Rat plasma glutathione peroxidase (GSH-Px) was purified 1,400-fold from rat serum by a combination of phenyl Sepharose, DEAE Sephacel, blue Sepharose and Sephacryl S-200 column chromatographies. The purified GSH-Px migrated as a single band corresponding to a molecular weight of 22,500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was used for the immunization of chickens to obtain a specific antibody and for determination of its amino acid sequence. Two overlapping cDNA clones for rat plasma GSH-Px were isolated from a placental cDNA library. The composite nucleotide sequence is 1,529 base-pairs long and encodes 226 amino acids. The deduced amino acid sequence completely coincided with the sequences of five individual peptide fragments derived from the purified plasma GSH-Px on digestion with lysyl endopeptidase. In order to identify the tissue(s) generating this plasma GSH-Px, immunoblot analysis was performed on homogenates prepared from 13 tissues. A single immunoreactive band of 22.5 kDa, corresponding to plasma GSH-Px, was detected for the kidney homogenate. A much fainter band was observed for the lung preparation, but liver, spleen, bone marrow, and other tissues examined were negative. Northern blot analysis further revealed that the expression level of the plasma GSH-Px gene was high in kidney and low in lung. No transcript was detected in liver or spleen. These results indicate that plasma GSH-Px is predominantly synthesized and secreted by renal cells.

Glutathione peroxidase (GPx) activity was determined in the plasma of 118 healthy persons, 18 nondialyzed patients with chronic renal failure (CRF), 20 patients on maintenance hemodialysis (HD), and 58 patients on continuous ambulatory peritoneal dialysis (CAPD). Serum creatinine levels in the nondialyzed CRF patients revealed a highly significant negative correlation (r = -0.71, p < 0.001) with plasma GPx activity. Immunoblot analysis revealed that the plasma (extracellular) GPx protein was reduced or undetectable in patients with low plasma GPx activity. Plasma GPx activities in the HD and CAPD patients were reduced to 44 and 23% (female), and to 70 and 45% (male) of the sex-matched control values, respectively. In contrast, erythrocyte (cellular) GPx activity was not decreased in the nondialyzed CRF patients and the dialyzed patients. Plasma selenium concentrations were within the normal range in these patient groups. These results indicate that the plasma GPx activity largely depends on renal function.

Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of chimeric mice respond to hypercaloric diets. As early as 4 weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatohepatitis selectively in the human graft, followed by pericellular fibrosis after 8 weeks of hypercaloric feeding. The PNPLA3 148M variant, either from a homozygous 148M human donor or overexpressed in a homozygous 148I donor background, caused microvesicular and more severe steatosis. In these livers hepatocytes displayed frequent ballooning degeneration, resulting in more active steatohepatitis than in 148I livers. We conclude that PNPLA3 148M in human hepatocytes exacerbates NAFLD. These models will facilitate mechanistic studies into human genetics associated with advanced fatty liver diseases.

No abstract

No abstract