[Urinary hexafluoroisopropanol in the assessment of occupational exposure to sevoflurane: methodologic features and critical points]. / L'esafluoroisopropanolo urinario nella valutazione dell'esposizione professionale a sevofluorano: aspetti metodologici e punti critici.

The increasing use of sevoflurane as anaesthetic leads to the need for finding a biological index to evaluate the occupational exposure in surgical activity. Several studies indicate that Hexafluoroisopropanol (HFIP) is a specific sevoflurane metabolite quickly glucuronidated and excreted as HFIP-glucuronide in the urine (HFIPu). Therefore the HFIP removal kinetics in occupational exposure and the correlation between sevoflurane exposure and HFIPu are poorly understood. We studied no. 86 operating room workers of Novara Hospital to evaluate the correlation between the sevoflurane individual exposure (SE) and the HFIPu at the end of the shift expressed in microgram/L (A-HFIPu) and in microgram/g creat. (C-HFIPu). Therefore, in the same group of subjects we evaluated the HFIPu in the urine sampled at 8.00 a.m. before the work. The correlation coefficient was R2 = 0.782 (p < 0.0001) for SE/A-HFIPu and R2 = 0.862 (p < 0.0001) for SE/C-HFIPu; HFIPu normalized for urinary creatinine (C-HFIPu) is an index more suitable than the A-HFIPu. Furthermore we concluded for the usefulness of pre-shift HFIPu.

Alterations of Na(+) homeostasis in hepatocyte reoxygenation injury.

Reperfusion injury represents an important cause of primary graft non-function during liver transplantation. However, the mechanism responsible for cellular damage during reoxygenation has not yet been completely understood. We have investigated whether changes in intracellular Na(+) distribution might contribute to cause hepatocyte damage during reoxygenation buffer after 24 h of cold storage. Hepatocyte reoxygenation resulted in a rapid increase in cellular Na(+) content that was associated with cytotoxicity. Na(+) accumulation and hepatocyte death were prevented by the omission of Na(+) from the incubation medium, but not by the addition of antioxidants. Blocking Na(+)/H(+) exchanger and Na(+)/HCO(3)(-) co-transporter by, respectively, 5-(N,N-dimethyl)-amiloride or omitting HCO(3)(-) from the reoxygenation medium significantly decreased Na(+) overload and cytotoxicity. Stimulation of ATP re-synthesis by the addition of fructose also lowered Na(+) accumulation and cell death during reoxygenation. A significant protection against Na(+)-mediated reoxygenation injury was evident in hepatocytes maintained in an acidic buffer (pH 6.5) or in the presence of glycine. The cytoprotective action of glycine or of the acidic buffer was reverted by promoting Na(+) influx with the Na(+)/H(+) ionophore monensin. Altogether, these results suggest that Na(+) accumulation during the early phases of reoxygenation might contribute to liver graft reperfusion injury.

DISTRIBUTION OF LIPID-SOLUBLE ANTIOXIDANTS IN LIPOPROTEINS FROM HEALTHY SUBJECTS. I. CORRELATION WITH PLASMA ANTIOXIDANT LEVELS AND COMPOSITION OF LIPOPROTEINS.

The concentration of five lipid-soluble antioxidants (gamma- and alpha-tocopherol, lycopene, beta-carotene and ubiquinol-10) was measured in plasma and very low-density, low-density and high-density lipoproteins (VLDL, LDL and HDL) isolated from young healthy normo- cholesterolemic subjects. Alpha-tocopherol was the exclusive antioxidant whose plasma concentration significantly correlated with the absolute concentration of total cholesterol (r =0.541, P<0.001). No correlation was found between plasma concentration and lipoprotein content of alpha-tocopherol and ubiquinol-10, whereas it reached statistically significant values for gamma-tocopherol, lycopene and beta-carotene. The alpha-tocopherol content in VLDL and HDL, but not in LDL, was strictly associated with the relative abundance of cholesterol and phospholipids in the lipoprotein particles. Moreover, the difference between alpha-tocopherol concentration in VLDL and LDL appeared to be strictly related to the differences in cholesterol, phospholipids and triglycerides. The percent distribution of the total plasma pool of antioxidant in each lipoprotein class revealed that gamma- and alpha-tocopherol were roughly equally distributed in LDL and HDL. On the other hand, lycopene, beta-carotene and ubiquinol-10 were preferentially sequestered in LDL. Finally, the absolute and relative concentration of alpha-tocopherol, but not that of other antioxidants, in HDL exhibited a statistically significant correlation with plasma HDL/LDL cholesterol ratio. These findings indicate that: (i) plasma concentrations of major lipid-soluble antioxidants are not always predictive of their levels in lipoproteins and that, within individual lipoprotein classes, (ii) the lipid composition, metabolism and relative plasma concentration may significantly affect their abundance. 2000 Academic Press@p$hr Copyright 2000 Academic Press.

DISTRIBUTION OF LIPID-SOLUBLE ANTIOXIDANTS IN LIPOPROTEINS FROM HEALTHY SUBJECTS. II. EFFECTS OF IN VIVO SUPPLEMENTATION WITH alpha-TOCOPHEROL.

The effects of orally supplemented dl -alpha-tocopherol on the plasma concentration of lipid-soluble antioxidants and their distribution in very-low-density, low-density and high-density lipoproteins (VLDL, LDL and HDL) was investigated in a cohort of control normocholesterolemic adult subjects receiving 600 mg alpha-tocopherol daily for 2 weeks. This regimen did not modify the plasma lipid profile (total, LDL and HDL cholesterol and triglycerides) and chemical composition of VLDL, LDL and HDL. Plasma concentration of alpha-tocopherol increased from 19.44+/-4.77 to 38.03+/-9.06 µm and this was associated with slight decrease in the concentration of gamma-tocopherol from 1.27+/-0.97 to 0.99+/-1.17 µm, without any significant changes of either lycopene and beta-carotene. Qualitatively similar changes were found in VLDL, LDL and HDL but the net increase of alpha-tocopherol in plasma did not correlate with the increase in alpha-tocopherol content in any of the lipoprotein types. Following supplementation, the percentage of total plasma alpha-tocopherol pool carried by VLDL increased from 20.97+/-6.07% to 33.57+/-6.97%, whereas it decreased from 41.85+/-7.02% to 36.36+/-5.69% in the case of LDL and from 37.17+/-6.04% to 30.05+/-4.88% in the case of HDL. The absolute and relative enrichment of alpha-tocopherol in either VLDL and LDL did not exhibit any statistically relevant correlation with the chemical composition of these lipoproteins in the different subjects investigated. On the other hand, the amount of alpha-tocopherol enriching the HDL particles was inversely related to the relative abundance of protein (r =0.449;P<0.05) and directly to the phospholipid/protein ratio (r =0.480, P<0.05). 2000 Academic Press@p$hr Copyright 2000 Academic Press.

The kinetics of copper-induced LDL oxidation depend upon its lipid composition and antioxidant content.

Copper promotes oxidation of human low-density lipoprotein (LDL) through molecular mechanisms that are still under investigation. We employed native human LDL, phospholipid-containing delipidated LDL ghosts, or trilinolein-reconstituted, phospholipid-containing LDL to investigate both LDL oxidation and the associated process of copper reduction. Both LDL ghosts and trilinolein-reconstituted LDL were devoid of antioxidants and were extremely susceptible to AAPH-induced oxidation but, paradoxically, were rather resistant to copper-mediated oxidation. The dynamic reduction of Cu(II) to Cu(I) was quantitatively decreased in LDL ghosts and in trilinolein-reconstituted LDL, also lacking the initial rapid reduction and the subsequent inhibition phases, due to the absence of endogenous antioxidants. Conversely, the rate of copper reduction was linear and likely due to lipid peroxides, either already present or formed during copper-induced oxidation. We suggest that copper undergoes redox transitions in LDL by utilizing reducing equivalents originating from endogenous antioxidants and/or from lipid peroxides in the LDL lipid core.

Distribution of lipid-soluble antioxidants in lipoproteins from healthy subjects. I. Correlation with plasma antioxidant levels and composition of lipoproteins.

The concentration of five lipid-soluble antioxidants (gamma- and alpha-tocopherol, lycopene, beta-carotene and ubiquinol-10) was measured in plasma and very low-density, low-density and high-density lipoproteins (VLDL, LDL and HDL) isolated from young healthy normo- cholesterolemic subjects. Alpha-tocopherol was the exclusive antioxidant whose plasma concentration significantly correlated with the absolute concentration of total cholesterol (r =0.541, P<0.001). No correlation was found between plasma concentration and lipoprotein content of alpha-tocopherol and ubiquinol-10, whereas it reached statistically significant values for gamma-tocopherol, lycopene and beta-carotene. The alpha-tocopherol content in VLDL and HDL, but not in LDL, was strictly associated with the relative abundance of cholesterol and phospholipids in the lipoprotein particles. Moreover, the difference between alpha-tocopherol concentration in VLDL and LDL appeared to be strictly related to the differences in cholesterol, phospholipids and triglycerides. The percent distribution of the total plasma pool of antioxidant in each lipoprotein class revealed that gamma- and alpha-tocopherol were roughly equally distributed in LDL and HDL. On the other hand, lycopene, beta-carotene and ubiquinol-10 were preferentially sequestered in LDL. Finally, the absolute and relative concentration of alpha-tocopherol, but not that of other antioxidants, in HDL exhibited a statistically significant correlation with plasma HDL/LDL cholesterol ratio. These findings indicate that: (i) plasma concentrations of major lipid-soluble antioxidants are not always predictive of their levels in lipoproteins and that, within individual lipoprotein classes, (ii) the lipid composition, metabolism and relative plasma concentration may significantly affect their abundance.

Distribution of lipid-soluble antioxidants in lipoproteins from healthy subjects. II. Effects of in vivo supplementation with alpha-tocopherol.

The effects of orally supplemented dl -alpha-tocopherol on the plasma concentration of lipid-soluble antioxidants and their distribution in very-low-density, low-density and high-density lipoproteins (VLDL, LDL and HDL) was investigated in a cohort of control normocholesterolemic adult subjects receiving 600 mg alpha-tocopherol daily for 2 weeks. This regimen did not modify the plasma lipid profile (total, LDL and HDL cholesterol and triglycerides) and chemical composition of VLDL, LDL and HDL. Plasma concentration of alpha-tocopherol increased from 19.44+/-4.77 to 38. 03+/-9.06 microm and this was associated with slight decrease in the concentration of gamma-tocopherol from 1.27+/-0.97 to 0.99+/-1.17 microm, without any significant changes of either lycopene and beta-carotene. Qualitatively similar changes were found in VLDL, LDL and HDL but the net increase of alpha-tocopherol in plasma did not correlate with the increase in alpha-tocopherol content in any of the lipoprotein types. Following supplementation, the percentage of total plasma alpha-tocopherol pool carried by VLDL increased from 20. 97+/-6.07% to 33.57+/-6.97%, whereas it decreased from 41.85+/-7.02% to 36.36+/-5.69% in the case of LDL and from 37.17+/-6.04% to 30.05+/-4.88% in the case of HDL. The absolute and relative enrichment of alpha-tocopherol in either VLDL and LDL did not exhibit any statistically relevant correlation with the chemical composition of these lipoproteins in the different subjects investigated. On the other hand, the amount of alpha-tocopherol enriching the HDL particles was inversely related to the relative abundance of protein (r =0.449;P<0.05) and directly to the phospholipid/protein ratio (r =0.480, P<0.05).

Ischemic preconditioning reduces Na(+) accumulation and cell killing in isolated rat hepatocytes exposed to hypoxia.

Short periods of ischemia followed up by reperfusion are known to protect the heart against injury caused by a subsequent sustained ischemia. This phenomenon, known as ischemic preconditioning, has also been recently shown to reduce ischemic liver damage, but the mechanisms involved are still unknown. By using isolated hepatocytes as an in vitro model of liver preconditioning, we have investigated the possible effect of preconditioning on intracellular pH and Na(+) homeostasis. Freshly isolated rat hepatocytes were preconditioned by 10 minutes of incubation under hypoxic conditions followed up by 10 minutes of reoxygenation and subsequently exposed to 90 minutes of hypoxia. Although preconditioning did not ameliorate adenosine triphosphate (ATP) depletion, preconditioned hepatocytes exhibited an increased resistance to cell killing during hypoxic incubation. Intracellular acidosis and Na(+) accumulation developing during hypoxia were appreciably reduced in preconditioned cells. The effects of preconditioning on intracellular pH, Na(+) homeostasis, and cytotoxicity were mimicked by stimulating protein kinase C (PKC) with 4beta-phorbol-12-myristate-13-acetate (PMA) or 1,2 dioctanoyl-glycerol (1,2 DOG). Conversely, inhibiting PKC with chelerythrine or blocking vacuolar proton ATPase (V-ATPase) with bafilomycin A(1) abolished the protection given by preconditioning or by PMA treatment on hypoxic acidosis, Na(+) overload, and hepatocyte killing. Similarly, the addition of Na(+) ionophore monensin also reverted the cytoprotection exerted by preconditioning. This indicated that ischemic preconditioning of isolated hepatocytes decreased cell killing during hypoxia by preventing intracellular Na(+) accumulation. We propose that, after preconditioning, the stimulation of PKC might activate proton extrusion through V-ATPase, thus, limiting intracellular acidosis and Na(+) overload promoted by Na(+)-dependent acid buffering systems.

When and why a water-soluble antioxidant becomes pro-oxidant during copper-induced low-density lipoprotein oxidation: a study using uric acid.

The inclusion of uric acid in the incubation medium during copper-induced low-density lipoprotein (LDL) oxidation exerted either an antioxidant or pro-oxidant effect. The pro-oxidant effect, as mirrored by an enhanced formation of conjugated dienes, lipid peroxides, thiobarbituric acid-reactive substances and increase in negative charge, occurred when uric acid was added late during the inhibitory or lag phase and during the subsequent extensive propagation phase of copper-stimulated LDL oxidation. The pro-oxidant effect of uric acid was specific for copper-induced LDL oxidation and required the presence of copper as either Cu(I) or Cu(II). In addition, it became much more evident when the copper to LDL molar ratio was below a threshold value of approx. 50. In native LDL, the shift between the antioxidant and the pro-oxidant activities was related to the availability of lipid hydroperoxides formed during the early phases of copper-promoted LDL oxidation. The artificial enrichment of isolated LDL with alpha-tocopherol delayed the onset of the pro-oxidant activity of uric acid and also decreased the rate of stimulated lipid peroxidation. However, previous depletion of alpha-tocopherol was not a prerequisite for unmasking the pro-oxidant activity of uric acid, since this became apparent even when alpha-tocopherol was still present in significant amounts (more than 50% of the original values) in LDL. These results suggest, irrespective of the levels of endogenous alpha-tocopherol, that uric acid may enhance LDL oxidation by reducing Cu(II) to Cu(I), thus making more Cu(I) available for subsequent radical decomposition of lipid peroxides and propagation reactions.

Different mechanisms are progressively recruited to promote Cu(II) reduction by isolated human low-density lipoprotein undergoing oxidation.

The kinetics of Cu(II) reduction and its relationship to the process of low density lipoprotein (LDL) oxidation were investigated in isolated human LDL incubated with CuSO4 by using the Cu(I) chelator and indicator dye bathocuproine disulfonate (BC). The inclusion of BC in the incubation medium containing isolated LDL and different concentrations of CuSO4 revealed a biphasic kinetics of Cu(II) reduction consisting of an early phase followed by a plateau phase and a subsequent extensive reduction phase. The amount of Cu(I) formed during the early phase, as well as the rate of its generation, were strictly dependent on both the level of Cu(II) available (saturation was observed at 20 and 50 microM CuSO4) and the concentration of alpha-tocopherol within native LDL particles. Artificial enrichment of LDL with different concentrations of alpha-tocopherol led to a parallel increase of both the amount of Cu(II) reduced and the rate of reduction. The late phase of Cu(II) reduction was strictly related to the availability of copper but was largely independent from alpha-tocopherol. Neither the amount of Cu(I) generated nor the rate of generation were saturated at concentrations of copper up to 100 microM. Comparable results were obtained by adding BC at different time-points to the LDL-copper mixture, in order to measure at the same time-points both the true rate of Cu(II) reduction and the generation of TBARS during the dynamic process of LDL oxidation. The rate of Cu(II) reduction was already high during the lag-phase of the LDL oxidation profile and progressively decreased as alpha-tocopherol concentration decreased. The subsequent increase in the rate of Cu(II) reduction paralleled the formation of TBARS during the extensive LDL oxidation phase. These results suggest that different mechanisms of Cu(II) reduction, namely alpha-tocopherol-dependent and independent (likely lipid peroxide-dependent), are progressively recruited during copper-promoted LDL oxidation.

Cu(I) availability paradoxically antagonizes antioxidant consumption and lipid peroxidation during the initiation phase of copper-induced LDL oxidation.

The incubation of isolated human low-density lipoprotein (LDL) with Cu(II) promoted extensive oxidation of both the lipid and protein moieties of the lipoprotein particle. When the Cu(II) to LDL molar ratio was equal or higher than 50, the removal of Cu(I) formed by the contemporary presence of the Cu(I) chelator bathocuproine disulphonate (BC) markedly accelerated the formation of end-products of lipid peroxidation. Moreover, the initial rate of Cu(II)-induced consumption of either endogenous antioxidants in LDL or free alpha-tocopherol in suspension was increased in the presence of BC, thus indicating that the continuous removal of Cu(I) enhanced both antioxidant consumption and LDL oxidation promoted by copper. Furthermore, the direct addition of Cu(I), together with Cu(II), to a suspension of isolated LDL efficiently delayed the onset of extensive lipid peroxidation and decreased the rate of antioxidant consumption. The latter effect, however, was detectable exclusively on LDL-associated alpha-tocopherol and not on alpha-tocopherol in suspension, thus suggesting that the competition between Cu(I) and Cu(II) was taking place at discrete sites associated with the LDL particle. Finally, the inclusion of Cu(I) in the incubation medium of LDL already undergoing extensive peroxidation did not inhibit but rather markedly stimulated the rate of peroxidation. Although apparently in contrast with the common view that Cu(I) and not Cu(II) is the real trigger species of LDL oxidation in the copper model, the results reported here indicate that the availability of Cu(I) during the initiation phase of LDL oxidation promoted by copper antagonizes both antioxidant consumption and the onset of extensive oxidation.