Comparative study of six sequential spectrophotometric methods for quantification and separation of ribavirin, sofosbuvir and daclatasvir: An application on Laboratory prepared mixture, pharmaceutical preparations, spiked human urine, spiked human plasma, and dissolution test.

In accordance with International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines, six novel, simple and precise sequential spectrophotometric methods were developed and validated for the simultaneous analysis of Ribavirin (RIB), Sofosbuvir (SOF), and Daclatasvir (DAC) in their mixture without prior separation steps. These drugs are described as co-administered for treatment of Hepatitis C virus (HCV). HCV is the cause of hepatitis C and some cancers such as liver cancer (hepatocellular carcinoma) and lymphomas in humans. These techniques consisted of several sequential steps using zero, ratio and/or derivative spectra. DAC was first determined through direct spectrophotometry at 313.7 nm without any interference of the other two drugs while RIB and SOF can be determined after ratio subtraction through five methods; Ratio difference spectrophotometric method, successive derivative ratio method, constant center, isoabsorptive method at 238.8 nm, and mean centering of the ratio spectra (MCR) at 224 nm and 258 nm for RIB and SOF, respectively. The calibration curve is linear over the concentration ranges of (6-42), (10-70) and (4-16) µg/mL for RIB, SOF, and DAC, respectively. This method was successfully applied to commercial pharmaceutical preparation of the drugs, spiked human urine, and spiked human plasma. The above methods are very simple methods that were developed for the simultaneous determination of binary and ternary mixtures and so enhance signal-to-noise ratio. The method has been successfully applied to the simultaneous analysis of RIB, SOF, and DAC in laboratory prepared mixtures. The obtained results are statistically compared with those obtained by the official or reported methods, showing no significant difference with respect to accuracy and precision at p = 0.05.

Metabolites characterization of a novel DPP-4 inhibitor, imigliptin in humans and rats using ultra-high performance liquid chromatography coupled with synapt high-resolution mass spectrometry.

Imigliptin has been reported as a novel dipeptidyl-peptidase-IV (DPP-4) inhibitor to treat type 2 Diabetes Mellitus (T2DM), and is currently being tested in clinical trials. In the first human clinical study, imigliptin was well tolerated and proved to be a potent DPP-4 inhibitor. Considering its potential therapeutic benefits and promising future, it is of great importance to study the metabolite profiles in the early stage of drug development. In the present study, a robust and reliable analytical method based on the ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) method combined with MassLynx software was established to investigate the characterization of metabolites of imigliptin in human and rat plasma, urine and feces after oral administration. As a result, a total of 9 metabolites were identified in humans, including 6, 9 and 8 metabolites in human plasma, urine, and feces, respectively. A total of 11 metabolites were identified in rats, including 7, 10 and 8 metabolites in rat plasma, urine, and feces, respectively. In addition, 6 of the metabolites detected in humans and rats were phase I metabolites, including demethylation, carboxylation, hydroxylation and dehydrogenation metabolites, and 5 of the metabolites were phase II metabolites, including acetylation and glucuronidation. There was no human metabolite detected compared to those in rats. The major metabolites detected in human plasma (M1 and M2) were products resulting from acetylation, and hydroxylation followed by dehydrogenation. M1 was the major metabolite in rat plasma. M2 and the parent drug were the major drug-related substances in human urine. The parent drug was the major drug-related substances in rat urine. M2, M5 (hydroxylation product) and M6 (2 × hydroxylation and acetylation product) were the predominant metabolites in human feces. M2 and M5 were the major metabolites in rat feces. In addition, renal clearance was the major route of excretion for imigliptin.

Concentrations of selected heterocyclic aromatic amines among US population aged ≥ 6 years: data from NHANES 2013-2014.

Data from National Health and Nutrition Examination Survey for US population aged ≥ 6 years for 2013-2014 were used to analyze data for four heterocyclic aromatic amines (HCAA), namely 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP), harman, and norharman. Data were analyzed separately for children aged 6-11 years (N = 416), adolescents aged 12-19 years (N = 475), adults aged 20-64 years (N = 1913), and seniors aged ≥ 65 years (N = 458). Adult males had lower concentrations of AαC and harman than adult females (1.44 vs. 2.22 pg/mL for AαC, p < 0.01 and 136.8 vs. 163.2 pg/mL for harman, p = 0.04). Racial/ethnic differences were observed in the adjusted concentrations of HCAAs. For adults, adjusted concentrations of HCAAs were lower for non-Hispanic Asians and Hispanics as compared to non-Hispanic blacks and whites. For example for AαC, the adjusted concentrations for non-Hispanic Asians, Hispanics, non-Hispanic blacks and whites were 1.16, 2.00, 2.37, and 2.16 pg/mL respectively. Adjusted concentrations of AαC were found to be lower among nonsmokers as compared to smokers for adolescents (0.34 vs. 1.32 pg/mL, p < 0.01), adults (0.40 vs. 7.91 pg/mL, p < 0.01), and seniors (0.30 vs. 4.29 pg/mL, p < 0.01). For both harman and norharman, adult nonsmokers had lower adjusted concentrations than smokers (125.7 vs. 177.6 pg/mL, p < 0.01 for harman, 296.1 vs. 421.6 pg/mL, p < 001, for norharman). Exposure to environmental tobacco smoke was found to be associated with higher concentrations of AαC among adolescents (p = 0.01) and adults (p = 0.01) and for harman (p = 0.01) and norharman (p = 0.01) among seniors. In conclusion, concentrations of selected HCAAs can be several fold higher among smokers as compared to nonsmokers and gender as well as race/ethnicity also affect the observed concentrations of HCAA.

Dietary intake of advanced glycation endproducts is associated with higher levels of advanced glycation endproducts in plasma and urine: The CODAM study.

BACKGROUND & AIMS: Advanced glycation endproducts (AGEs) are formed by the reaction between reducing sugars and proteins. AGEs in the body have been associated with several age-related diseases. High-heat treated and most processed foods are rich in AGEs. The aim of our study was to investigate whether dietary AGEs, are associated with plasma and urinary AGE levels. METHODS: In 450 participants of the Cohort on Diabetes and Atherosclerosis Maastricht study (CODAM study) we measured plasma and urine concentrations of the AGEs Nε-(carboxymethyl)lysine (CML), Nε-(1-carboxyethyl)lysine (CEL) and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) using UPLC-MS/MS. We also estimated dietary intake of CML, CEL and MG-H1 with the use of a dietary AGE database and a food frequency questionnaire (FFQ). We used linear regression to investigate the association between standardized dietary AGE intake and standardized plasma or urinary AGE levels, after adjustment for age, sex, glucose metabolism status, waist circumference, kidney function, energy- and macro-nutrient intake, smoking status, physical activity, alcohol intake, LDL-cholesterol and markers of oxidative stress. RESULTS: We found that higher intake of dietary CML, CEL and MG-H1 was associated with significantly higher levels of free plasma and urinary CML, CEL and MG-H1 (ßCML = 0.253 (95% CI 0.086; 0.415), ßCEL = 0.194 (95% CI 0.040; 0.339), ßMG-H1 = 0.223 (95% CI 0.069; 0.373) for plasma and ßCML = 0.223 (95% CI 0.049; 0.393), ßCEL = 0.180 (95% CI 0.019; 0.332), ßMG-H1 = 0.196 (95% CI 0.037; 0.349) for urine, respectively). In addition, we observed non-significant associations of dietary AGEs with their corresponding protein bound plasma AGEs. CONCLUSION: We demonstrate that higher intake of dietary AGEs is associated with higher levels of AGEs in plasma and urine. Our findings may have important implications for those who ingest a diet rich in AGEs.

[Biomonitoring in workers exposed to pesticides: development and application of method detecting imidacloprid in urine].

Imidacloprid is a relatively new insecticide in neonicotinoids chemical class with neuroactivity in insects, being one of the most widely used insecticides in the world. For biomonitoring in workers exposed to pesticides, the authors designed a method detecting low levels of Imidacloprid in urine of operators, based on tandem liquid mass-spectrometry with ionization source--electrostatic dispersion (positive ionization) in multi-reaction monitoring regime with subsidiary ion (mass/charge) 209 for quantitative assessment and ion 175.1 for confirmation onion ratio. The study incorporated diurnal urine, about 100 ml of average sample was frozen and kept at temperature -20C for analysis. Before extraction, the sample was unfrozen, an aliquot of 5 ml was selected, diluted with 5 ml of 0.1% formic acid. The substance was concentrated out of the urine samples via solid-phase extraction with application of cartridges based on octadecylsilane, eluition--1 ml of methanol. Lower limit of Imidacloprid detection in urine is 0.02 ng/ml, of the quantitative assessment--0.1 ng/ ml, linear range of concentrations measured 0.1-10 ng/ml. The method was tested for monitoring in workers exposed to Imidacloprid preparations in natural conditions of pesticides application in agriculture, with various processing technologies. Imidacloprid was identified in urine of two professional operators after work in seed treatment and the subsequent seeding at lower limit of detection (0.02 ng/ml) and 0.34 ng/ml.

Phenethyl isothiocyanate and indole-3-carbinol from cruciferous vegetables, but not furanocoumarins from apiaceous vegetables, reduced PhIP-induced DNA adducts in Wistar rats.

SCOPE: We previously showed that apiaceous but not cruciferous vegetables reduced DNA adducts formed by 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) in rats. Here, we report the effects of the putative chemopreventive phytochemicals from these vegetables on PhIP metabolism and DNA adduct formation. METHODS AND RESULTS: Rats received three supplemented diets: P + I (phenethyl isothiocyanate and indole-3-carbinol), furanocoumarins (FC, 5-methoxypsoralen, 8-methoxypsoralen, and isopimpinellin), and combination (P + I and FC). Phytochemical supplementation matched the levels in vegetables fed in our previous study. After 6 days, rats were injected with PhIP (10 mg/kg body wt) and killed after 24-h urine collection. Compared to the control, P + I increased activity of hepatic cytochrome P450 (CYP) 1A1 (10.1-fold), CYP1A2 (3.62-fold), and sulfotransferase 1A1 (2.70-fold). The combination diet also increased CYP1A1 and CYP1A2 activity. Urinary metabolomics revealed that PhIP metabolite profiles generally agreed with biotransformation enzyme activities. P + I and combination diets reduced PhIP-DNA adducts by 43.5 and 24.1%, respectively, whereas FC had no effect on adducts, compared to the control diet. CONCLUSION: Effects of phytochemicals on metabolic outcomes and markers of carcinogenesis might differ from fresh vegetables, thus limiting the inferences that one can draw from the effects of purified phytochemicals on the health benefits of the vegetables from which they derive.

Micellar electrokinetic chromatographic method for the dabrafenib determination in biological samples.

Two different micellar electrokinetic chromatographic methods to determine dabrafenib in urine and serum, both using borate buffer (pH 9.2, 20 mM) and SDS as separation electrolyte, are developed and validated. The analyses were carried out in a fused-silica capillary of 75 µm of internal diameter and total length of 47 and 37 cm for urine and serum determination, respectively. The detection of the target compound was performed at 227 nm in urine samples and at 251 nm in serum samples. The linearity range was from 1 to 21 mg/L of dabrafenib in urine and from 2 to 40 mg/L in serum. In all cases, inter- and intraday RSDs were <4%. Sample preparation of serum samples consists of an only step of 1:1 dilution with water before its injection in the electrophoretic system. These simple, sensitive, accurate, and cost-effective methods can be used in routine clinical practice to monitor dabrafenib concentrations in urine and serum of metastatic melanoma skin cancer patients.

Short-term effects of dietary advanced glycation end products in rats.

Dietary advanced glycation end products (AGE) formed during heating of food have gained interest as potential nutritional toxins with adverse effects on inflammation and glucose metabolism. In the present study, we investigated the short-term effects of high and low molecular weight (HMW and LMW) dietary AGE on insulin sensitivity, expression of the receptor for AGE (RAGE), the AGE receptor 1 (AGER1) and TNF-α, F2-isoprostaglandins, body composition and food intake. For 2 weeks, thirty-six Sprague-Dawley rats were fed a diet containing 20% milk powder with different proportions of this being given as heated milk powder (0, 40 or 100%), either native (HMW) or hydrolysed (LMW). Gene expression of RAGE and AGER1 in whole blood increased in the group receiving a high AGE LMW diet, which also had the highest urinary excretion of the AGE, methylglyoxal-derived hydroimidazolone 1 (MG-H1). Urinary excretion of N ε-carboxymethyl-lysine increased with increasing proportion of heat-treated milk powder in the HMW and LMW diets but was unrelated to gene expression. There was no difference in insulin sensitivity, F2-isoprostaglandins, food intake, water intake, body weight or body composition between the groups. In conclusion, RAGE and AGER1 expression can be influenced by a high AGE diet after only 2 weeks in proportion to MG-H1 excretion. No other short-term effects were observed.

Pharmacokinetics and Urinary Excretion Mechanism of Orteronel (TAK-700), A Novel 17,20-Lyase Inhibitor, in Animals.

Orteronel is newly identified as a selective 17,20-lyase inhibitor for an agent for castration resistant prostate cancer. The absorption and disposition of [(14)C]orteronel were investigated in rats and monkeys. Orteronel was extensively excreted into rat and monkey urine in an unchanged form after oral administration. The unbound based renal clearances in rats and monkeys were greater than the respective glomerular filtration rates (GFR), suggesting that urinary tubular secretion plays an important role in the renal excretion of orteronel. Therefore, the uptake of [(14)C]orteronel was investigated using rat kidney slices to estimate the contribution of carrier-mediated transport on the urinary tubular secretion. The uptake study using rat kidney slices suggested that the transport of orteronel from the blood circulation to the kidney was mediated by a digoxin sensitive transport system represented by Oatp4c1 and non-saturable components. Furthermore, the saturable component accounted for a limited fraction of the total renal uptake by rat kidney slices. These results suggested that non-saturable uptake mainly contributed to the renal excretion of orteronel in rats.

Histidine decarboxylase and urinary methylimidazoleacetic acid in gastric neuroendocrine cells and tumours.

AIM: To study histidine decarboxylase (HDC) expression in normal and neoplastic gastric neuroendocrine cells in relationship to the main histamine metabolite. METHODS: Control tissues from fundus (n = 3) and corpus (n = 3) mucosa of six patients undergoing operations for gastric adenocarcinoma, biopsy and/or gastric surgical specimens from 64 patients with primary gastric neuroendocrine tumours (GNETs), as well as metastases from 22 of these patients, were investigated using conventional immunohistochemistry and double immunofluorescence with commercial antibodies vs vesicular monoamine transporter 2 (VMAT-2), HDC and ghrelin. The urinary excretion of the main histamine metabolite methylimidazoleacetic acid (U-MeImAA) was determined using high-performance liquid chromatography in 27 of the 64 patients. RESULTS: In the gastric mucosa of the control tissues, co-localization studies identified neuroendocrine cells that showed immunoreactivity only to VMAT-2 and others with reactivity only to HDC. A third cell population co-expressed both antigens. There was no co-expression of HDC and ghrelin. Similar results were obtained in the foci of neuroendocrine cell hyperplasia associated with chronic atrophic gastritis type A and also in the tumours. The relative incidence of the three aforementioned markers varied in the tumours that were examined using conventional immunohistochemistry. All of these GNETs revealed both VMAT-2 and HDC immunoreactivity, and their metastases showed an immunohistochemical pattern and frequency similar to that of their primary tumours. In four patients, increased U-MeImAA excretion was detected, but only two of the patients exhibited related endocrine symptoms. CONCLUSION: Human enterochromaffin-like cells appear to partially co-express VMAT-2 and HDC. Co-expression of VMAT-2 and HDC might be required for increased histamine production in patients with GNETs.

Can creatine supplementation form carcinogenic heterocyclic amines in humans?

There is a long-standing concern that creatine supplementation could be associated with cancer, possibly by facilitating the formation of carcinogenic heterocyclic amines (HCAs). This study provides compelling evidence that both low and high doses of creatine supplementation, given either acutely or chronically, does not cause a significant increase in HCA formation. HCAs detection was unrelated to creatine supplementation. Diet was likely to be the main factor responsible for HCAs formation after either placebo (n = 6) or creatine supplementation (n = 3). These results directly challenge the recently suggested biological plausibility for the association between creatine use and risk of testicular germ cell cancer. Creatine supplementation has been associated with increased cancer risk. In fact, there is evidence indicating that creatine and/or creatinine are important precursors of carcinogenic heterocyclic amines (HCAs). The present study aimed to investigate the acute and chronic effects of low- and high-dose creatine supplementation on the production of HCAs in healthy humans (i.e. 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-(1,6-dimethylfuro[3,2-e]imidazo[4,5-b])pyridine (IFP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx)). This was a non-counterbalanced single-blind crossover study divided into two phases, in which low- and high-dose creatine protocols were tested. After acute (1 day) and chronic supplementation (30 days), the HCAs PhIP, 8-MeIQx, IFP and 4,8-DiMeIQx were assessed through a newly developed HPLC-MS/MS method. Dietary HCA intake and blood and urinary creatinine were also evaluated. Out of 576 assessments performed (from 149 urine samples), only nine (3 from creatine and 6 from placebo) showed quantifiable levels of HCAs (8-MeIQx: n = 3; 4,8-DiMeIQx: n = 2; PhIP: n = 4). Individual analyses revealed that diet rather than creatine supplementation was the main responsible factor for HCA formation in these cases. This study provides compelling evidence that both low and high doses of creatine supplementation, given either acutely or chronically, did not cause increases in the carcinogenic HCAs PhIP, 8-MeIQx, IFP and 4,8-DiMeIQx in healthy subjects. These findings challenge the long-existing notion that creatine supplementation could potentially increase the risk of cancer by stimulating the formation of these mutagens.

Apiaceous vegetable consumption decreases PhIP-induced DNA adducts and increases methylated PhIP metabolites in the urine metabolome in rats.

BACKGROUND: Heterocyclic aromatic amines, such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), are carcinogenic compounds produced during heating of protein-containing foods. Apiaceous vegetables inhibit PhIP-activating enzymes, whereas cruciferous vegetables induce both PhIP-activating and -detoxifying enzymes. OBJECTIVE: We investigated the effects of these vegetables, either alone or combined, on PhIP metabolism and colonic DNA adduct formation in rats. METHODS: Male Wistar rats were fed cruciferous vegetables (21%, wt:wt), apiaceous vegetables (21%, wt:wt), or a combination of both vegetables (10.5% wt:wt of each). Negative and positive control groups were fed an AIN-93G diet. After 6 d, all groups received an intraperitoneal injection of PhIP (10 mg · kg body weight(-1)) except for the negative control group, which received only vehicle. Urine was collected for 24 h after the injection for LC-tandem mass spectrometry metabolomic analyses. On day 7, rats were killed and tissues processed. RESULTS: Compared with the positive control, cruciferous vegetables increased the activity of hepatic PhIP-activating enzymes [39.5% and 45.1% for cytochrome P450 (CYP) 1A1 (P = 0.0006) and CYP1A2 (P < 0.0001), respectively] and of uridine 5'-diphospho-glucuronosyltransferase 1A (PhIP-detoxifying) by 24.5% (P = 0.0267). Apiaceous vegetables did not inhibit PhIP-activating enzymes, yet reduced colonic PhIP-DNA adducts by 20.4% (P = 0.0496). Metabolomic analyses indicated that apiaceous vegetables increased the relative abundance of urinary methylated PhIP metabolites. The sum of these methylated metabolites inversely correlated with colonic PhIP-DNA adducts (r = -0.43, P = 0.01). We detected a novel methylated urinary PhIP metabolite and demonstrated that methylated metabolites are produced in the human liver S9 fraction. CONCLUSIONS: Apiaceous vegetables did not inhibit the activity of PhIP-activating enzymes in rats, suggesting that the reduction in PhIP-DNA adducts may involve other pathways. Further investigation of the importance of PhIP methylation in carcinogen metabolism is warranted, given the inverse correlation of methylated PhIP metabolites with a biomarker of carcinogenesis and the detection of a novel methylated PhIP metabolite.

Diminished reliability of tryptase as risk indicator of mastocytosis in older overweight subjects.

BACKGROUND: Currently, measurement of serum tryptase level is the most commonly used test to estimate the need for bone marrow biopsy in patients suspected to have indolent systemic mastocytosis (ISM). Yet tryptase levels do not solely reflect the mast cell load and can be elevated by overweight, older age, and impaired renal function. The influence of these factors on urinary methylhistamine (MH) and methylimidazole acetic acid (MIMA) is still unknown. OBJECTIVE: We investigated the impact of age, body mass index (BMI), and kidney function on the diagnostic accuracy of tryptase, MH, and MIMA to select the most optimal test indicating the necessity of a bone marrow biopsy in ISM-suspected patients. METHODS: Retrospective data analysis of all adults in whom bone marrow investigations were performed because of high clinical suspicion and/or elevated tryptase, MH, or MIMA. RESULTS: 194 subjects were included. ISM was present in 112 and absent in 82 subjects (non-ISM). Tryptase was elevated by age and body weight in non-ISM subjects and by BMI in ISM subjects; however, these factors did not influence MH or MIMA. In the total study population, the diagnostic accuracy of tryptase, MH, and MIMA were comparable (area under the curve 0.80, 0.80, and 0.83). In subjects >50 years with a BMI >25 kg/m(2), the diagnostic accuracy of MIMA was higher compared with that of tryptase (area under the curve 0.93 vs 0.74; P = .011). CONCLUSION: In ISM-suspected patients >50 years with a BMI of >25 kg/m(2), MIMA has a greater value compared with tryptase in estimating the need for bone marrow biopsy.

Etamicastat, a new dopamine-ß-hydroxylase inhibitor, pharmacodynamics and metabolism in rat.

Despite the importance of sympathetic nervous system in pathophysiological mechanisms of cardiac heart failure and essential hypertension, therapy specifically targeting the sympathetic nervous system is currently underutilized. Etamicastat is a novel dopamine-ß-hydroxylase (DBH) inhibitor that is oxidized into BIA 5-965 and deaminated followed by oxidation to BIA 5-998, which represents 13% of total etamicastat and quantified metabolites. However, the primary metabolic pathway of etamicastat in rats was found to be the N-acetylation (BIA 5-961), which represents 44% of total etamicastat and quantified metabolites. Trace amounts of BIA 5-961 de-sulfated and S-glucuronide were also detected. All the main metabolites of etamicastat inhibited DBH with IC50 values of 306 (228, 409), 629 (534, 741), 427 (350, 522) nM for BIA 5-965, BIA 5-998 and BIA 5-961, respectively. However, only etamicastat (IC50 of 107 (94; 121) nM) was able to reduce catecholamine levels in sympathetic nervous system innervated peripheral tissues, without effect upon brain catecholamines. Quantitative whole body autoradiography revealed a limited transfer of etamicastat related radioactivity to brain tissues and the mean recovery of radioactivity was ~90% of the administered radioactive dose, eliminated primarily via renal excretion over 5 days. The absolute oral bioavailability of etamicastat was 64% of the administered dose. In conclusion, etamicastat is a peripheral selective DBH inhibitor mainly N-acetylated in the aminoethyl moiety and excreted in urine. Etamicastat main metabolites inhibit DBH, but only etamicastat demonstrated unequivocal pharmacological effects as a DBH inhibitor with impact upon the activity of the sympathetic nervous system under in vivo conditions.

Higher mast cell load decreases the risk of Hymenoptera venom-induced anaphylaxis in patients with mastocytosis.

BACKGROUND: Increased basal serum tryptase (bsT) levels are a well-described risk factor for Hymenoptera venom-induced anaphylaxis (HVAn) in patients allergic to Hymenoptera venom. Increased bsT levels might also indicate the presence of mastocytosis. In this study we evaluated whether the risk of HVAn increases with increasing mast cell load in patients with mastocytosis. METHODS: Consecutive patients with different subtypes of mastocytosis (n = 329) admitted to the University Medical Center Groningen were retrospectively assessed. As markers for mast cell load, levels of both bsT and the urinary histamine metabolites methylhistamine and methylimidazole acetic acid (MIMA) were used. RESULTS: In the entire patient group, irrespective of disease subtype and Hymenoptera venom exposure, HVAn prevalence gradually increased with increasing marker levels to a maximum of 36% to 47% at a bsT level of 28.0 µg/L, a methylhistamine level of 231.0 µmol/mol creatinine, and a MIMA level of 2.7 mmol/mol creatinine but decreased thereafter with a further increase in these levels. In patients with indolent systemic mastocytosis with a history of Hymenoptera venom exposure after age 15 years or greater (n = 152), MIMA and age at the most recent Hymenoptera sting were independent predictors for HVAn (odds ratios of 0.723 [P = .001] and 1.062 [P < .001], respectively). CONCLUSIONS: In patients with mastocytosis, HVAn prevalence does not increase constantly with increasing levels of mast cell load parameters: after a gradual increase to a maximum of near 50%, it decreases with a further increase in these levels. In the indolent systemic mastocytosis population, all mast cell load markers were independent negative predictors of HVAn. These findings suggest a complex pathophysiologic association between mast cell load and HVAn risk in patients with mastocytosis.

Biomonitoring of dietary heterocyclic amines and metabolites in urine by liquid phase microextraction: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a possible biomarker of exposure to dietary PhIP.

Heterocyclic amines (HCAs) are mutagenic/carcinogenic compounds formed at ng/g levels during frying meat or fish. The effect of the normal intake of dietary HCAs in humans and their involvement in the etiology of cancer are currently unknown. In this work, a new extraction method, liquid phase microextraction (LPME) with hollow fibers, and LC-MS/MS have been used for the first time to determine HCAs and metabolites in nonspiked human urine following a single meal of chicken cooked at 180 °C for 6 min. The total intake of HCAs was estimated to be 6 µg, of which 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) accounted for about 1 µg. The concentrations of PhIP in nonhydrolyzed urine samples ranged from 11.7 to 59.4 pg/g. The total amount of PhIP in urine ranged between 9.3 and 21.1 ng, which corresponds to 0.91-2.1% of the ingested PhIP. In addition, the urine levels of 4'-OH-PhIP (2-amino-1-methyl-6-(4'-hydroxy)phenylimidazo[4,5-b]pyridine) and 5-OH-PhIP (2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine) also showed a narrow variation between the samples. The analysis of urine samples after acid hydrolysis did not give additional information but showed a notable increase in norharman in some cases. The obtained results suggest PhIP in urine as a possible biomarker of exposure to HCAs and the LPME and LC-MS/MS method as an appropriate strategy to biomonitor HCAs in urine.

Impact of aqueous doash extract on urinary mutagenicity in rats exposed to heterocyclic amines.

Doash (Origanum majorana) is an herbaceous plant found commonly in Saudi Arabia. It is used as a food flavor and a folk remedy to treat a number of diseases. The 2-amino-3-methylimidazo[4,5-f] quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are the most abundant of the heterocyclic amine carcinogens present in cooked food. In the present study, the potential of doash tea to influence carcinogen metabolism was investigated indirectly using heterocyclic amines as model mutagens, IQ and PhIP. Results obtained showed that doash tea had no influence on body weight in both the studies. Rats were treated with different doses of IQ (1, 3, 5 and 10 mg/kg) or PhIP (1, 5, 10 and 20 mg/kg). The selected dosage was 5 mg/kg for both heterocyclic amines. Results obtained revealed that rats treated with doash tea and given a single dose of the heterocyclic amines, whether for 1 day (short-term) or for 1 month (long term), showed a statistically significant decrease in their excretion of indirect mutagens (IQ or PhIP). Following treatment of the rats with a single oral dose of IQ or PhIP, the highest mutagenic activity determined in the presence of an activation system was excreted in the urine after 24 h, with much lower levels of mutagencity being excreted during subsequent elimination from the body. No mutagenicity was observed in the absence of an activation system that is direct-acting mutagenicity using (IQ and PhIP). Statistical analysis revealed that, in comparison with the control group, the aqueous doash extract significantly reduced the mutagenic response after 24 h. It was concluded that doash extract significantly decreased the excretion of mutagens in comparison with the control group (water only).

Comparability and repeatability of methods for estimating the dietary intake of the heterocyclic amine contaminant 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP).

Inconsistent risk estimates for dietary heterocyclic amine (HCA) exposure and cancers may be due to differences in exposure assessment methods and the associated measurement error. We evaluated repeatability and comparability of intake estimates of the HCA 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP) among two food frequency questionnaire (FFQ) collections, three diary collections, and three measurements of urinary PhIP and its metabolites in 36 non-smokers in Baltimore, Maryland, during 2004-2005. Collections spanned ∼9 months. Method repeatability was characterised with intraclass correlation coefficients (ICCs). Comparability among methods was assessed with Spearman correlation coefficients. Within-subject variability in PhIP intake was comparably high across all methods (ICCs of 0.20, 0.30, and 0.15 for FFQ, diary, and creatinine-adjusted urinary PhIP, respectively). Mean diary-based PhIP intake and mean urinary PhIP concentration were strongly correlated when restricting the analysis to participants with at least one non-zero diary-based estimate of PhIP intake (n = 15, r = 0.75, p = 0.001), but not in the full study population (n = 36, r = 0.18, p = 0.28). Mean PhIP intake from the FFQ was not associated with that either based on the diary or urinary PhIP separately, but was modestly correlated with a metric that combined the diary- and biomarker-based approaches (r = 0.30, p = 0.08). The high within-subject variability will result in significantly attenuated associations if a single measure is used to estimate exposure within an epidemiologic study. Improved HCA assessment tools, such as a combination of methods or validated biomarkers that capture long term exposure, are needed.

Pharmacokinetics, distribution and excretion of YM155 monobromide, a novel small-molecule survivin suppressant, in male and pregnant or lactating female rats.

YM155 monobromide is a novel small-molecule survivin suppressant. The pharmacokinetics, distribution and excretion of YM155/[14C]YM155 were investigated using males and pregnant or lactating female rats after a single intravenous bolus administration. For the 0.1, 0.3 and 1 mg/kg YM155 doses given to male rats, increases in area under the plasma concentration-time curves were approximately proportional to the increase in the dose level. After administering [14C]YM155, radioactivity concentrations in the kidney and liver were highest among the tissues in both male and pregnant rats: e.g. 14.8- and 5.24-fold, respectively, and higher than in plasma at 0.1 h after dosing to male rats. The YM155 concentrations in the brain were lowest: 25-fold lower than in plasma. The transfer of radioactivity into fetuses was low (about 2-fold lower than in plasma). In lactating rats, the radioactivity was transferred into milk at a level 8- to 21-fold higher than for plasma. Radioactivity was primarily excreted in feces (64.0%) and urine (35.2%). The fecal excretion was considered to have occurred mainly by biliary excretion and partly by secretion across the gastrointestinal membrane from the blood to the lumen.