Phospholipid hydroperoxide cysteine peroxidase activity of human serum albumin.
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Human serum albumin (HSA) reduced the phospholipid hydroperoxide, 1-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-l-3-phosphatidylcholine (PLPC-OOH) to the corresponding hydroxy-derivative with a high apparent affinity (Km=9. 23+/-0.95 microM). Removal of bound lipid during purification increased this activity. At physiological concentration, HSA reduced the phospholipid hydroperoxide in the absence of a cofactor. However, in the presence of a cofactor (reductant), the rate of the reaction was increased. All of the major aminothiols in plasma could act as reductants, the best being the most abundant, cysteine (Km=600+/-80 microM). For every nanomole of PLPC-OOH reduced by HSA, 1.26 nmol of cystine was formed, indicating a reaction stoichiometry of 1 mol PLPC-OOH to 2 mol cysteine. We used chemical modification to determine which amino acid residues on HSA were responsible for the activity. Oxidation of thiol group(s) by N-ethylmaleimide led to a reduction in the rate of activity, whereas reduction of thiols by either dithiothreitol or the angiotensin-converting enzyme inhibitor, captopril, increased the activity. Both N-ethylmaleimide-modified HSA and dithiothreitol-treated HSA exhibited increased apparent affinities for PLPC-OOH. For a range of preparations of albumin with different modifications, the activity on PLPC-OOH was dependent on the amount of free thiol groups on the albumin (correlation coefficient=0.91). Patients with lowered albumin concentrations after septic shock showed lowered total plasma thiol concentrations and decreased phospholipid hydroperoxide cysteine peroxidase (PHCPx) activities. These results therefore show for the first time that HSA exhibits PHCPx activity, and that the majority of the activity depends on the presence of reduced thiol group(s) on the albumin. (+info)
Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury.
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Heme oxygenase (HO) catalyzes the conversion of heme to carbon monoxide, iron, and biliverdin, which is immediately reduced to bilirubin (BR). Two HO active isozymes exist: HO1, an inducible heat shock protein, and HO2, which is constitutive and highly concentrated in neurons. We demonstrate a neuroprotective role for BR formed from HO2. Neurotoxicity elicited by hydrogen peroxide in hippocampal and cortical neuronal cultures is prevented by the phorbol ester, phorbol 12-myristate 13-acetate (PMA) via stimulation of protein kinase C. We observe phosphorylation of HO2 through the protein kinase C pathway with enhancement of HO2 catalytic activity and accumulation of BR in neuronal cultures. The neuroprotective effects of PMA are prevented by the HO inhibitor tin protoporphyrin IX and in cultures from mice with deletion of HO2 gene. Moreover, BR, an antioxidant, is neuroprotective at nanomolar concentrations. (+info)
Genomic structure of the canalicular multispecific organic anion-transporter gene (MRP2/cMOAT) and mutations in the ATP-binding-cassette region in Dubin-Johnson syndrome.
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Dubin-Johnson syndrome (DJS) is an autosomal recessive disease characterized by conjugated hyperbilirubinemia. Previous studies of the defects in the human canalicular multispecific organic anion transporter gene (MRP2/cMOAT) in patients with DJS have suggested that the gene defects are responsible for DJS. In this study, we determined the exon/intron structure of the human MRP2/cMOAT gene and further characterized mutations in patients with DJS. The human MRP2/cMOAT gene contains 32 exons, and it has a structure that is highly conserved with that of another ATP-binding-cassette gene, that for a multidrug resistance-associated protein. We then identified three mutations, including two novel ones. All mutations identified to date are in the cytoplasmic domain, which includes the two ATP-binding cassettes and the linker region, or adjacent putative transmembrane domain. Our results confirm that MRP2/cMOAT is the gene responsible for DJS. The finding that mutations are concentrated in the first ATP-binding-cassette domain strongly suggests that a disruption of this region is a critical route to loss of function. (+info)
Soccer players under regular training show oxidative stress but an improved plasma antioxidant status.
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Physical activity is known to induce oxidative stress in individuals subjected to intense exercise. In this study, we investigated the lipoprotein profile and the plasma antioxidant status in a group of soccer players engaged in a regular training programme. As was expected for aerobic exercise, high-density lipoprotein-cholesterol (HDL-C) and HDL3-C levels were significantly increased in the sportsmen (P<0.05). Total plasma antioxidant capacity was 25% higher in sportsmen than in controls (P<0.005). Accordingly, plasma hydrosoluble antioxidant levels (ascorbic acid and uric acid) were found to be significantly elevated in the soccer players (P<0.005). In addition, these subjects showed high concentrations of alpha-tocopherol in plasma compared with controls (P<0.005). Furthermore, an increase in plasma superoxide dismutase activity was also observed in relation to exercise (P<0.01). The elevation in plasma activities of antioxidant enzymes and the higher levels of free radical scavengers of low molecular mass may compensate the oxidative stress caused by physical activity. High levels of high-density lipoprotein in plasma may offer additional protection by inhibiting low-density lipoprotein oxidation and thus liposoluble antioxidant consumption. Therefore, soccer players under regular training show an improved plasma antioxidant status in comparison to sedentary controls. (+info)
Functional heterogeneity of UDP-glucuronosyltransferase as indicated by its differential development and inducibility by glucocorticoids. Demonstration of two groups within the enzyme's activity towards twelve substrates.
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1. UDP-glucuronosyltransferase activity towards 12 substrates has been assessed in rat liver during the perinatal period. 2. Between days 16 and 20 of gestation, enzyme activities towards the substrates 2-aminophenol, 2-aminobenzoate, 4-nitrophenol, 1-naphthol, 4-methylumbelliferone and 5-hydroxytryptamine (the 'late foetal' group) surge to reach adult values, while activities towards bilirubin, testosterone, beta-oestradiol, morphine, phenolphthalein, and chloramphenicol (the 'neonatal' group) remain negligible or at less than 10% of adult values. 3. By the second postnatal day, enzyme activities towards the neonatal group have attained, or approached adult values. 4. Dexamethasone precociously stimulates in 17-day foetal liver in utero transferase activities in the late foetal, but not the neonatal group. A similar inductive pattern is found for 15-day foetal liver in organ culture. 5. It is suggested that foetal glucocorticoids, whose synthesis markedly increases between days 16 and 20 of gestation, are responsibile for triggering the simultaneous surge of all the hepatic UDP-glucuronosyltransferase activities in the late foetal group. The neonatal group of activities apparently require a different or additional stimulus for their appearance. 6. The relationship of these two groups of transferase activities to other similar groups observed during induction by xenobiotics and enzyme purification is discussed. (+info)
Role of bilirubin overproduction in revealing Gilbert's syndrome: is dyserythropoiesis an important factor?
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Gilbert's syndrome was diagnosed in 37 patients with unconjugated hyperbilirubinaemia without overt haemolysis or structural liver abnormality, who had a marked reduction in hepatic bilirubin UDP-glucuronosyltransferase activity (B-GTA) (as compared with that of 23 normal subjects). No significant correlation existed in these patients between serum bilirubin level and the values of B-GTA, thus suggesting that factors other than a low B-GTA must influence the degree of hyperbilirubinaemia in Gilbert's syndrome. Studies of 51Cr erythrocyte survival and 59Fe kinetics in 10 unselected patients demonstrated slight haemolysis in eight, whereas mild ineffective erythropoiesis was suggested in all from a low 24-hour incorporation of radioactive iron into circulating red cells. This overproduction of bilirubin resulting from mild haemolysis and perhaps dyserythropoiesis might reflect only an extreme degree of the normal situation. It certainly contributes to the hyperbilirubinaemia of Gilbert's syndrome and may play a major role in the manifestation of this condition. (+info)
Unconjugated bilirubin exhibits spontaneous diffusion through model lipid bilayers and native hepatocyte membranes.
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The liver is responsible for the clearance and metabolism of unconjugated bilirubin, the hydrophobic end-product of heme catabolism. Although several putative bilirubin transporters have been described, it has been alternatively proposed that bilirubin enters the hepatocyte by passive diffusion through the plasma membrane. In order to elucidate the mechanism of bilirubin uptake, we measured the rate of bilirubin transmembrane diffusion (flip-flop) using stopped-flow fluorescence techniques. Unconjugated bilirubin rapidly diffuses through model phosphatidylcholine vesicles, with a first-order rate constant of 5.3 s-1 (t(1)/(2) = 130 ms). The flip-flop rate is independent of membrane cholesterol content, phospholipid acyl saturation, and lipid packing, consistent with thermodynamic analyses demonstrating minimal steric constraint to bilirubin transmembrane diffusion. The coincident decrease in pH of the entrapped vesicle volume supports a mechanism whereby the bilirubin molecule crosses the lipid bilayer as the uncharged diacid. Transport of bilirubin by native rat hepatocyte membranes exhibits kinetics comparable with that in model vesicles, suggesting that unconjugated bilirubin crosses cellular membranes by passive diffusion through the hydrophobic lipid core. In contrast, there is no demonstrable flip-flop of bilirubin diglucuronide or bilirubin ditaurate in phospholipid vesicles, yet these compounds rapidly traverse isolated rat hepatocyte membranes, confirming the presence of a facilitated uptake system(s) for hydrophilic bilirubin conjugates. (+info)
Auxiliary partial orthotopic liver transplantation for Crigler-Najjar syndrome type I.
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OBJECTIVE: To determine if auxiliary partial orthotopic liver transplantation (APOLT) has the long-term potential to correct the underlying abnormality in Crigler-Najjar syndrome type 1 (CNS1) without the need for total liver replacement. BACKGROUND: Orthotopic liver transplantation has been used successfully to replace the defective enzyme in CNS1. Experimental studies have shown that only 1% to 2% of the normal hepatocyte mass is needed for bilirubin conjugation. If APOLT corrects the underlying metabolic abnormality, it has the advantage of preserving the native liver, which would serve as a "safety net" should the graft fail, and there is the potential for gene therapy in the future with possible withdrawal of immunosuppression. METHODS: Seven APOLT procedures were performed in six recipients with CNS1. Median age at transplantation was 10.5 years. Six transplants were performed as a left auxiliary liver transplant, and one was performed as a right auxiliary liver transplant. Median serum bilirubin level at transplantation was 320 micromol/L. All patients required 12 to 16 hours of phototherapy daily before the transplant to maintain serum bilirubin levels between 250 and 350 micromol/L. RESULTS: Median serum bilirubin level was 50 micromol/L at day 5 after the transplant and 23 micromol/L at a median follow-up of 32 months. In four children, early severe acute rejection developed, requiring conversion to tacrolimus; one underwent a second transplant for chronic rejection and graft atrophy but died from lymphoproliferative disease 6 months after the second transplant. CONCLUSIONS: This report shows that APOLT is technically feasible and provides adequate hepatocyte mass to correct the underlying metabolic abnormality in CNS1. (+info)