Effect of orlistat on postprandial lipemia, NMR lipoprotein subclass profiles and particle size.

Evidence suggests that metabolic phenomena during postprandial lipemia may be important in the pathogenesis of atherosclerosis. Both lipid concentrations and lipoprotein subclass patterns may be important cardiovascular risk modifiers. The pancreatic lipase inhibitor orlistat reduces fat absorption by 30% and is used for the treatment of overweight and obesity. We evaluated the effect of orlistat on postprandial lipemia and lipoprotein particle distribution after moderate-and high-fat meals in healthy volunteers. In this double-blind, randomized, cross-over study, 10 healthy young men received orlistat 120 mg plus a high-fat meal (HFO), orlistat plus a moderate-fat meal (MFO) or placebo plus a high-fat meal (HFP). Plasma triacylglycerol, glucose, insulin, and free fatty acids were measured at baseline (fasting) and postprandially for 8h. Lipoprotein subclass profile was assessed by nuclear magnetic resonance spectroscopy. The 8h postprandial mean triacylglycerol area under the curve (AUC) was significantly lower with MFO and HFO (0.79 versus 1.33 mmol/lh) versus HFP (4.33 mmol/lh; p=0.02). Mean change in large VLDL subclass concentration during the 4-8h and mean VLDL size after 8h was significantly lower with HFO and MFO versus HFP (p<0.001). Small HDL particle concentration decreased significantly with HFP versus MFO or HFO (p<0.001). There was no significant difference in postprandial concentrations of glucose, insulin or free fatty acids on the different regimens. The lowering of postprandial triacylglycerol AUC, shorter postprandial lipemia, lower concentration of large triacylglycerol-rich particles and decrease of VLDL particle size supports the hypothesis of a less atherogenic postprandial lipoprotein profile following orlistat ingestion.

Detection of a novel exon 4 low-density lipoprotein receptor gene deletion in a swiss family with severe familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal dominant disease which results in 2-3-fold elevated cholesterol levels and in accelerated atherosclerosis. FH is caused by small mutations or larger rearrangements in the low-density lipoprotein receptor (LDLR). Here, we report that screening the LDLR gene in a Swiss family (n = 15) with clinical symptoms of FH by combined single strand conformation polymorphism and long-distance PCR identified a novel 1.3 kb deletion in the LDLR. The deletion eliminated exon 4 of the LDLR presumably by recombination between two identical 25 bp repeats present in intron 3 and 4. The 25 bp sequence in intron 3 is part of an Alu repeat, whereas no homology to Alu repeats was found for the intron 4 region. This 1.3 kb LDLR deletion allele cosegregated with elevated cholesterol levels over three generations. Even on high-dose statin therapy, carriers of the deletion averaged 1.6 times higher cholesterol levels and 1.9 times higher apolipoprotein B-100 (apoB-100) levels than non-carriers who had lipid and apoB-100 levels within the range of the Swiss population. Most affected members of the first and second generation of this family had experienced a first myocardial infarction (MI) before the age of 55 years and most LDLR gene deletion carriers older than 40 years showed severe coronary artery disease (CAD). Hence, we conclude that deletion of exon 4 in the LDLR gene drastically decreases low-density lipoprotein binding leading to severe hypercholesterolemia.

Mass spectrometric analysis of human transferrin in different body fluids.

In this study, we present a versatile new procedure for the analysis of transferrin and its isoforms isolated from human body fluids such as serum, plasma, and cerebrospinal fluid. This method is based on a three-step procedure: (i) isolation of transferrins using anion-exchange chromatography with UV detection; (ii) concentration of the transferrin fraction; (iii) detection of the transferrins with liquid chromatography-electrospray mass spectrometry. Pre-analytical sample procedures can be omitted and no immunoaffinity columns or transferrin-specific immunoassays were used. Anticoagulants such as heparin, EDTA, citrate, and oxalate do not interfere with our analysis. According to their respective molecular masses, up to ten different isoforms of transferrin could be identified in a serum sample from a patient with a congenital disorder of glycosylation type Ia (CDG-Ia). The method was successfully applied to different pathological samples from patients with CDG-Ia, CDG-Ib, CDG-Ic, CDG-Ie, CDG-If, and CDG-IIa. Additionally, samples from alcohol consumers that were found with turbidimetric immunoassay to contain increased levels of carbohydrate-deficient transferrin were analyzed.

Coffee acutely increases sympathetic nerve activity and blood pressure independently of caffeine content: role of habitual versus nonhabitual drinking.

BACKGROUND: Coffee is the most abundantly consumed stimulant worldwide. However, its cardiovascular safety remains controversial. Possible health hazards have been related to its main ingredient, caffeine. Activation of the sympathetic nervous system by coffee may enhance cardiovascular risk; however, it is unclear whether this effect of coffee is related to caffeine or other substance(s) also contained in decaffeinated coffee. METHODS AND RESULTS: In 15 healthy volunteers (6 habitual and 9 nonhabitual coffee drinkers) arterial blood pressure (BP), heart rate, and muscle sympathetic nervous activity (MSA) were continuously recorded before and after drinking a triple espresso or a decaffeinated triple espresso or after intravenous administration of caffeine (250 mg) or placebo (saline) in the same subjects. There was a significant time x condition interaction for the intravenous caffeine and placebo conditions for MSA, with caffeine showing a significant increase in MSA at 60 minutes (53.2+/-14.1% total activity) and the placebo group showing no effect. A similar significant time effect was found for coffee drinking (54.1+/-22.5% total activity). Habitual and nonhabitual coffee drinkers demonstrated similar changes in MSA and BP after intravenous caffeine, whereas coffee drinking increased BP in nonhabitual drinkers only, despite comparable increases of MSA and plasma caffeine levels. Nonhabitual coffee drinkers showed similar activation of MSA and BP after caffeine infusion, coffee, or decaffeinated coffee. CONCLUSIONS: Acutely, coffee and caffeine induced comparable increases in MSA and BP in nonhabitual coffee drinkers, whereas habitual coffee drinkers exhibited lack of BP increase despite MSA activation to coffee. Because decaffeinated coffee also increases BP and MSA in nonhabitual drinkers, ingredients other than caffeine must be responsible for cardiovascular activation.

Rapid detection of the CCR2-V64I, CCR5-A59029G and SDF1-G801A polymorphisms by tetra-primer PCR.

OBJECTIVE: To develop tetra-primer PCR assays for detection of the CCR2-V64I, CCR5-A59029G and SDF1-G801A polymorphisms associated with HIV pathogenesis. DESIGN AND METHODS: For each assay, two primers for the amplification of the gene locus are combined in one tube with two primers for the subsequent allele specific amplification (ASA). In the first set of cycles, pre-amplification of the gene region of interest is ensured by the gene specific primers. In the second set of cycles, lowering the annealing temperature allows ASA on the newly produced template. RESULTS: Analysis of 90 DNA samples resulted in allele frequencies for CCR2-V64I, CCR5-A59029G and SDF1-G801A which are similar to other Caucasian cohorts. Furthermore, re-analysis of sequenced genomic DNA by tetra-primer PCR analysis (7-11 times) always showed identical results. CONCLUSION: Our set of single-tube assays allows rapid and reproducible genotyping of the CCR2-V64I, CCR5-A59029G and SDF1-G801A polymorphisms. These inexpensive but accurate assays are valuable for screening these polymorphisms in cohorts of HIV-infected patients.

Single-step assays to analyze CYP2D6 gene polymorphisms in Asians: allele frequencies and a novel *14B allele in mainland Chinese.

BACKGROUND: Cytochrome P450-dependent monooxygenase 2D6 (CYP2D6) activity can be estimated by investigating the metabolism of model drugs or by genotyping the most common CYP2D6 alleles. For Caucasians, the CYP2D6 allele frequencies are well investigated, and single-step assays are available for genotyping, whereas allele analysis in mainland Chinese is limited. METHODS: Two tetra-primer assays and one allele-specific amplification assay were developed to easily genotype the CYP2D6 alleles *8, *10, and *14 previously detected in Asians. Applying these assays in combination with established single-tube assays, we analyzed 223 DNA samples from Chinese volunteers for the CYP2D6 alleles *3, *4, *5, *6, *8, *10, and *14 and for duplication of CYP2D6. RESULTS: Six different alleles were detected in mainland Chinese. The most frequent mutant allele was the intermediate metabolizer allele, CYP2D6*10, with a prevalence of 51.3%, followed by the poor metabolizer alleles CYP2D6*5 (7.2%) and a novel variant of CYP2D6*14. This novel *14B allele (2.0%) differs from the *14 allele by the absence of the C188T substitution and by the additional G1749C substitution. Furthermore, six duplication alleles of CYP2D6 were detected, including one duplication of the *10 allele (*10X2). CONCLUSIONS: The CYP2D6 allele frequencies in mainland Chinese shows some genetic diversity compared with Chinese from other regions: a novel *14B allele, a slightly higher frequency of the *5 allele, and a slightly lower frequency of the *10 allele than in most other Chinese populations.