The fraction of nitric oxide in exhaled gas (FENO) is decreased after exposure to hyperoxia in vivo, although the mechanisms for this decrease is not clear. A key co-factor for nitric oxide synthase (NOS), tetrahydrobiopterin (BH4), has been shown to be oxidized in vitro when exposed to hyperoxia. We hypothesized that the decrease of FENO is due to decreased enzymatic generation of NO due to oxidation of BH4. The present study was performed to investigate the relationship between levels of FENO and plasma BH4 following hyperoxic exposure in humans. Two groups of healthy subjects were exposed to 100% oxygen for 90 minutes. FENO was measured before and 10 minutes (n = 13) or 60 minutes (n = 14) after the exposure. Blood samples were collected at the same time points for quantification of biopterin levels (BH4, BH2 and B) using LC-MS/MS. Each subject was his or her own control, breathing air for 90 minutes on a separate day. Hyperoxia resulted in a 28.6 % decrease in FENO 10 minutes after exposure (p < 0.001), confirming previous findings. Moreover, hyperoxia also caused a 14.2% decrease in plasma BH4 (p = 0.012). No significant differences were observed in the group measured 60 minutes after exposure. No significant correlation was found between the changes in FENO and BH4 after the hyperoxic exposure (r = 0.052, p = 0.795), this might be due to the recovery of BH4 being faster than the recovery of FENO.

Nitric oxide (NO) may protect against gas bubble formation and risk of decompression sickness. We have previously shown that the crucial co-factor tetrahydrobiopterin (BH4) is oxidized in a dose-dependent manner when exposed to hyperoxia similar to diving conditions but with minor effects on the NO production by nitric oxide synthase. By manipulating the intracellular redox state, we further investigated the relationship between BH4 levels and production of NO in human endothelial cells (HUVECs). HUVECs were cultured with and without ascorbic acid (AA) and the glutathione (GSH) synthesis inhibitor buthionine sulfoximine, prior to hyperoxic exposure. The levels of biopterins and GSH were determined in cell lysates while the production of NO was determined in intact cells. Omitting AA resulted in a 91% decrease in BH4 levels (0.49 ± 0.08 to 0.04 ± 0.01 pmol/10⁶ cells, p⟨0.001) at 20 kPa oxygen (O2), and 88% decrease (0.24 ± 0.03 to 0.03 ± 0.01 pmol/10⁶ cells, p=0.01) after exposure to 60 kPa O2. The NO generation was decreased by 23% (74.5 ± 2.2 to 57.3 ± 5.6 pmol/min/mg protein, p⟨0.001) at 20 kPa O2, but no significant change was observed at 60 kPa O2. GSH depletion had no effects on the NO generation. No correlation was found between NO generation and the corresponding intracellular BH4 concentration (p=0.675, r=-0.055) or the BH4 to BH2 ratio (p=0.983, r=0.003), determined across 18 in vitro experiments. Decreased BH4 in HUVECs, due to hyperoxia or lack of ascorbic acid, does not imply corresponding decreases in NO generation.

Purpose:Nitric oxide (NO) has been shown to protect against bubble formation and the risk of decompression sickness. We hypothesize that oxidation of tetrahydrobiopterin (BH4) leads to a decreased production of NO during simulated diving.Methods:Human umbilical vein endothelial cells (HUVEC) were exposed to hyperoxia or simulated diving for 24 hours. The levels of biopterins (BH4, BH2 and B) were determined by LC-MS/MS, and the production of NO by monitoring the conversion of L-arginine to L-citrulline.Results:Exposure to hyperoxia decreased BH4 in a dose-dependent manner; by 48 ± 15% following exposure to 40 kPa O2 (P⟨0.001 vs. control at 20 kPa O2), and 70 ± 16% following exposure to 60 kPa O2. Exposure to 40 kPa O2 decreased NO production by 25 ± 9%, but there was no further decrease when increasing oxygen exposure to 60 kPa (25 ± 10%). No additional effects of simulated diving were observed, indicating no additive or synergistic effects of hyperbaria and hyperoxia on the BH4 level or NO generation.Conclusion:NO generation in intact human endothelial cells was decreased by simulated diving, as well as by hyperoxic exposure, while BH4 levels seem to be affected only by hyperoxia. Hence, the results suggest that BH4 is not the sole determinant of NO generation in HUVEC.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) and metabolite (2',2'-difluoro-2'-deoxyuridine, dFdU) quantification is warranted for individualized treatment strategies. Analyte stability is crucial for the validity of such quantification. We therefore studied the impact of the time interval from blood sampling to separation of plasma on gemcitabine stability. Blood from gemcitabine-treated patients was drawn into tetrahydrouridine (THU)-spiked heparin and ethylenediaminetetraacetic acid tubes and kept on ice until separation. Plasma was separated sequentially up to 24 h after sampling and dFdC and dFdU were quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). The change in plasma concentrations over time was compared with the highest imprecision for concentrations above the lower limit of quantification of the LC-MS/MS method. Analyte concentrations decreased slightly over time, but for samples stored for 4 h on ice, the decline was smaller than the expected analytical imprecision. After 24 h, the maximum decline was 14.0%, which exceeded the expected analytical imprecision. dFdC and dFdU stabilities were acceptable for at least 4 h when THU-spiked whole blood samples were kept on ice. This is within the scope of routine sampling procedures. Further, variations in separation time intervals within this time frame are negligible when interpreting drug concentrations.

Quantification of endogenous nucleotides is of interest for investigation of numerous cellular biochemical processes, such as energy metabolism and signal transduction, and may also be applied in cancer and antiretroviral therapies in which nucleoside analogues are used. For these purposes we developed and validated a sensitive and high accuracy ion-pair liquid chromatography tandem mass spectrometry (IP LC-MS/MS) method for simultaneous quantification of eight endogenous nucleotides (ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, dTTP) and 2',2'-difluoro-2'-deoxycytidine triphosphate (dFdCTP), an intracellular metabolite of the nucleoside analogue gemcitabine. The assay was validated using 200μL aliquots of peripheral blood mononuclear cell (20×10(6)cells/ml, 4×10(6)cells) extracts, pretreated with activated charcoal and spiked with unlabeled nucleotides, deoxynucleotides and dFdCTP. Analytes were extracted by simple precipitation with cold 60% methanol containing isotope labeled internal standards and separated on a porous graphitic carbon column. For method validation, the concentration ranges were: 0.125-20.8pmol injected for deoxynucleotides, 0.25-312.5pmol injected for dFdCTP and 5-3200pmol injected for nucleotides. The highest coefficients of variation (CV) were 12.1% for within run assay and 11.4% for between run assay, both representing the precision at the lowest analyte concentrations. The method was applied to monitor dFdCTP and changes in endogenous nucleotides in patients who were receiving gemcitabine infusions.

Nitric oxide (NO) seems to be related to bubble formation and endothelial dysfunction resulting in decompression sickness. Bubble formation can be affected by aerobic exercise or manipulating NO. A prior heat stress (HS) has been shown to confer protection against decompression sickness in rats. An important question was if the oxidative environment experienced during diving limits the availability of the nitric oxide synthase (NOS) cofactor tetrahydrobiopterin (BH4). Human endothelial cells were used to investigate how HS and simulated diving affected NO synthesis and defense systems such as heat shock protein 70 (HSP70) and glutathione (GSH). BH4 was measured using a novel LC–MS/MS method and NOS by monitoring the conversion of radiolabeled l-arginine to l-citrulline. Increased pO2 reduced BH4 levels in cells in a dose-dependent manner independently of high pressure. This effect may result in decreased generation of NO by NOS. The BH4 decrease seemed to be abolished when cells were exposed to HS prior to hyperoxia. NOS enzyme was unaffected by increased pO2 but substantially reduced after HS. The BH4 level seemed to a minor extent to be dependent upon GSH and probably to a higher degree dependent on other antioxidants such as ascorbic acid. A simulated dive at 60 kPa O2 had a potentiating effect on the heat-induced HSP70 expression, whereas GSH levels were unaffected by hyperoxic exposure. HS, hyperoxia, and dive affected several biochemical parameters that may play important roles in the mechanisms protecting against the adverse effects of saturation diving.

A simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method was developed for the quantification of tetrahydrobiopterin (BH4), dihydrobiopterin (BH2), and biopterin (B) in human umbilical vein endothelial cells (HUVECs). Freshly prepared cell samples were treated with a mixture consisting of 0.2M trichloroacetic acid (TCA) and a cocktail of various antioxidants in order to precipitate proteins and other cellular components and to stabilize red/ox conditions in the lysates. Chromatography of the cell lysates was performed on a Poroshell 120 SB-C18 column (2.7μm, 150×2.1mm) using a stepwise gradient elution made from two mobile phases. Quantification was performed on a triple quadrupole mass spectrometer employing electrospray ionization with the operating conditions as multiple reaction monitoring (MRM) at positive ion mode. Total chromatographic run time was 23min. The method was validated for analysis in HUVECs, and the limits of quantification were 1nM for BH4 and BH2 and 2.5nM for B. Standard curves were linear in the concentration ranges of 1 to 100nM for BH4 and BH2 and 2.5 to 100nM for B. The current study reports a novel method for the simultaneous and direct quantification of BH4, BH2, and B in a single injection.

Seasonal and ontogenetic variation in egg buoyancy (egg specific gravity; rho) (n = 63) and nucleotide content (n = 46) were examined for wild-caught Atlantic (Barents Sea) cod (Gadus morhua) held in captivity over two successive spawning seasons, i.e. each female (n = 5) was studied both as recruit and repeat spawner. All eggs were naturally spawned and fertilized, and incubated under optimal condition in flow-through aquaria. Egg diameter and egg dry weight declined steadily during the spawning period, while stage-specific rho was approximately constant between egg batches (typically around 15 in total). Within each egg batch, i.e. during egg incubation, rho significantly decreased from the time of gastrulation to before hatching, accompanied by increased contents of ATP and ADP. Altogether, we found that adenylate energy charge (EC) (EC = ([ATP] + 0.5 [ADP])/([ATP] + [ADP] + [AMP]) positively affected egg buoyancy (P = 0.013) in concert with egg developmental stage (P<0.001) and egg diameter (P=0.014) (LMM). The presently studied eggs were considered to be in good quality and showed generally very high fertilization rates. Although the number of analyzed females in this complex repeated measurement experiment was limited due to logistic restrains, it can be expected that cod eggs in the field would show comparably similar trends in p and levels of nucleotides. (c) 2012 Elsevier B.V. All rights reserved.

A study was conducted to assess the effect of substituting high levels of dietary fish oil (FO) and fishmeal (FM) for vegetable oil (VO) and plant protein (PP) on the intestinal arachidonic acid (AA) cascade in the carnivorous fish species Atlantic salmon. Four diets were fed to salmon over a period of 12 months, including a control FMFO diet, with varying replacements of plant-derived ingredients: 80 % PP and 35 % VO; 40 % PP and 70 % VO; 80 % PP and 70 %VO. Subsequently, fish were examined pre- (0 h) and post- (1 h) acute stress for blood parameters and intestinal bioactive lipidic mediators of inflammation (prostaglandins). Plasma cortisol responses were greatest in the FMFO group, while 80 % PP and 70 % VO fish exhibited increased plasma chloride concentrations. The n -3: n -6 PUFA ratio in intestinal glycerophospholipids from 70 % VO groups significantly decreased in both proximal and distal regions due to elevated levels of 18 : 2 n -6 and the elongation/desaturation products 20 : 2 n -6 and 20 : 3 n -6. Increases in n -6 PUFA were not concomitant with increased AA, although the AA:EPA ratio did vary significantly. The 40 % PP and 70 % VO diet produced the highest intestinal AA:EPA ratio proximally, which coincided with a trend in elevated levels of PGF 2α , PGE 2 and 6-keto-PGF 1α in response to stress. PGE 2 predominated over PGF 2α and 6-keto-PGF 1α (stable metabolite of PGI 2 ) with comparable concentrations in both intestinal regions. Cyclo-oxygenase-2 (COX-2) mRNA expression was an order of magnitude higher in distal intestine, compared with proximal, and was significantly up-regulated following stress. Furthermore, the 80 % PP and 70 % VO diet significantly amplified proximal COX-2 induction post-stress. Results demonstrate that high replacements with plant-derived dietary ingredients can enhance COX-2 induction and synthesis of pro-inflammatory eicosanoids in the intestine of salmon in response to acute physiological stress.

A highly selective and sensitive method for the determination of 30 meta- and para-substituted alkylphenols from phenol (C0) to nonylphenol (C9) in biota is described. Dichloromethane extracts of spiked cod liver and muscle samples are cleaned up by gel permeation chromatography, derivatised with pentafluorobenzoyl chloride and analysed by gas chromatography-mass spectrometry with negative-ion chemical ionisation. Quantification is done with isotope dilution of five internal standards of different chain length. The detection limits were in the low microg/kg levels. There were encountered problems with background levels of 4-nonylphenol. 4-Nonylphenol isomers were found in a number of plastic and rubber products used in the laboratory.