Chronic rhinitis and stress: the possible culprits of midfacial segment pain.

BACKGROUND: Bilateral symmetrical pain in the midfacial region without evidence of sinonasal disease is termed midfacial segment pain (MSP), about which little is known. The present study explored the prevalence of facial pain and the risk factors for MSP. METHODS: We analysed cross-sectional data from the Korea National Health and Nutrition Examination Survey (KNHANES). Those who reported facial pain or pressure lasting at least three months with no evidence of a sinonasal disease on nasal endoscopy were considered to have MSP. The participants were categorised according to the presence of facial pain and chronic rhinosinusitis. Basic demographic data and medical conditions, including hypertension, diabetes mellitus, and dyslipidemia, were compared between subject groups. We also evaluated psychological stress, depressive episodes, and suicidal thoughts, as well as physician-diagnosed nasal diseases, including chronic rhinitis and symptomatic nasal septal deviation. Univariate and multivariate logistic regression analyses were performed to determine risk factors for MSP. RESULTS: Of 31,999 participants, the prevalence of facial pain was 0.59%. A total of 58 (0.18%) respondents had MSP, of whom 40 (73.5%) were female. On univariate analysis, female sex, chronic rhinitis, and psychological stress were more prevalent in the subjects with MSP than the control subjects. However, in the multivariate analysis, only chronic rhinitis and psychological stress remained significant, while the female sex exhibited only marginal significance. CONCLUSION: Chronic rhinitis and psychological stress may be significant risk factors for MSP.

HLA-DQA1-HLA-DRB1 polymorphism is a major predictor of azathioprine-induced pancreatitis in patients with inflammatory bowel disease.

BACKGROUND: Azathioprine (AZA)-induced pancreatitis is an unpredictable and dose-independent adverse event affecting 2%-7% of patients with inflammatory bowel disease (IBD) patients treated with AZA. There are no tools in clinical practice to identify at-risk individuals; however, a genome wide association study (GWAS) identified a strong association between the Class II HLA gene region polymorphism (rs2647087) and thiopurine-induced pancreatitis. AIM: To independently confirm the findings of the GWAS in an IBD cohort, to evaluate its utility in clinical practice and to offer a novel AZA treatment algorithm for IBD based on pharmacogenomic principles. METHODS: A retrospective cohort study evaluated 373 AZA-exposed IBD patients from a tertiary care academic centre in London, Canada. Due to the limited number of patients taking mercaptopurine (MP), such patients were not included this cohort. All subjects underwent screening for the single nucleotide polymorphism (SNP) rs2647087 mapped to the HLA-DQA1*02:01-HLA-DRB1*07:01 haplotype and were sub-divided based on the presence (n = 13) or absence (n = 360) of an AZA-induced pancreatitis diagnosis. The risk of AZA-induced pancreatitis was assessed based on rs2647087 genotype. RESULTS: The risk of pancreatitis during AZA-therapy was highly predictable and genotype dependent: 0.53% for wild type (A/A), 4.25% (OR = 4.19, 95% CI 1.02-36.45, P = 0.044) for heterozygous (A/C), and 14.63% (OR = 15.83, 95% CI 3.80-145.26, P = 0.0001) for homozygous variant (C/C) patients. CONCLUSIONS: The class II HLA region (at rs2647087) is an important marker of AZA-induced pancreatitis risk. We propose a simple and clinically implementable algorithm based on rs2647087 and TPMT genotypes for AZA selection and dosing for patients with IBD.

Genetic Predictors of Azathioprine Toxicity and Clinical Response in Patients with Inflammatory Bowel Disease.

BACKGROUND: Thiopurines (Azathioprine (AZA) and 6-Mercaptopurine (6-MP) are considered a well-established therapy for patients with Inflammatory Bowel Disease (IBD) including ulcerative colitis (UC) and Crohn's Disease (CD). However, nearly 20% of patients discontinue thiopurines due to adverse events. Functional polymorphisms of several enzymes involved in the metabolism of thiopurines have been linked with toxicity. The clinical value of variant carriers such as TPMT, ITPA and GSTs in predicting toxicity and adverse events for IBD patients treated with thiopurines remains to be clarified. OBJECTIVES: To determine if variation in TPMT, ITPA and GST genotypes can predict adverse effects such as neutropenia, pancreatitis, liver enzyme elevation, as well as clinical response for patients with IBD treated with thiopurines. METHODS: Patients known to have IBD and treated with AZA or 6MP were enrolled. Adverse effects were calculated and their correlation with TPMT, ITPA and GST genotypes was evaluated. Further, the correlation between clinical response and TPMT, ITPA and GST genotypes were assessed. RESULTS: A total of 53 patients were enrolled. 16/53 patients (28.6%) responded to AZA therapy. 17 patients experienced adverse events with 10 having to discontinue treatment. Three patients (5.4%) developed severe myelosuppression (WBC< 2.0 or neutrophils <1.0). Loss of function TPMT genotype was associated with adverse events (OR 3.64, 95% CI 0.55 - 24.23, p=0.0313). ITPA and GST polymorphisms were not associated with toxicity. GSTM1 deletion was associated with poor clinical response to therapy (OR 3.75, 95% CI 0.940 - 14.97, p=0.1028), however, neither TPMT*3A nor ITPA polymorphisms were associated with clinical response. CONCLUSION: In addition to TPMT for adverse events, genotyping for GSTM1 appears to predict clinical response in IBD patients treated with thiopurines.

External validation of the prognostic index in acute paraquat poisoning.

OBJECTIVE: Some studies have evaluated the prognostic indicators associated with acute paraquat (PQ) poisoning. In this study, we externally validated the Yamaguchi index, which showed a good prognostic relevance in predicting the outcome of PQ poisoning. METHODS: A retrospective analysis of 297 patients was performed. The Yamaguchi index was calculated using the following equation: Eq1 = (K(+) × HCO3(-))/(Creatinine × 0.088)(mEq/L) against time from PQ ingestion (T). The patients were divided into three groups: group A: Eq1 > 1500 - 399 × log T, group B: 930 - 399 × log T < Eq1 ≤ 1500 - 399 × log T, and group C: Eq1 ≤ 930 - 399 × log T). RESULTS: The overall mortality rate was 65.3% (194 of 297). The mortality rates of the three groups stratified by the Yamaguchi index were 7.1% (2 of 28), 22.4% (15 of 67), and 87.6% (177 of 202). The area under the receiver-operating characteristic curve for predicting mortality from the external validation of the Yamaguchi index was 0.842 (95% confidence interval: 0.795-0.882). CONCLUSION: The Yamaguchi index is a reliable prognostic factor and could be helpful in predicting mortality due to PQ poisoning.

OATP1B1 and tumour OATP1B3 modulate exposure, toxicity, and survival after irinotecan-based chemotherapy.

BACKGROUND: Treatment of advanced and metastatic colorectal cancer with irinotecan is hampered by severe toxicities. The active metabolite of irinotecan, SN-38, is a known substrate of drug-metabolising enzymes, including UGT1A1, as well as OATP and ABC drug transporters. METHODS: Blood samples (n=127) and tumour tissue (n=30) were obtained from advanced cancer patients treated with irinotecan-based regimens for pharmacogenetic and drug level analysis and transporter expression. Clinical variables, toxicity, and outcomes data were collected. RESULTS: SLCO1B1 521C was significantly associated with increased SN-38 exposure (P<0.001), which was additive with UGT1A1*28. ABCC5 (rs562) carriers had significantly reduced SN-38 glucuronide and APC metabolite levels. Reduced risk of neutropenia and diarrhoea was associated with ABCC2-24C/T (odds ratio (OR)=0.22, 0.06-0.85) and CES1 (rs2244613; OR=0.29, 0.09-0.89), respectively. Progression-free survival (PFS) was significantly longer in SLCO1B1 388G/G patients and reduced in ABCC2-24T/T and UGT1A1*28 carriers. Notably, higher OATP1B3 tumour expression was associated with reduced PFS. CONCLUSIONS: Clarifying the association of host genetic variation in OATP and ABC transporters to SN-38 exposure, toxicity and PFS provides rationale for personalising irinotecan-based chemotherapy. Our findings suggest that OATP polymorphisms and expression in tumour tissue may serve as important new biomarkers.

Clinical performance of bleeding risk scores for predicting major and clinically relevant non-major bleeding events in patients receiving warfarin.

BACKGROUND: Oral anticoagulant therapy is associated with an increased risk of hemorrhage, which can be assessed by bleeding risk scores. We evaluated the performance of five validated scores for predicting major and clinically relevant non-major bleeding events in patients receiving warfarin. METHODS AND RESULTS: We conducted an ambispective, single-center cohort study of 321 consecutive patients enrolled in an academic anticoagulation clinic. The following scores were calculated: modified Outpatient Bleeding Risk Index, Contemporary Bleeding Risk Model, HEMORR(2)HAGES (Hepatic or Renal Disease, Ethanol Abuse, Malignancy, Older Age, Reduced Platelet Count or Function, Re-Bleeding, Hypertension, Anemia, Genetic Factors, Excessive Fall Risk and Stroke), ATRIA (Anticoagulation and Risk Factors in Atrial Fibrillation), and HAS-BLED (Hypertension, Abnormal Renal/Liver Function, Stroke, Bleeding History or Predisposition, Labile International Normalized Ratio, Elderly, Drugs/Alcohol). Main outcomes were major bleeding and a composite of major plus clinically relevant non-major bleeding. Incidence rates for all group were 3.8 (95% confidence interval [CI] 2.0-6.4) and 11.9 (95% CI 8.6-16.4) events per 100 patient-years for major bleeding and major plus clinically relevant non-major bleeding, respectively. Agreement among the five scores was low to moderate (Kendall's tau-b coefficients 0.22-0.54). For major bleeding, the c-statistics ranged from 0.606 to 0.735, whereas for major plus clinically relevant non-major bleeding, they ranged from 0.549 to 0.613. For all scores, the 95% CI for the c-statistics crossed 0.5 or was very close. Among high-risk patients, the hazard ratios for major bleeding ranged from 0.90 to 39.01, whereas for major plus clinically relevant non-major bleeding, they ranged from 1.52 to 8.71. For intermediate-risk patients, no score, except the Contemporary Bleeding Risk Model, produced statistically significant hazard ratios. CONCLUSION: The scores demonstrated poor agreement and low to moderate discriminatory ability. General clinical implementation of these scores cannot be recommended yet.

The transfer of pravastatin in the dually perfused human placenta.

HMG-CoA reductase inhibitors (statins) are contraindicated during pregnancy. However, it has been suggested that the hydrophilic property of pravastatin prevents its placental transfer to the fetus, explaining neutral effects observed in controlled studies. Using the ex-vivo placental perfusion model, placental transfer of pravastatin (50 ng/ml) was determined. The mean maximum fetal concentration was 4.4 ng/ml. The transfer of pravastatin's across the placenta appears to be limited and slow. Combined with its rapid elimination half-life of 2 h and 50% protein binding, the transfer of pravastatin from maternal to fetal compartments is substantially more limited than observed in the perfusion experiments.

Pharmacokinetic profiles for oral and subcutaneous methotrexate in patients with Crohn's disease.

BACKGROUND: Methotrexate (MTX) is administered subcutaneously to Crohn's Disease (CD) patients. There are very few studies evaluating the use of oral (PO) MTX in CD. A drug and its pharmaceutical alternative are equivalent (bioequivalence) when the bioavailability of the alternative falls within 80-125% of the bioavailability of the standard (US Food and Drug Administration - FDA). AIM: To compare the pharmacokinetic (PK) profiles of PO and subcutaneous (SC) MTX in CD patients to determine the bioequivalence of these two routes. METHODS: Eleven patients received a PO and an SC MTX dose (25 mg) separated by one week over a two-week interval. Blood samples were collected at specified times over a 24-h period for each patient on two separate days. MTX plasma levels were obtained using sensitive mass spectrometry. Areas under the curve (AUC) were compared between the two routes. RESULTS: The mean AUC values were 3375 ng/mL × h (PO MTX) and 3985 ng/mL × h (SC MTX). The mean AUC ratio (PO/SC) was 0.86 (0.62-1.08). This correlates with a relative PO bioavailability of 86% in comparison to SC. The 90% confidence interval for the mean AUC (PO/SC) ratio is (0.785, 0.929). There were no adverse events. CONCLUSIONS: The mean MTX AUC (PO/SC) in these patients falls outside the 90% confidence interval for the bioequivalence limit. SC MTX is more bioavailable than PO MTX; however, the mean relative MTX bioavailability (PO/SC) nearly met the FDA bioequivalence standard and PO MTX could be proposed in responders who would prefer this route.

In vitro and in vivo assessment of renal drug transporters in the disposition of mesna and dimesna.

Mesna and its dimer, dimesna, are coadministered for mitigation of ifosfamide- and cisplatin-induced toxicities, respectively. Dimesna is selectively reduced to mesna in the kidney, producing its protective effects. In vitro screens of uptake and efflux transporters revealed saturable uptake by renal organic anion transporters OAT1, OAT3, and OAT4. Efflux transporters breast cancer resistance protein; multidrug and toxin extrusion 1 (MATE1); multidrug resistance proteins MRP1, MRP2, MRP4, and MRP5; and P-glycoprotein (Pgp) significantly reduced dimesna accumulation. Further investigation demonstrated that renal apical efflux transporters MATE1, MRP2, and Pgp were also capable of mesna efflux. Administration of OAT inhibitor probenecid to healthy subjects significantly increased combined mesna and dimesna plasma exposure (91% ± 34%) while decreasing the renal clearance due to net secretion (67.0% ± 12.7%) and steady-state volume of distribution (45.2% ± 13.4%). Thus, the kidney represents a significant sink of total mesna, whereas function of renal drug transporters facilitates clearance in excess of glomerular filtration rate and likely the presence of active mesna in the urine. Loss of renal transporter function due to genetic variability or drug-drug interactions may decrease the efficacy of chemoprotectants, increasing the risk of ifosfamide- and cisplatin-induced toxicities.

Drug transporters in drug efficacy and toxicity.

Drug transporters are now widely acknowledged as important determinants governing drug absorption, excretion, and, in many cases, extent of drug entry into target organs. There is also a greater appreciation that altered drug transporter function, whether due to genetic polymorphisms, drug-drug interactions, or environmental factors such as dietary constituents, can result in unexpected toxicity. Such effects are in part due to the interplay between various uptake and efflux transporters with overlapping functional capabilities that can manifest as marked interindividual variability in drug disposition in vivo. Here we review transporters of the solute carrier (SLC) and ATP-binding cassette (ABC) superfamilies considered to be of major importance in drug therapy and outline how understanding the expression, function, and genetic variation in such drug transporters will result in better strategies for optimal drug design and tissue targeting as well as reduce the risk for drug-drug interactions and adverse drug responses.

Disposition of atorvastatin, rosuvastatin, and simvastatin in oatp1b2-/- mice and intraindividual variability in human subjects.

Response to statin therapy is often unpredictable because of variability in metabolism and transport. In the recently created organic anion transporting-polypeptide 1b2 (Oatp1b2/Slco1b2)-null mice, the investigators found significantly lower liver-to-plasma ratios compared with controls for atorvastatin (16.0 ± 5.1 vs 43.5 ± 13.7, P = .002) and rosuvastatin (15.2 ± 3.3 vs 28.4 ± 9.3, P = .03), but not simvastatin (5.2 ± 1.1 vs 6.3 ± 2.9, P = .49), following tail vein injection of 1 mg/kg of each drug. In addition, the investigators examined intraindividual variation in atorvastatin, rosuvastatin, and simvastatin pharmacokinetics in healthy human subjects in a crossover study design. Areas under the plasma concentration-time curve of atorvastatin and simvastatin acid were significantly related (Spearman r = 0.68; P = .035), whereas rosuvastatin profile was not related to atorvastatin or simvastatin exposure. Together, these results in mice and humans demonstrate that predictability of exposure to one statin based on another is dependent on the specific statin pairs and the context in which they are compared.

Environmental and genetic factors affecting transport of imatinib by OATP1A2.

The bioavailability of orally administered imatinib is >90%, although the drug is monocationic under the acidic conditions in the duodenum. In vitro, we found that imatinib is transported by the intestinal uptake carrier organic anion transporting polypeptide (OATP1A2) and that this process is sensitive to pH, rosuvastatin, and genetic variants. However, in a study in patients with cancer, imatinib absorption was not associated with OATP1A2 variants and was unaffected by rosuvastatin. These findings highlight the importance of verifying in a clinical setting the drug-transporter interactions observed in in vitro tests.

Induction of intestinal P-glycoprotein by St John's wort reduces the oral bioavailability of talinolol.

St John's wort (SJW) is known to induce cytochrome P450 (CYP) 3A4 and P-glycoprotein through pregnane X-receptor activation. Our study evaluated the effects of long-term SJW administration on oral and intravenous pharmacokinetics of the nonmetabolized in vivo probe of P-glycoprotein, talinolol, in relation to intestinal P-glycoprotein expression. In a controlled, randomized study (N=9), the pharmacokinetics of oral (50 mg) and intravenous talinolol (30 mg) was determined before and after 12 days SJW (900 mg daily, Jarsin 300). Duodenal biopsies were taken and MDR1 genotypes assessed. SJW reduced the oral talinolol bioavailability by 25% (P=0.049) compared with water control. A 93% increase in oral clearance (P=0.177) and a 31% reduction in area under the serum concentration time curve (AUC; P=0.030) were observed. Renal and nonrenal clearance (CLNR), elimination half-life, peak serum drug concentration (Cmax), and time to reach Cmax were not significantly altered. After intravenous talinolol, SJW affected only CLNR (35% increase compared with water, P=0.006). SJW increased MDR1 messenger ribonucleic acid (mRNA) as well as P-glycoprotein levels in the duodenal mucosa. Subjects with the combined MDR1 genotype comprising 1236C>T, 2677G>T/A, and 3435C>T polymorphisms had lower intestinal MDR1 mRNA levels and displayed an attenuated inductive response to SJW as assessed by talinolol disposition. Long-term SJW decreased talinolol AUC with a corresponding increase in intestinal MDR1 expression, suggesting that SJW has a major inductive effect on intestinal P-glycoprotein. Interestingly, the magnitude of induction appeared to be affected by MDR1 genotype.

Naringin is a major and selective clinical inhibitor of organic anion-transporting polypeptide 1A2 (OATP1A2) in grapefruit juice.

We showed previously that grapefruit and orange juices inhibited human enteric organic anion-transporting polypeptide (OATP)1A2 in vitro and lowered oral fexofenadine bioavailability clinically. Inhibition of OATP1A2 transport by flavonoids in grapefruit (naringin) and orange (hesperidin) was conducted in vitro. Two randomized, crossover, pharmacokinetic studies were performed clinically. In one study, 120 mg of fexofenadine was ingested with 300 ml grapefruit juice, an aqueous solution of naringin at the same juice concentration (1,200 microM), or water. In the other study, fexofenadine was administered with grapefruit juice, with or 2 h before aqueous suspension of the particulate fraction of juice containing known clinical inhibitors of enteric CYP3A4, but relatively low naringin concentration (34 microM), or with water. Naringin and hesperidin's half-maximal inhibitions were 3.6 and 2.7 microM, respectively. Fexofenadine area under the plasma drug concentration-time curves (AUCs) with grapefruit juice and naringin solution were 55% (P<0.001) and 75% (P<0.05) of that with water, respectively. Fexofenadine AUCs with grapefruit juice and particulate fractions were 57% (P<0.001), 96% (not significant (NS)), and 97% (NS) of that with water, respectively. Individuals tested in both studies (n=9 of 12) had highly reproducible fexofenadine AUC with water (r(2)=0.85, P<0.001) and extent of reduction of it with grapefruit juice (r(2)=0.72, P<0.01). Naringin most probably directly inhibited enteric OATP1A2 to decrease oral fexofenadine bioavailability. Inactivation of enteric CYP3A4 was probably not involved. Naringin appears to have sufficient safety, specificity, and sensitivity to be a clinical OATP1A2 inhibitor probe. Inherent OATP1A2 activity may be influenced by genetic factors. This appears to be the first report of a single dietary constituent clinically modulating drug transport.

Intestinal drug transporter expression and the impact of grapefruit juice in humans.

The goals of this study were to assess the extent of human intestinal drug transporter expression, determine the subcellular localization of the drug uptake transporter OATP1A2, and then to assess the effect of grapefruit juice consumption on OATP1A2 expression relative to cytochrome P450 3A4 and MDR1. Expression of drug uptake and efflux transporters was assessed using human duodenal biopsy samples. Fexofenadine uptake by different transporters was measured in a transporter-transfected cell line. We investigated the influence of grapefruit juice on pharmacokinetics of orally administered fexofenadine. The effect of grapefruit juice on the expression of intestinal transporters was determined using real-time polymerase chain reaction and Western blot analysis. In the duodenum of healthy volunteers, an array of CYP enzymes as well as uptake and efflux transporters was expressed. Importantly, uptake transporters thought to be liver-specific, such as OATP1B1 and 1B3, as well as OATP2B1 and 1A2 were expressed in the intestine. However, among OATP transporters, only OATP1A2 was capable of fexofenadine uptake when assessed in vitro. OATP1A2 colocalized with MDR1 to the brush border domain of enterocytes. Consumption of grapefruit juice concomitantly or 2 h before fexofenadine administration was associated with reduced oral fexofenadine plasma exposure, whereas intestinal expression of either OATP1A2 or MDR1 remained unaffected. In conclusion, an array of drug uptake and efflux transporters are expressed in the human intestine. OATP1A2 is likely the key intestinal uptake transporter for fexofenadine absorption whose inhibition results in the grapefruit juice effect. Although short-term grapefruit juice ingestion was associated with reduced fexofenadine availability, OATP1A2 or MDR1 expression was unaffected.

Organic anion-transporting polypeptide (OATP) transporter family and drug disposition.

Drug transporters are increasingly recognized as a key determinant of drug disposition. Recent studies have revealed that targeted expression of drug uptake and efflux transporters to specific cell membrane domains allows for the efficient directional movement of many drugs in clinical use. While the role of certain efflux transporters such as MDR1 (P-glycoprotein) in drug disposition has been extensively studied, emerging evidence suggests that uptake transporters may also be important to the intestinal absorption and renal or hepatic elimination of drugs. Members of the organic anion-transporting polypeptide (OATP) family of drug uptake transporters have been found capable of transporting a large array of structurally divergent drugs. Moreover, expression of OATP isoforms in the gastrointestinal tract, liver and kidney, as well as at the level of the blood-brain barrier, has important implications for our understanding of the factors governing drug absorption, elimination and tissue penetration.

A common beta1-adrenergic receptor polymorphism (Arg389Gly) affects blood pressure response to beta-blockade.

BACKGROUND: A common polymorphism of the beta(1)-adrenergic receptor Arg389Gly markedly affects function in vitro, but little is known about its in vivo significance. METHODS AND RESULTS: Resting and exercise hemodynamic responses were measured in subjects homozygous for Arg389 (n = 21) or Gly389 (n = 13) alleles before and 3 hours after administration of a beta-blocker, atenolol. Demographic characteristics and atenolol concentrations were similar in the two genotypic groups. Genotype had a marked effect on resting hemodynamic responses to atenolol, with Arg389-homozygous subjects having a larger decrease in resting systolic blood pressure (8.7 +/- 1.3 mm Hg versus 0.2 +/- 1.7 mm Hg, P < .001) and mean arterial blood pressure (7.2 +/- 1.0 mm Hg versus 2.0 +/- 1.7 mm Hg, P = .009). Attenuation of exercise-induced hemodynamic responses by atenolol was not affected by genotype. CONCLUSIONS: There is reduced sensitivity of Gly389 homozygotes to a beta-adrenergic receptor antagonist, and this polymorphism may be an important determinant of variability in response to beta-blockade.

In-vivo effects of Glu298Asp endothelial nitric oxide synthase polymorphism.

Endothelial nitric oxide synthase catalyses the formation of the vasodilator nitric oxide, a major regulator of vascular tone. The Asp298 polymorphism of the nitric oxide synthase gene is associated with altered function and expression of the enzyme in vitro and myocardial infarction and coronary artery spasm in vivo. We examined the effect of the Glu298Asp polymorphism on: (1) local vascular responses to phenylephrine, acetylcholine, glyceryl trinitrate and prostaglandin E1 in the dorsal hand vein; (2) changes in forearm blood flow during mental stress, a measure of nitric oxide-mediated effect on resistance vessels; (3) excretion of urinary nitrite/nitrate as a measure of total body nitric oxide production; and (4) F2-isoprostane metabolite, a measure of oxidative stress, in healthy Glu298 (n = 12) and Asp298 (n = 13) homozygotes. There were no significant differences in acetylcholine dose responses (P = 0.29) in Glu298 and Asp298 homozygotes. Responses to glyceryl trinitrate, prostaglandin E1 and the alpha-adrenergic agonist phenylephrine did not differ by genotype. Forearm blood flow was similar at rest and increased significantly (from 7.5 ml/min/100 ml to 12.2 ml/min/100 ml; P = 0.003), but similarly (P = 0.2), during mental stress in both genotypes. Asp298 homozygotes excreted significantly less nitrate/nitrite than Glu298 homozygotes (nitrate + nitrite/creatinine ratio 0.05 +/- 0.01 vs. 0.09 +/- 0.01, respectively; P < 0.005). Urinary F2-isoprostane metabolite excretion did not differ (Glu298, 2.04 +/- 0.25 ng/mg creatinine; Asp298, 1.85 +/- 0.37 ng/mg creatinine; P = 0.7). We conclude that in healthy volunteers the Glu298Asp polymorphism affects endogenous nitric oxide production without affecting nitric oxide-mediated vascular responses. This polymorphism may only have clinical significance in the presence of endothelial dysfunction.