Ocfentanil testing in hair from a fatality case: Comparative analysis of a lock of hair versus a single hair fiber.

In clinical and forensic toxicology, hair analysis offers a larger window for detecting drug exposure than blood or urine. Drug measurements are generally carried out using a segmented lock of hair, but few articles report the use of a single hair to document drug exposure. Nevertheless, single hair analysis can be very useful, particularly if only small amounts of biological matrices are available. More data on analyzing new synthetic opioids (NSOs) in hair are needed to help interpretation in future cases. In this study, segmental single hair analysis is compared with segmental hair lock analysis to document an ocfentanil-related death. The hair lock and single hair analyses were performed using the LC-MS/MS method after decontamination and incubation. Ocfentanil (OcF) concentrations ranged from 42 to 150 pg/mg in the segmented hair lock, depending on the segments. The hair lock and single hair analyses showed similar results: the highest concentrations were measured in the first two centimeters and decreased from root to tip. The similar profiles obtained from both the lock of hair and the single hair demonstrate the relevance of single hair analysis in cases where very few data are available. This article describes OcF concentrations in an authentic hair sample after a documented intake of this molecule in a fatality.

CYP2C9, SLCO1B1, SLCO1B3, and ABCB11 polymorphisms in patients with bosentan-induced liver toxicity.

Bosentan is an endothelin receptor antagonist used as a first-line treatment in pulmonary arterial hypertension (PAH). Its main adverse effect is a dose-dependent liver toxicity. CYP2C9*2 has recently been shown to be associated with hepatotoxicity in PAH patients. We conducted a nested case-control study to further explore the relationship between functional polymorphisms of gene products involved in bosentan pharmacokinetics (OATP1B1, OATP1B3, and CYP2C9) or hepatobiliary transporters affected by bosentan (ABCB11) and bosentan-induced liver toxicity.

Impact of flexible insulin therapy on blood glucose variability, oxidative stress and inflammation in type 1 diabetic patients: the VARIAFIT study.

AIMS: HbA1c only partially predicts vascular risk in patients with type 1 diabetes (T1D), and a role for blood glucose variability (BGV) is a matter of debate. For this reason, this study investigated the impact of an educational programme of flexible insulin therapy (FIT) on BGV and oxidative stress. METHODS: Tests were conducted on 30 adult T1D patients in a prospective, single-centre trial at baseline (M0), and at 3 and 6 months (M3 and M6, respectively) of the FIT programme to determine BGV, as reflected by mean amplitude of glycaemic excursions (MAGE), low blood glucose index (LBGI), lability index (LI), average daily risk range (ADRR), glycaemic lability (scored by two diabetologists), urinary leukotriene E4 (LTE4), 11-dehydro-thromboxane B2 (TXB2) and 8-iso-prostaglandin F2α (PGF2). RESULTS: HbA1c (7.7 ± 0.9%), ADRR, MAGE, LBGI and LI did not change from M0 to M3 and M6, although ADRR and LBGI significantly improved at M3 and M6 in patients with the highest baseline indices (≥ 40 and ≥ 5, respectively). TXB2 declined at M6 (832 ± 625 vs. 633 ± 972 pg/mg; P=0.048), whereas LTE4 and PGF2 remained stable. ADRR showed the strongest correlation with glycaemic lability scores at all visits (r≥0.84, P<0.0001). CONCLUSION: A FIT educational programme improved BGV only in patients with the highest baseline variability, and led to no changes in HbA1c, while ADRR closely correlated with glycaemic lability score. Our data do not support a relationship between BGV and oxidative stress in T1D patients, although the impact of variability on TXB2 deserves further investigation (ClinicalTrials.gov NCT00973492).

Cutaneous iontophoresis of treprostinil in systemic sclerosis: a proof-of-concept study.

Ischemic digital ulcer (DU) is a serious complication of systemic sclerosis (SSc). Intravenous prostanoids are the only approved treatment for active DUs, but they induce dose-limiting side effects and require hospitalization. Our objective was to evaluate the effect of iontophoresis (a noninvasive drug delivery method) of treprostinil in SSc patients. Three studies were conducted: a pharmacokinetic study in 12 healthy volunteers showed that peak dermal concentration was reached at 2 hours, whereas plasma treprostinil was undetected. Then, a placebo-controlled, double-blind incremental dose study assessed the effect of treprostinil on digital skin blood flow in 22 healthy subjects. The effect of the highest dose was then compared with that of placebo in 12 SSc patients. Treprostinil significantly increased skin blood flow in healthy subjects (P = 0.006) and in SSc patients (P = 0.023). In conclusion, digital iontophoresis of treprostinil is feasible, is well tolerated, and increases digital skin perfusion. It could be tested as a treatment for SSc-related DUs.

Therapeutic drug monitoring of posaconazole in allogeneic hematopoietic stem cell transplantation patients who develop gastrointestinal graft-versus-host disease.

Posaconazole (PCZ) is the latest triazole antifungal agent that has been approved for prophylaxis of invasive aspergillosis in high-risk immunocompromised patients, such as allogeneic hematopoietic stem cell transplantation patients, who develop graft-versus-host disease (GVHD). PCZ has high interindividual variability with regard to its plasma drug trough concentrations (C(min)). Moreover, the concentration-efficiency relationship remains to be better characterized in prophylaxis. To determine the variability factors in plasma drug concentrations, the PCZ C(min) and clinical parameters (localization of GVHD, presence of diarrhea, and diagnosis of invasive aspergillosis) were collected retrospectively in 29 consecutive allogeneic hematopoietic stem cell transplantation patients who developed GVHD and were receiving prophylactic PCZ (200 mg, 3 times/day, for ≥7 days). Blood samples were analyzed at steady state to determine the PCZ C(min) by liquid chromatography-tandem mass spectrometry. The average PCZ C(min) was 1.28 ± 0.82 mg/liter (mean ± standard deviation; n = 292 dosages), with an intraindividual variability of 49% and an interindividual variability of 64%. Twenty percent of C(min)s were below 0.7 mg/liter, which is considered the threshold of efficacy by the Food and Drug Administration. The patients who had gastrointestinal (GI) GVHD experienced a 24% reduction in the posaconazole C(min), compared with those with other localizations of GVHD. This decrease reached 33% when patients presented with diarrhea due to GI GVHD or an infectious etiology. PCZ C(min)s were 26% lower when invasive aspergillosis was declared. These data demonstrate that GI disturbances affect drug concentrations. Thus, therapeutic monitoring of PCZ can be used to detect low drug concentrations, possibly resulting in a lack of efficacy of invasive aspergillosis prophylaxis.

Urinary leukotriene E4 concentrations as a potential marker of inflammation in dogs with inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) and food-responsive diarrhea (FRD) are chronic enteropathies of dogs (CCE) that currently can only be differentiated by their response to treatment after exclusion of other diseases. In humans, increased urinary concentrations of leukotriene E4 (LTE4) have been associated with active IBD. OBJECTIVES: To evaluate urinary LTE4 concentrations in dogs with IBD, FRD, and healthy controls, and to assess correlation of urinary LTE4 concentrations with the canine IBD activity index (CIBDAI) scores. ANIMALS: Eighteen dogs with IBD, 19 dogs with FRD, and 23 healthy control dogs. METHODS: In this prospective study, urine was collected and CIBDAI scores were calculated in client-owned dogs with IBD and those with FRD. Quantification of LTE4 in urine was performed by liquid chromatography-tandem mass spectrometry and corrected to creatinine. RESULTS: Urinary LTE4 concentrations were highest in dogs with IBD (median 85.2 pg/mg creatinine [10th-90th percentiles 10.9-372.6]) followed by those with FRD (median 31.2 pg/mg creatinine [10th-90th percentiles 6.2-114.5]) and control dogs (median 21.1 pg/mg creatinine [10th-90th percentiles 9.1-86.5]). Urinary LTE4 concentrations were higher in dogs with IBD than in control dogs (P = .011), but no significant difference between IBD and FRD was found. No correlation was found between urinary LTE4 concentrations and CIBDAI. CONCLUSIONS AND CLINICAL IMPORTANCE: The higher urinary LTE4 concentrations in dogs with IBD suggest that cysteinyl leukotriene pathway activation might be a component of the inflammatory process in canine IBD. Furthermore, urinary LTE4 concentrations are of potential use as a marker of inflammation in dogs with CCE.

Leukotriene B4: early mediator of atherosclerosis in obstructive sleep apnoea?

Severity of oxygen desaturation is predictive of early atherosclerosis in obstructive sleep apnoea (OSA). Leukotriene (LT)B(4) is a lipid mediator involved in atherogenesis. In 40 non-obese OSA patients, free of a cardiovascular history, and 20 healthy volunteers, the following were evaluated: 1) LTB(4) production by polymorphonuclear leukocytes (PMNs) stimulated with A23187; and 2) the relationships between LTB(4) production and both OSA severity and infraclinical atherosclerosis markers. The effect of continuous positive airway pressure (CPAP) on LTB(4) production was also studied. An overnight sleep study was followed by first-morning blood sampling. Isolated PMNs were stimulated with A23187 in order to induce LTB(4) production, which was measured by liquid chromatography-tandem mass spectrometry. Carotid intima-media thickness (IMT) and luminal diameter were measured in subset groups of 28 OSA patients and 11 controls. LTB(4) production was increased in OSA patients compared with controls. LTB(4) levels correlated with the mean and minimal arterial oxygen saturation (S(a,O(2))). LTB(4) production correlated with luminal diameter data in patients with a mean S(a,O(2)) of < or = 94% but not with IMT. Lastly, CPAP significantly reduced LTB(4) production by 50%. Leukotriene B(4) production is increased in obstructive sleep apnoea in relation to oxygen desaturation. Leukotriene B(4) could promote early vascular remodelling in moderate-to-severe hypoxic obstructive sleep apnoea patients.

5-lipoxygenase expression and activity in aorta from streptozotocin-induced diabetic rats.

We previously reported an activation of the 5-lipoxygenase pathway in aorta from streptozotocin-induced diabetic rats. The aim of this study was to investigate whether this activation was associated with an increased expression of 5-lipoxygenase, an increased cysteinyl leukotriene (CysLT) production in response to arachidonic acid or calcium ionophore A23187 and/or a hypersensitivity of the aorta to CysLTs in streptozotocin-induced diabetic rats. In aorta from diabetic and control rats, reverse transcriptase-PCR and western blot analysis with a specific 5-lipoxygenase antibody provided evidence for the presence of 5-lipoxygenase in aorta. However, the expression of 5-lipoxygenase was not significantly different between diabetic and control rats. Challenge by A23187 (10 microM) and arachidonic acid (10 microM and 0.1 mM) with or without A23187 (10 micromol/l) induced a significant increase of CysLT release (measured by enzyme immunoassay) that was in the same range in aorta from control and diabetic rats. In contrast, aortas from diabetic rats showed a greater sensitivity to LTC4 and LTD4 contractile effects. These data suggested that the activation of the 5-lipoxygenase pathway previously reported in streptozotocin-induced diabetic rats could be explained by an augmented sensitivity to CysLTs of the diabetic aorta.

Chronic intermittent hypoxia increases infarction in the isolated rat heart.

Coronary heart disease is frequently associated with obstructive sleep apnea syndrome and treating obstructive sleep apnea appears to significantly improve the outcome in coronary heart disease. Thus we have developed a rat model of chronic intermittent hypoxia (IH) to study the influence of this condition on myocardial ischemia-reperfusion tolerance and on functional vascular reactivity. Wistar male rats were divided in three experimental groups (n = 12 each) subjected to chronic IH (IH group), normoxia (N group), or control conditions (control group). IH consisted of repetitive cycles of 1 min (40 s with inspired O(2) fraction 5% followed by 20 s normoxia) and was applied for 8 h during daytime, for 35 days. Normoxic cycles were applied in the same conditions, inspired O(2) fraction remaining constant at 21%. On day 36, mean arterial blood pressure (MABP) was measured before isolated hearts were submitted to an ischemia-reperfusion protocol. The thoracic aorta and left carotid artery were also excised for functional reactivity studies. MABP was not significantly different between the three experimental groups. Infarct sizes (in percent of ventricles) were significantly higher in IH group (46.9 +/- 3.6%) compared with N (26.1 +/- 2.8%) and control (21.7 +/- 2.1%) groups. Vascular smooth muscle function was similar in aorta and carotid arteries from all groups. The endothelium-dependent relaxation in response to acetylcholine was also similar in aorta and carotid arteries from all groups. Chronic IH increased heart sensitivity to infarction, independently of a significant increase in MABP, and did not affect vascular reactivity of aorta and carotid arteries.

Effects of L-arginine and sodium nitroprusside on the spontaneous contractility of human non-pregnant uterus.

BACKGROUND: The aim of this study was to investigate, in isolated human non-gravid myometrium, the involvement of the cyclic guanosine monophosphate (cGMP) pathway in nitric oxide (NO) induced relaxation. METHODS: Strips of human myometrium from hysterectomized women were suspended in organ baths for recording of isometric tension. Cumulative concentration-response curves for L-arginine and sodium nitroprusside were performed in the presence of methylene blue (10 micromol/l) or vehicle (control). The effect of increasing concentrations of 8-bromo-cGMP on uterine spontaneous contraction was also studied. RESULTS: L-arginine and sodium nitroprusside induced a concentration-dependent decrease in the amplitude of the myometrial spontaneous contractions. Pre-treatment with methylene blue enhanced the inhibitory effect of L-arginine and sodium nitroprusside on myometrial spontaneous contractions. In addition, 8-bromo-cGMP had no effect on spontaneous contractions in human myometrium. CONCLUSIONS: These data provide evidence that L-arginine and sodium nitroprusside inhibit the spontaneous contractions of the non-pregnant human uterus through a cGMP independent pathway.

[Leukotrienes and 12-HETE: key mediators of angiotensin II-mediated vascular effects.Rol in hypertension]. / Leucotriènes et 12-HETE: modulateurs des effets vasculaires de l'angiotensine II. Implication dans l'hypertension artérielle.

Arachidonic acid metabolism-derived products are key mediators of angiotensin II-mediated vascular effects. The modulatory effect of cyclooxygenase derived products--in particular thromboxane A2 and prostaglandin H2--in angiotensin II-mediated vascular effects is well established. In contrast, few studies have assessed the involvement of lipoxygenase-derived products in the vascular effects of angiotensin II. Cysteinyl leukotrienes (5-lipoxygenase-derived products) and 12-hydroxyeicosatetraenoic acids (12-HETE) (12-lipoxygenase-derived products) are potent proinflammatory and vasomotor mediators. Their biosynthesis is increased in various models of hypertension. In addition, compelling evidence has suggested that they might contribute to the vasoconstrictor, hypertrophic and mitogenic effects of angiotensin II. The demonstration of their contribution to angiotensin II-mediated vascular effects may explain, at least in part, the vascular inflammatory complications associated with hypertension.

Cysteinyl leukotrienes modulate angiotensin II constrictor effects on aortas from streptozotocin-induced diabetic rats.

Angiotensin II (Ang II) is a vasopressor peptide involved in the pathogenesis of cardiovascular diseases associated with diabetes mellitus. We have previously reported that the 5-lipoxygenase-derived products, particularly the cysteinyl leukotrienes (CysLTs), are involved in Ang II-induced contraction. In this study, we demonstrated that CysLTs contribute to the contraction elicited by Ang II in isolated aortas from streptozotocin-induced diabetic (SS) rats but not from insulin-treated diabetic rats, fructose-fed rats, or control rats. In an organ bath, pretreatment with the 5-lipoxygenase inhibitor (AA861, 10 micromol/L) reduced by 37.6+/-8.2% and 30.1+/-10.9% the Ang II-induced contractions in intact and endothelium-denuded aortic rings, respectively, from SS rats. In contrast, the CysLT(1) receptor antagonist (MK571, 1 micromol/L) or the dual CysLT(1)/CysLT(2) receptor antagonist (BAY-u9773, 0.1 micromol/L) did not affect Ang II-induced contraction. In addition, Ang II induced a 6.2+/-1.5-fold increase in CysLT release through the stimulation of the Ang II type 1 receptor. Furthermore, the urinary excretion of leukotriene E(4) was increased in SS rats (leukotriene E(4), 13.7+/-2.9 ng/24 h [SS rats, n=10] versus 1.5+/-0.5 ng/24 h [control rats, n=6]; P<0.0004). These data suggest the activation of the 5-lipoxygenase pathway in SS rats and the involvement of 5-lipoxygenase-derived products, particularly the CysLTs, in Ang II-induced contraction in aortas from SS rats through stimulation of CysLT receptors different from the well-characterized CysLT(1) or CysLT(2) receptor.

Increased lipid peroxidation in patients with pulmonary hypertension.

Isoprostanes are chemically stable lipid peroxidation products of arachidonic acid, the quantification of which provides a novel approach to the assessment of oxidative stress in vivo. The main objective of this study was to quantify the urinary levels of isoprostaglandin F(2alpha) type III (iPF(2alpha)-III), an F(2)-isoprostane, in patients with pulmonary hypertension (PHT) in comparison with healthy controls. The secondary objective was to test whether baseline iPF(2alpha)-III levels correlate to the reversibility of pulmonary hypertension in response to inhaled NO challenge. Urinary iPF(2alpha)-III levels were measured by gas chromatography-mass spectrometry in 25 patients with PHT, 14 of whom were investigated for response to inhaled NO challenge. Urinary iPF(2alpha)-III levels in PHT patients (225 +/- 27 pmol/mmol creatinine) were 2.3 times as high as in controls (97 +/- 7 pmol/mmol creatinine, p < 0.001). The mean pulmonary arterial pressure variation and the pulmonary vascular resistance variation in response to inhaled NO were correlated to basal iPF(2alpha)-III levels. This study shows that oxidative stress is increased in patients with pulmonary hypertension. Furthermore, iPF(2alpha)-III levels inversely correlate to pulmonary vasoreactivity. These observations are consistent with the hypothesis that free radical generation is involved in PHT pathogenesis.

Formation of isoprostanes in children with type IIa hypercholesterolemia.

F2-isoprostanes are stable lipid peroxidation products of arachidonic acid and their quantification provides a novel approach to the assessment of oxidative stress in vivo. F2-isoprostanes are present in increased amounts in adult hypercholesterolemia, but no data exist concerning children. We investigated urinary isoprostaglandin F2, type III production as an index of lipid peroxidation in 15 children presenting with type IIa hypercholesterolemia (serum total cholesterol, 290 [SD +/- 70] mg/dl; low-density lipoprotein cholesterol, 210 [SD +/- 90] mg/dl) compared with 15 sex- and age-paired control children (serum total cholesterol, 160 [SD +/- 20] mg/dl). Urinary levels of isoprostaglandin F2alpha type III were measured by gas chromatography mass spectrometry. Urinary concentrations did not differ significantly in hypercholesterolemic children compared with control children (84.7 [SD +/- 37] vs. 96 [SD +/- 35] pmol/mmol creatinine, respectively). No significant correlation was found with total cholesterol, low-density-lipoprotein and high-density-lipoprotein cholesterol, and apolipoprotein B and A1 serum levels. F2-isoprostane urinary levels in children with type IIa hypercholesterolemia do not differ from those of age- and sex-matched control children and are not correlated to blood lipid parameters, suggesting that hypercholesterolemia is not associated with increased lipid peroxidation in childhood.

Determination of isoprostaglandin F2alpha type III in human urine by gas chromatography-electronic impact mass spectrometry. Comparison with enzyme immunoassay.

F2-Isoprostanes are stable lipid peroxidation products of arachidonic acid, the quantification of which provides an index of oxidative stress in vivo. We describe a method for analysing isoprostaglandin F2alpha type III (15-F2t-IsoP) in biological fluids. The method involves solid-phase extraction on octadecyl endcapped and aminopropyl cartridges. After conversion to trimethylsilyl ester trimethylsilyl ether derivatives, isoprostaglandin F2alpha type III is analysed by mass spectrometry, operated in electronic impact selected ion monitoring mode. We have compared enzyme immunoassay (EIA; Cayman, Ann Arbor, MI, USA) to this method with 30 human urine aliquots following the same extraction procedure in order to determine the agreement between both methods. Isoprostaglandin F2alpha type III concentrations determined with gas chromatography-mass spectrometry (GC-MS) did not agree with those determined with EIA. Our results suggest that GC-MS and EIA do not measure the same compounds. As a consequence, comparison of clinical results using GC-MS and EIA should be avoided.

Isoprostaglandin E2 type-III (8-iso-prostaglandin E2) evoked contractions in the human internal mammary artery.

E2-isoprostanes are recently discovered compounds that are produced in vivo from free radical-catalysed peroxidation of arachidonic acid. One such compound whose formation is favoured by this mechanism is isoprostaglandin E2 type III (iPE2-III, also named 8-iso-prostaglandin E2 or 15-E2t-isoprostaglandin). The aim of this study was to evaluate the vasomotor properties of iPE2-III in isolated human internal mammary artery. In organ bath, iPE2-III was approximately 10 times more potent than isoprostaglandin F2alpha-III and 27 times more potent than prostaglandin E2, whereas both isoprostaglandin F3alpha-III and 15-epi-isoprostaglandin F2alpha-II induced weak contractions. The responses to iPE2-III were inhibited in a concentration-dependent manner by the thromboxane A2 receptor antagonist GR 32191 (3.10(-9) to 3.10(-7) M). Indomethacin, a cyclooxygenase inhibitor and phosphoramidon, an endothelin converting enzyme inhibitor, did not affect iPE2-III response. These data shows that iPE2-III is a more potent vasoconstrictor of human internal mammary arteries than isoprostaglandin F2alpha-III. These effects are mediated by TP receptors, but involve neither cyclooxygenase products nor endothelins. iPE2-III production may induce more pronounced vasomotor effects than isoprostaglandin F2alpha-III in situations of oxidative stress, and in particular may modulate internal mammary artery tone following coronary bypass surgery.

Enhanced in vivo lipid peroxidation in scleroderma spectrum disorders.

OBJECTIVE: A new family of prostaglandin F2 isomers called F2-isoprostanes, produced by free radical peroxidation of arachidonic acid, has recently been described in vivo. Its quantification has been suggested to be a reliable measure of oxidant injury in vivo. The purpose of this study was to investigate urinary F2-isoprostane formation as an index of lipid peroxidation in scleroderma spectrum disorders. METHODS: Urine samples were obtained from 52 patients with systemic sclerosis (SSc; n = 37) or undifferentiated connective tissue diseases (UCTD; n = 15) and from 20 healthy volunteers. Urinary isoprostaglandin F2alpha type III (iPF2alpha-III) and 11-dehydro thromboxane B2 (11-dehydroTXB2) concentrations were determined using enzyme immunoassays. RESULTS: The urinary concentration of iPF2alpha-III was approximately twice as high in patients (mean +/- SEM 229+/-16 pmoles/mmoles creatinine) as in controls (116+/-9 pmoles/mmoles creatinine) (P < 0.0001). However, the urinary concentration of iPF2alpha-III was not significantly different among patients with UCTD, limited SSc, and diffuse SSc (mean +/- SEM 221+/-27 versus 245+/-32 versus 220+/-25 pmoles/mmoles creatinine, respectively). No significant correlation was found between the urinary concentrations of iPF2alpha-III and 11-dehydroTXB2. CONCLUSION: This study provides evidence of enhanced lipid peroxidation in both SSc and UCTD, and suggests a rationale for antioxidant treatment of SSc. Formation of F2-isoprostanes has to be investigated as a means for the evaluation of such therapy.