of low density lipoproteins in patients with Type 2 diabetes mellitus a LDL pre-oxidative condition?

Aims The study aimed to evaluate whether low density lipoprotein (LDL) in diabetic patients is more glycated and susceptible to oxidation than in non‐diabetic subjects and investigated the hypothesis that LDL glycation is associated with an increased plasma concentration of LDL– (a circulating electronegatively charged LDL), proposed as an index of in vivo oxidation.

Effects of gemfibrozil on insulin sensitivity and on haemostatic variables in hypertriglyceridemic patients.

In order to assess the efficacy of gemfibrozil on lipid and haemostatic parameters in patients with plurimetabolic syndrome, a multicenter double-blind placebo controlled, parallel study was carried out in 56 patients with primary hypertriglyceridemia and glucose intolerance. These patients had elevated PAI activity and antigen and t-PA antigen levels at rest and after venous occlusion. Gemfibrozil reduced plasma triglyceride levels (P<0.001), whereas it increased free fatty acids (P<0.05) and high density lipoprotein cholesterol levels (P<0.05). In those patients reaching normalization of plasma triglyceride levels (triglyceride reduction > or =50%) (n=15), insulin levels (P<0.05) as well as the insulin resistance index were reduced by gemfibrozil treatment, suggesting an improvement of the insulin resistance index in this patient subgroup. Gemfibrozil treatment did not affect plasma fibrinolysis or fibrinogen levels, despite marked reduction of plasma triglycerides and improvement of the insulin sensitivity associated with triglyceride normalization.

Physiologic variables affecting thromboxane B2 production in human whole blood

Thrombin-induced thromboxane (TX) A2 production in whole blood, as reflected by serum TXB2 measurements, has proven to be a simple and reproducible capacity-related index of platelet TXA2 production ex vivo. In the present study we have determined the influence of a number of physiologic variables on serum TXB2 measurements performed by radioimmunoassay in a group of 177 subjects undergoing evaluation for atherosclerosis risk factors. Serum TXB2 averaged 300 +/- 108 ng/ml in the whole group, with a normal distribution. No statistically significant correlation was found between serum TXB2 and the continuous variables examined (age, BMI, blood pressure, cigarettes, serum cholesterol, triglyceride, glucose and wine consumption) except for the number of platelets. Platelet TXB2 production did not differ between men and women (296 +/- 119 vs 302 +/- 100 ng/ml). We conclude that, within the limits of the examined variables, serum TXB2 is an easily measurable and highly reproducible capacity index primarily related to platelet cyclooxygenase and TX-synthase activity, which can be used for monitoring drug- or disease-induced changes of these enzyme activities.

Recurrent mutations of the apolipoprotein A-I gene in three kindreds with severe HDL deficiency

Two siblings with high density lipoprotein (HDL) deficiency and no plasma apolipoprotein A-I (Apo A-I) were found to be homozygous for a cytosine deletion in exon 3 of Apo A-I gene (c.85 del C, Q5FsX11). This mutation causes a frameshift leading to a premature stop codon and abolishes the synthesis of Apo A-I. Although both siblings had corneal opacifications and planar xanthomas, only one of them had premature coronary artery disease, probably as the result of mildly elevated LDL levels. In two other unrelated subjects HDL deficiency was due to heterozygosity for a nucleotide substitution in exon 4 of Apo A-I gene (c.494 T>G, L141R). Both Apo A-I mutations were reported previously in an Italian kindred which included compound heterozygotes and simple heterozygotes. We investigated all carriers of these mutations in the three kindreds and in the one previously reported. Plasma Apo A-I and HDL-C levels were lower in the mutation carriers than in non-carrier family members. These levels, however, were lower in L141R carriers than in carriers of c.85 del C. Haplotype analysis performed using several polymorphisms suggested that both the c.85 del C and L141R are likely to be recurrent mutations.