Assessment of urinary metabolite excretion after rat acute exposure to perfluorooctanoic acid and other peroxisomal proliferators.

Perfluorooctanoic acid (PFOA) is a persistent environmental contaminant. Activation of the peroxisome proliferator activated receptor alpha (PPARα) resulting from exposure to PFOA has been extensively studied in rodents. However, marked differences in response to peroxisome proliferators prevent extrapolation of rodent PPARα activation to human health risks and additional molecular mechanisms may also be involved in the biological response to PFOA exposure. To further explore the potential involvement of such additional pathways, the effects of PFOA exposure on urinary metabolites were directly compared with those of other well-known PPARα agonists. Male rats were administered PFOA (10, 33, or 100 mg/kg/d), fenofibrate (100 mg/kg/d), or di(2-ethylhexyl) phthalate (100 mg/kg/d) by gavage for 3 consecutive days and allowed to recover for 4 days, and overnight urine was collected. Greater urinary output was observed exclusively in PFOA-treated rats as the total fraction of PFOA excreted in urine increased with the dose administered. Assessment of urinary metabolites (ascorbic acid, quinolinic acid, 8-hydroxy-2'-deoxyguanosine, and malondialdehyde) provided additional information on PFOA's effects on hepatic glucuronic acid and tryptophan-nicotinamide adenine dinucleotide (NAD) pathways and on oxidative stress, whereas increased liver weight and palmitoyl-CoA oxidase activity indicative of PPARα activation and peroxisomal proliferation persisted up to day five after the last exposure.

Urinary globotriaosylsphingosine-related biomarkers for Fabry disease targeted by metabolomics.

Fabry disease is a lysosomal storage disorder caused by deficiency of α-galactosidase A, resulting in glycosphingolipid accumulation in organs and tissues, including plasma and urine. Two disease-specific Fabry biomarkers have been identified and quantified in plasma and urine: globotriaosylceramide (Gb(3)) and globotriaosylsphingosine (lyso-Gb(3)). The search continues for biomarkers that might be reliable indicators of disease severity and response to treatment. The main objective of this study was to target other urinary biomarkers using a time-of-flight mass spectrometry metabolomic approach. Urinary metabolites of 63 untreated Fabry patients and 59 controls were analyzed. A multivariate statistical analysis performed on a subset of male samples revealed seven novel Fabry biomarkers in urine, all lyso-Gb(3) analogues having modified sphingosine moieties. The empirical formulas of the sphingosine modifications were determined by exact mass measurements (- C(2)H(4), - C(2)H(4) + O, - H(2), - H(2) + O, + O, + H(2)O(2), + H(2)O(3)). We evaluated the relative concentration of lyso-Gb(3) and its seven analogues by measuring area counts for each analogue in all Fabry patients. All samples were normalized to creatinine. We found higher concentrations for males with Fabry disease compared to females. None of these biomarkers were detected in controls. To our knowledge, this is the first time that lyso-Gb(3)-related Fabry disease biomarkers are detected in urine.

Determination of glycated and acetylated hemoglobins in cord blood by time-of-flight mass spectrometry.

The characterization of cord blood hemoglobin at the molecular level is a daunting challenge because hemoglobin F (HbF) and hemoglobin A (HbA) coexist in neonatal blood. We developed and validated a method using electrospray time-of-flight mass spectrometry (ES-TOF-MS) that measures, in a single analysis, relative levels of glycated and acetylated hemoglobin and allows the calculation of relative proportions of HbA, HbF(0), and HbF(1) in cord blood. Specific sections of acquired spectra were deconvoluted using a maximum entropy-based approach to true mass scale spectra. Mass precisions were less than 3 ppm with similar accuracies. Intra-interday precisions for α- and γ-chain glycation levels were 2.10%/3.72% and 2.75%/6.79%, respectively. The linearity of the α-chain glycation response was excellent (r(2) = 0.9990). We performed sample analysis on 39 cord blood specimens and found that the glycated α- and γ-chain levels were 2.27 ± 0.21% and 2.38 ± 0.29%, respectively, while the acetylated (G)γ and (A)γ-chain levels were 8.48 ± 0.53% and 7.14 ± 0.74%, respectively. We observed three types of HbF distinguishable by the intensities of γ-chain variants. Two-thirds of cord blood specimens were classified as HbF(I) with an intensity ratio (G)γ/(A)γ of 1.90 ± 0.12. For HbF(II) type (10/39 neonates), the intensity ratio of (G)γ/(A)γ was 3.71 ± 0.28. For three neonates with HbF(III), no (A)γ-chain was detected.

Efficient analysis of urinary glycosaminoglycans by LC-MS/MS in mucopolysaccharidoses type I, II and VI.

Mucopolysaccharidoses (MPSs) are complex storage disorders caused by specific lysosomal enzyme deficiencies, resulting in the accumulation of glycosaminoglycans (GAGs) in urine, plasma, as well as in various tissues. We devised and validated a straightforward, but accurate and precise tandem mass spectrometry methodology coupled to high performance liquid chromatography (LC-MS/MS) for the quantification of GAGs in urine. The method is applicable to the investigation of patients with MPS I, II, and VI, by quantifying dermatan sulfate (DS) and heparan sulfate (HS) in urine. We analyzed urine samples from 28 MPS patients, aged 1 to 42 years, and 55 control subjects (41 days to 18 years old). Levels of DS and HS in urine from healthy controls of all ages were below the limit of quantification. The levels of DS and HS in urine from 6 treated patients with MPS I were lower than in 6 untreated patients in DS (0.7-45 vs 9.3-177 mg/mmol creat) and HS (0-123 mg/mmol creatinine vs 38-418 mg/mmol creatinine); similar results were obtained for 9 patients with MPS II and 7 patients with MPS VI. Analyses were performed on as little as 250 µL of urine. Methanolysis took 75 min per sample; the total analysis run time for each LC-MS/MS injection was 8 min. Results indicate that the method is applicable to a wide variety of situations in which high accuracy and precision are required, including the evaluation of the effectiveness of existing and emerging treatments.

Ghrelin agonist (TZP-101): safety, pharmacokinetics and pharmacodynamic evaluation in healthy volunteers: a phase I, first-in-human study.

The authors evaluate the human safety, tolerability, pharmacokinetics, and pharmacodynamics of TZP-101, an agonist of the hGHS-R1a (ghrelin) receptor. Healthy subjects were randomized to either single-dose TZP-101 (20-600 microg/kg) or placebo by 30-minute intravenous infusion. Subjects underwent continuous cardiac monitoring, 12-lead electrocardiograms, and assessment for orthostatic hypotension, injection site tolerability, vital signs, and adverse events during the 24-hour postdose period. Blood and urine samples were collected for pharmacokinetic/pharmacodynamic assessment for 24 hours. Forty-eight subjects randomly received 1 of 6 TZP-101 doses or placebo. TZP-101 was well tolerated, with single episodes each of headache, lower abdominal pain, diarrhea, and dizziness. At the highest dose, 2 subjects experienced bradycardia. All events were self-limited. Mean arterial blood pressure and heart rate decreased from baseline approximately 45 to 60 minutes after infusion start at higher doses. No other significant changes were observed. Pharmacokinetic analysis revealed less than dose-proportional behavior of drug with low clearance (approximately 7 mL/h/kg), small volume of distribution (approximately 114 mL/kg), and half-life values of approximately 13 hours, which were independent of dose. Pharmacodynamic analyses suggested TZP-101, at doses as low as 40 microg/kg, expressed activity at the receptor. TZP-101 displayed a promising pharmacokinetic, pharmacodynamic, and safety profile for use in gastrointestinal motility disorders.

Efficient parallel synthesis of macrocyclic peptidomimetics.

A new method for solid phase parallel synthesis of chemically and conformationally diverse macrocyclic peptidomimetics is reported. A key feature of the method is access to broad chemical and conformational diversity. Synthesis and mechanistic studies on the macrocyclization step are reported.

Identification of the hydroxamate siderophore ferricrocin in Cladosporium cladosporioides.

The hydroxamate siderophore ferricrocin was identified in Cladosporium cladosporioides growth medium by solid phase extraction and ultra high pressure liquid chromatography coupled to a time of flight mass spectrometer (UHPLC/QTOF-MS). Both desferricrocin and ferricrocin were detected in the extracellular medium assisted by high resolution mass spectrometry. This is the first identification of a hydroxamate siderophore in Cladosporium cladosporioides. This finding emphasizes the common meaning of ferricrocin in fungi.

LC-MS/MS analysis of plasma lyso-Gb3 in Fabry disease.

BACKGROUND: Fabry disease is a complex, multisystemic and clinically heterogeneous disease, with elevated excretion of globotriaosylceramide (Gb(3)) and globotriaosylsphingosine (lyso-Gb(3)) accumulating in biological fluids caused by deficiency of the enzyme, lysosomal α-galactosidase A. Our aims were to propose a tandem mass spectrometry fragmentation mechanism for lyso-Gb(3), to develop and validate a simple, and robust methodology for the measurement of plasma lyso-Gb(3) using LC-MS/MS in large Fabry cohorts and in controls. Response to treatment was also evaluated. METHOD: A solid-phase extraction procedure was used to process plasma samples. The 1-ß-D-glucosylsphingosine (GSG) internal standard was chosen for its commercial availability. A liquid chromatography method was devised to allow the co-elution of the GSG internal standard with lyso-Gb(3), thus compensating for system variability and reducing the matrix effect. A multiple reaction monitoring method was developed, working in positive electrospray ionization. RESULTS: The validation of the method provided good accuracy and precision: intraday and interday biases of less than 8% and 5%, respectively, and intraday and interday CVs of <12% and 7%, respectively. Limit of detection was 0.7 nmol/l and limit of quantification was 2.5 nmol/l. Plasma samples were stable for up to 6h at room temperature, 48 h at 4 °C, and 20 weeks at -20 °C. Regarding untreated Fabry patients, the mean lyso-Gb(3) concentrations were 170 nmol/l for males and 9.7 nmol/l for females, and for treated patients, 40.2 nmol/l for males and 7.5 nmol/l for females. CONCLUSION: A robust LC-MS/MS methodology is presented for plasma lyso-Gb(3) quantification.

Effect of alipogene tiparvovec (AAV1-LPL(S447X)) on postprandial chylomicron metabolism in lipoprotein lipase-deficient patients.

BACKGROUND: Lipoprotein lipase-deficient (LPLD) individuals display marked chylomicronemia and hypertriglyceridemia associated with increased pancreatitis risk. The aim of this study was to determine the effect of i.m. administration of an adeno-associated viral vector (AAV1) for expression of LPL(S447X) in muscle (alipogene tiparvovec, AAV1-LPL(S447X)) on postprandial chylomicron metabolism and on nonesterified fatty acid (NEFA) and glycerol metabolism in LPLD individuals. METHODOLOGY: In an open-label clinical trial (CT-AMT-011-02), LPLD subjects were administered alipogene tiparvovec at a dose of 1 × 10(12) genome copies per kilogram. Two weeks before and 14 wk after administration, chylomicron metabolism and plasma palmitate and glycerol appearance rates were determined after ingestion of a low-fat meal containing (3)H-palmitate, combined with (continuous) iv infusion of [U-(13)C]palmitate and [1,1,2,3,3-(2)H]glycerol. PRINCIPAL FINDINGS: After administration of alipogene tiparvovec, the triglyceride (TG) content of the chylomicron fraction and the chylomicron-TG/total plasma TG ratio were reduced throughout the postprandial period. The postprandial peak chylomicron (3)H level and chylomicron (3)H area under the curve were greatly reduced (by 79 and 93%, 6 and 24 h after the test meal, respectively). There were no significant changes in plasma NEFA and glycerol appearance rates. Plasma glucose, insulin, and C-peptide also did not change. CONCLUSIONS/SIGNIFICANCE: Intramuscular administration of alipogene tiparvovec resulted in a significant improvement of postprandial chylomicron metabolism in LPLD patients, without inducing large postprandial NEFA spillover.

An improved method for glycosaminoglycan analysis by LC-MS/MS of urine samples collected on filter paper.

BACKGROUND: Mucopolysaccharidoses are complex lysosomal storage disorders caused by any of eleven different enzyme deficiencies resulting in the accumulation of substrates, mainly glycosaminoglycans (GAGs), in various tissues and biological fluids. METHOD: We developed and validated a urine filter paper methodology for the analysis of GAGs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for mucopolysaccharidoses type I, type II and type VI patients. We focused on 2 objectives: first, its applicability to high-risk screening, and secondly, to facilitate the collection and shipping of samples to reference centers as part of diagnostic investigation, as well as from treated patients needing to be monitored for assessment of the efficacy of treatment. GAGs in urine dried onto filter paper were extracted and subjected to methanolysis to obtain the repeating disaccharides of the molecules. We devised a multiple reaction monitoring method in positive electrospray ionization mode. RESULTS: The use of deuterated internal standards for dermatan sulfate (DS) and heparan sulfate (HS) reduced a troubling matrix effect. The resulting CVs were <14%. Linearity assessment showed Pearson correlation coefficients of 0.999 and 0.997, for DS and HS, respectively. The stability on filter paper was good for DS and HS for up to 6 weeks at various temperatures. CONCLUSION: We devised a robust and efficient LC-MS/MS methodology for GAGS quantification in urine dried on filter paper and subjected to environmental conditions likely to be encountered during collection, storage and shipping of specimens from referring physicians to medical centers.

A deficit in peripheral serotonin levels in major depressive disorder but not in chronic widespread pain.

OBJECTIVES: It has been proposed that serotonin dysfunctions underlie the pathophysiology of various mood disorders (including major depressive disorder, MDD) and chronic pain conditions characterized by deficient pain inhibition, such as fibromyalgia (FM). There is reliable data showing that serotonin disturbances are involved in the pathophysiology of MDD. However, in the case of FM, results published so far are less consistent. Therefore, the current cross-sectional study sought to measure plasma serotonin levels in FM patients, MDD patients, and healthy controls (HC). METHODS: Twenty-nine FM patients, 17 MDD patients, and 57 HC were recruited who did not differ in terms of age, sex, and the presence or absence of a regular menstrual cycle. Plasma samples were analysed with mass spectrometry. RESULTS: Serotonin levels were decreased in MDD patients, relative to FM patients and HC. Post hoc analyses showed that serotonin levels were decreased in FM patients taking antidepressants, relative to HC, but not in drug-free FM patients. Moreover, serotonin levels were negatively correlated with mood symptoms across groups. DISCUSSION: Our results further confirm that MDD is associated with decreased serotonin levels, but that serotonin levels are not altered in FM per se, and suggest that 5-Hydroxytryptamine is related to mood symptoms in these patient groups. Our results also suggest that the taking of antidepressant is a major confound to consider when studying serotonin functioning in FM. The long-term use of antidepressants in FM may lead to serotonin depletion. Conversely, serotonin depletion may be before the taking of antidepressants in FM.

Optimization of the potency and pharmacokinetic properties of a macrocyclic ghrelin receptor agonist (Part I): Development of ulimorelin (TZP-101) from hit to clinic.

High-throughput screening of Tranzyme Pharma's proprietary macrocycle library using the aequorin Ca2+-bioluminescence assay against the human ghrelin receptor (GRLN) led to the discovery of novel agonists against this G-protein coupled receptor. Early hits such as 1 (Ki=86 nM, EC50=134 nM) though potent in vitro displayed poor pharmacokinetic properties that required optimization. While such macrocycles are not fully rule-of-five compliant, principally due to their molecular weight and clogP, optimization of their pharmacokinetic properties proved feasible largely through conformational rigidification. Extensive SAR led to the identification of 2 (Ki=16 nM, EC50=29 nM), also known as ulimorelin or TZP-101, which has progressed to phase III human clinical trials for the treatment of postoperative ileus. X-ray structure and detailed NMR studies indicated a rigid peptidomimetic portion in 2 that is best defined as a nonideal type-I' ß-turn. Compound 2 is 24% orally bioavailable in both rats and monkeys. Despite its potency, in vitro and in gastric emptying studies, 2 did not induce growth hormone (GH) release in rats, thus demarcating the GH versus GI pharmacology of GRLN.

Increased postprandial nonesterified fatty acid appearance and oxidation in type 2 diabetes is not fully established in offspring of diabetic subjects.

BACKGROUND: It has been proposed that abnormal postprandial plasma nonesterified fatty acid (NEFA) metabolism may participate in the development of tissue lipotoxicity and type 2 diabetes (T2D). We previously found that non-diabetic offspring of two parents with T2D display increased plasma NEFA appearance and oxidation rates during intravenous administration of a fat emulsion. However, it is currently unknown whether plasma NEFA appearance and oxidation are abnormal during the postprandial state in these subjects at high-risk of developing T2D. METHODOLOGY: Palmitate appearance and oxidation rates and glycerol appearance rate were determined in eleven healthy offspring of two parents with T2D (positive family history, FH+), 13 healthy subjects without first-degree relatives with T2D (FH-) and 12 subjects with T2D at fasting, during normoglycemic hyperinsulinemic clamp and during continuous oral intake of a standard liquid meal to achieve steady postprandial NEFA and triacylglycerols (TG) without and with insulin infusion to maintain similar glycemia in all three groups. PRINCIPAL FINDINGS: Plasma palmitate appearance and oxidation were higher at fasting and during the clamp conditions in the T2D group (all P<0.05). In the postprandial state, palmitate appearance, oxidative and non oxidative rates were all elevated in T2D (all P<0.05) but not in FH+. Both T2D and FH+ displayed elevated postprandial TG vs. FH- (P<0.001). Acute correction of hyperglycemia during the postprandial state did not affect these group differences. Increased waist circumference and BMI were positively associated with elevated postprandial plasma palmitate appearance and oxidation. CONCLUSIONS/SIGNIFICANCE: Postprandial plasma NEFA intolerance observed in subjects with T2D is not fully established in non-diabetic offspring of both parents with T2D, despite the presence of increased postprandial plasma TG in the later. Elevated postprandial plasma NEFA appearance and oxidation in T2D is observed despite acute correction of the exaggerated glycemic excursion in this group.

How well does urinary lyso-Gb3 function as a biomarker in Fabry disease?

BACKGROUND: Fabry disease is characterized by accumulation of glycosphingolipids, such as globotriaosylceramide (Gb(3)), in many tissues and body fluids. A novel plasma biomarker, globotriaosylsphingosine (lyso-Gb(3)), is increased in patients with the disease. Until now, lyso-Gb(3) was not detectable in urine, possibly because of the presence of interfering compounds. METHODS: We undertook to: 1) characterize lyso-Gb(3) in urine; 2) develop a method to quantitate urinary lyso-Gb(3) by mass spectrometry; 3) evaluate urinary lyso-Gb(3) as a potential biomarker for Fabry disease; and 4) determine whether lyso-Gb(3) is an inhibitor of α-galactosidase A activity. We analyzed urinary lyso-Gb(3) from 83 Fabry patients and 77 healthy age-matched controls. RESULTS: The intraday and interday bias and precision of the method were <15%. Increases in lyso-Gb(3)/creatinine correlated with the concentrations of Gb(3) (r(2)=0.43), type of mutations (p=0.0006), gender (p<0.0001) and enzyme replacement therapy status (p=0.0012). Urine from healthy controls contained no detectable lyso-Gb(3). Lyso-Gb(3) did not inhibit GLA activity in dried blood spots. Increased urinary excretion of lyso-Gb(3) of Fabry patients correlated well with a number of indicators of disease severity. CONCLUSION: Lyso-Gb(3) is a reliable independent biomarker for clinically important characteristics of Fabry disease.