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Numerous studies on molluscs have been carried out to clarify the physiological roles of haemolymph serum proteins and haemocytes. However, little is known about the presence and functional role of the serum metabolites. In this study, Nuclear Magnetic Resonance (NMR) was used to assess whether changes of the metabolic profile of Mytilus galloprovincialis haemolymph may reflect alterations of the physiological status of the organisms due to environmental stressors, namely copper and temperature. Mussel haemolymph was taken from the posterior adductor muscle after a 4-day exposure to ambient (16 °C) or high temperature (24 °C) and in the absence or presence (5 μg/L, 20 μg/L, or 40 μg/L) of sublethal copper (Cu2+). The total glutathione (GSH) concentration in the haemolymph of both control and treated mussels was minimal, indicating the absence of significant contaminations by muscle intracellular metabolites due to the sampling procedure. In the 1H-NMR spectrum of haemolymph, 27 metabolites were identified unambiguously. The separate and combined effects of exposure to copper and temperature on the haemolymph metabolic profile were assessed by Principal Component Analysis (PCA) and Ranking-PCA multivariate analysis. Changes of the metabolomic profile due to copper exposure at 16 °C became detectable at a dose of 20 μg/L copper. Alanine, lysine, serine, glutamine, glycogen, glucose and protein aliphatics played a major role in the classification of the metabolic changes according to the level of copper exposition. High temperature (24 °C) and high copper levels caused a coherent increase of a common set of metabolites (mostly glucose, serine, and lysine), indicating that the metabolic impairment due to high temperature is enforced by the presence of copper. Overall, the results demonstrate that, as for human blood plasma, the analysis of haemolymph metabolites represents a promising tool for the diagnosis of pollutant-induced stress syndrome in marine mussels.

Summary Protection against oxidative damage caused by excessive reactive oxygen species (ROS) by an antioxidant network is essential for the health of tissues, especially in the cardiovascular system. Here, we identified a gene with important antioxidant features by analyzing a null allele of zebrafish ubiad1, called barolo (bar). bar mutants show specific cardiovascular failure due to oxidative stress and ROS-mediated cellular damage. Human UBIAD1 is a nonmitochondrial prenyltransferase that synthesizes CoQ10 in the Golgi membrane compartment. Loss of UBIAD1 reduces the cytosolic pool of the antioxidant CoQ10 and leads to ROS-mediated lipid peroxidation in vascular cells. Surprisingly, inhibition of eNOS prevents Ubiad1-dependent cardiovascular oxidative damage, suggesting a crucial role for this enzyme and nonmitochondrial CoQ10 in NO signaling. These findings identify UBIAD1 as a nonmitochondrial CoQ10-forming enzyme with specific cardiovascular protective function via the modulation of eNOS activity., Graphical Abstract Highlights ► UBIAD1 is a Golgi prenyltransferase ► UBIAD1 contributes to the nonmitochondrial pool of CoQ10 ► UBIAD1 protects cardiovascular tissues from NOS-dependent oxidative damage ► UBIAD1 is a target for therapeutic strategies by limiting the side effects of statins, UBIAD1 is identified as a nonmitochondrial CoQ10 biosynthetic enzyme required for oxidative damage protection. Through CoQ10 synthesis, UBIAD1 modulates endothelial nitric oxide synthase activity and nitric oxide signaling necessary for cardiovascular development and homeostasis.

A new assay for the measurement of the antioxidant capacity of biomolecules by high resolution 19F-NMR spectroscopy is presented here. This method is based on the use of trifluoroacetanilidic detectors, namely trifluoroacetanilide, N-(4-hydroxyphenyl)-trifluoroacetamide and 2-hydroxy-4-trifluoroacetamidobenzoic acid. Upon hydroxyl radical attack, such fluorinated detectors yield trifluoroacetamide and trifluoroacetic acid that can be quantitatively determined by 19F-NMR spectroscopy. Trifluoroacetamide was found to be a reliable reporter of hydroxyl radical attack on the fluorinated detectors, whereas N-(4-hydroxyphenyl)-trifluoroacetamide was found to be the most sensitive detector amongst the ones considered. Therefore, N-(4-hydroxyphenyl)-trifluoroacetamide has been used in competition experiments to assess the antioxidant capacity of a number of low and high molecular weight antioxidants. The antioxidant capacity of a given compound has been scaled in terms of an adimensional parameter, kF, that represents the ratio between the scavenger abilities of the fluorinated detector and the competitor. kF values obtained for low-molecular-mass compounds fall in the range 0.17 < kF < 1.5 and are in good agreement with second order rate constants (k2OH) for the reaction of the antioxidant with hydroxyl radicals. The kF value for serum albumin is much larger (46.9) than that predicted from the reported k2OH value. This finding supports the view that the protein can very effectively scavenge hydroxyl radicals as well as secondary radicals. Human blood serum showed that its antioxidant capacity is even higher than that shown by aqueous solutions of albumin at physiologic concentration suggesting a further contribution from other macromolecular serum components.

Complexes of the cyanomercury cation with various polypyrazolylborato ligands of the type HB(pz)3 · Hg-CN or pzB(pz)3 · Hg-CN (pz = pyrazolyl or substituted pyrazolyl) have been synthesised and characterised by IR, conductivity, 1H, 13C, and 199Hg NMR spectroscopy. The crystal structure of the cyanomercury hydridotris(1H-3,5-dimethylpyrazol-1-yl)borate has been resolved (space group Pī with a = 7.863(3), b = 11.157(5), c = 13.117(5)A; α = 89.32(3), β = 78.31(3), γ = 79.13(4)o, V = 1106.22A3, Z = 2) showing a distorted tetrahedral coordination around Hg. The tris(pyrazolyl)borato complexes contain four-coordinated Hg and are rigid in solution at r.t., while the tetrakis(pyrazolyl)borates are fluxional. The 15N-CPMAS-NMR spectrum of the pzTp · HgCN derivative suggests a tetracoordination around mercury in this complex in the solid state.

The solid state and solution NMR spectra of the tetrakis(pyrazol-l-yl) borate (L a ) and tris(3,5-dimethylpyrazol-l-yl) hydroborate (L b ) ligands and their complexes with MeHg II and RSHg II (R = Me, Et) are reported. The solid state 13 C and 15 N solid state NMR spectra of the MeHgL a complex are consistent with the reported X-ray structure. The close similarity of the spectral data shown by MeHgL a and MeSHgL a suggests that the two species have the same structure. In order to confirm this hypothesis, the structure of MeSHgL a has been determined by single crystal X-ray diffraction. The chemical shift anisotropy (CSA) of 199 Hg resonance is too large in these T-shaped complexes and does not allow the observation of the 199 Hg resonance under cross polarisation-magic angle spinning (CPMAS) experimental mode. On the other hand 199 Hg CPMAS NMR spectra were obtained for the complexes with the L b ligand. The reduced 199 Hg CSA value together with the information gained from the 13 C and 15 N CPMAS NMR spectra suggest a tetra coordination around mercury in these complexes. The tight co-ordination mode shown by complexes with the L b ligand is reflected in a decrease in the thiol intermolecular exchange rate in solution. This decreased ligand lability allows the detection of 3 J Hg-H couplings (inside HgSCH 3 and HgSCH 2 CH 3 moieties) in the low temperature NMR limiting spectra.