Variants of the

Clinical efficacy of the antiplatelet drug clopidogrel is hampered by its variable biotransformation into the active metabolite. The variability in the clinical response to clopidogrel treatment has been attributed to genetic factors, but the specific genes and mechanisms underlying clopidogrel bioactivation remain unclear. Using in vitro metabolomic profiling techniques, we identified paraoxonase-1 (PON1) as the crucial enzyme for clopidogrel bioactivation, with its common Q192R polymorphism determining the rate of active metabolite formation. We tested the clinical relevance of the PON1 Q192R genotype in a population of individuals with coronary artery disease who underwent stent implantation and received clopidogrel therapy. PON1 QQ192 homozygous individuals showed a considerably higher risk than RR192 homozygous individuals of stent thrombosis, lower PON1 plasma activity, lower plasma concentrations of active metabolite and lower platelet inhibition. Thus, we identified PON1 as a key factor for the bioactivation and clinical activity of clopidogrel. These findings have therapeutic implications and may be exploited to prospectively assess the clinical efficacy of clopidogrel.

Context Regular intake of cocoa-containing foods is linked to lower cardiovascular mortality in observational studies. Short-term interventions of at most 2 weeks indicate that high doses of cocoa can improve endothelial function and reduce blood pressure (BP) due to the action of the cocoa polyphenols, but the clinical effect of low habitual cocoa intake on BP and the underlying BP-lowering mechanisms are unclear.Objective To determine effects of low doses of polyphenol-rich dark chocolate on BP.Design, Setting, and Participants Randomized, controlled, investigatorblinded, parallel-group trial involving 44 adults aged 56 through 73 years (24 women, 20 men) with untreated upper-range prehypertension or stage 1 hypertension without concomitant risk factors. The trial was conducted at a primary care clinic in Germany between January 2005 and December 2006.Intervention Participants were randomly assigned to receive for 18 weeks either 6.3 g (30 kcal) per day of dark chocolate containing 30 mg of polyphenols or matching polyphenol-free white chocolate.Main Outcome Measures Primary outcome measure was the change in BP after 18 weeks. Secondary outcome measures were changes in plasma markers of vasodilative nitric oxide (S-nitrosoglutathione) and oxidative stress (8-isoprostane), and bioavailability of cocoa polyphenols. ResultsFrom baseline to 18 weeks, dark chocolate intake reduced mean (SD) systolic BP by −2.9 (1.6) mm Hg (PϽ.001) and diastolic BP by −1.9 (1.0) mm Hg (PϽ.001) without changes in body weight, plasma levels of lipids, glucose, and 8-isoprostane. Hypertension prevalence declined from 86% to 68%. The BP decrease was accompanied by a sustained increase of S-nitrosoglutathione by 0.23 (0.12) nmol/L (PϽ.001), and a dark chocolate dose resulted in the appearance of cocoa phenols in plasma. White chocolate intake caused no changes in BP or plasma biomarkers.Conclusions Data in this relatively small sample of otherwise healthy individuals with above-optimal BP indicate that inclusion of small amounts of polyphenol-rich dark chocolate as part of a usual diet efficiently reduced BP and improved formation of vasodilative nitric oxide.

Catecholaminergic signaling regulates various physiological functions, such as blood pressure 1 and is implicated in drug dependence, affective disorders and male aggressive behavior 2,3 . The actions of released catecholamines are terminated by sodium-driven, high-affinity transporters in the plasma membrane of the releasing neurons 4,5 and by a corticosteronesensitive, low-affinity, high-capacity extraneuronal transport system 6 , originally named uptake 2 , found in sympathetically innervated tissues 7 and in central nervous system glia 8 . Here we report the molecular identification and pharmacological characterization of the extraneuronal catecholamine transporter, which is unrelated to the family of sodium-driven neuronal monoamine transporters 5 .Extraneuronal uptake is closely related to the non-neuronal metabolism of catecholamines 7 by catechol-O-methyltransferase (COMT), which exists almost exclusively in non-neuronal cells 9 , and monoamine oxidases (MAO-A and MAO-B). Extraneuronal transport is the predominant pathway for terminating the actions of circulating adrenaline and noradrenaline 10 . Although neuronal and extraneuronal uptake compete for released catecholamines, these transport systems have distinct pharmacological profiles, that is, affinity for substrates and sensitivity to various drugs. Overlap in the antagonist sensitivity between extraneuronal catecholamine uptake and apical renal transport of organic cations by OCT2 (ref. 11) raised the possibility that the extraneuronal transporter might belong to the recently defined family of amphiphilic solute facilitators (ASF) 12 . Degenerate oligonucleotides were derived from common sequence motifs of the ASF family and used for PCR on cDNA from Caki-1 cells, a human kidney carcinoma cell line known to express extraneuronal noradrenaline transport 13 . Amplicons of the expected sizes were isolated by ultraviolet-protected gel electrophoresis, cloned and sequenced. A fragment with similarity to members of the ASF family was identified. This fragment, in northern analysis of Caki-1 mRNA, detected a single band with a length of approximately 3.4 kb. The full-length cDNA of the corresponding transporter was assembled from a Caki-1 cDNA library clone and a fragment from inverse PCR. Because of its functional characteristics, we named this new transporter EMT (extraneuronal transporter for monoamine transmitters).Amino-acid sequence analysis identified EMT as a new member of the ASF transporter family 12 . No proteins highly homologous to EMT are yet known. EMT is similar to the OCT and OAT proteins (renal transporters for organic cations and organic anions), with identity (similarity) scores of about 50% (70%) and 32% (55%), respectively. EMT consists of 556 amino acids ( Fig. 1) with 12 putative transmembrane segments. To determine whether the functional properties of EMT match the characteristics of extraneuronal transport of catecholamines, the EMT cDNA was inserted into the expression vector pcDNA3 and transfected into 293 cells, a cell line fro...