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J W Chisholm

Publications and source records attributed to J W Chisholm.

9 recordsLinked to original sources
PubMed2000

Accumulation of cholestatic lipoproteins in ANIT-treated human apolipoprotein A-I transgenic rats is diminished through dose-dependent apolipoprotein A-I activation of LCAT.

Administration of alpha-naphthylisothiocyanate (ANIT) to rats induces changes to plasma lipids consistent with cholestasis. We have previously shown (J. Lipid Res. 37 (1996) 1086) that animals treated with ANIT accumulate large amounts of free cholesterol (FC) and phospholipid (PL)-rich cholestatic lipoproteins in the LDL density range by 48 h. This lipid was cleared by 120 h through apparent movement into HDL with concomitant cholesteryl ester (CE) production. It was hypothesised that the clearance was mediated through the movement of the PL and FC into apolipoprotein A-I (apo A-I) containing lipoproteins followed by LCAT esterification to form CE. To test this hypothesis, rats overexpressing various amounts of human apo A-I (TgR[HuAI] rats) were treated with ANIT (100 mg/kg) and the effect of plasma apo A-I concentration on plasma lipids and lipoprotein distribution was examined. In untreated TgR[HuAI] rats, human apo A-I levels were strongly correlated to plasma PL (r(2)=0. 94), FC (r(2)=0.93) and CE (r(2)=0.90), whereas in ANIT-treated TgR[HuAI] rats, human apo A-I levels were most strongly correlated to CE levels (r(2)=0.80) and an increased CE/FC ratio (r(2)=0.62) and the movement of cholestatic lipid in the LDL to HDL. Since LCAT activity was not affected by ANIT treatment, these results demonstrate that the ability of LCAT to esterify the plasma FC present in cholestatic liver disease is limited by in vivo apo A-I activation of the cholestatic lipid and not by the catalytic capacity of LCAT.

1-Naphthylisothiocyanate↗
PubMed1999

Classical LCAT deficiency resulting from a novel homozygous dinucleotide deletion in exon 4 of the human lecithin: cholesterol acyltransferase gene causing a frameshift and stop codon at residue 144.

Lecithin: cholesterolacyltransferase (LCAT) transacylates the fatty acid at the sn-2 position of lecithin to the 3beta-OH group of cholesterol forming lysolecithin and the majority of cholesteryl ester found in plasma. LCAT participates in the reverse cholesterol transport pathway in man where it esterifies tissue-derived cholesterol following efflux from peripheral cells into HDL. Only 38 unique mutations in the human LCAT gene have been reported worldwide. Our French female proband presented with corneal opacity and no detectable plasma LCAT activity using either endogenous or exogenous assays. Her total plasma cholesterol and HDL cholesterol were low (2.34 mmol/l and 0.184 mmol/l, respectively) with a very high cholesterol/cholesteryl ester molar ratio (10.9:1). Plasma triglycerides were 0.470 mmol/l with low apo B (40.5 mg/dl), apo A-I (14.7 mg/dl), apo A-II (6.8 mg/dl) and apo E (2.1 mg/dl) levels. Plasma lipoprotein analysis by ultracentrifugation showed very low HDL concentrations and a characteristic shift of the lipoprotein profile towards larger, less dense particles. No proteinuria, renal dysfunction or signs of atherosclerosis were noted at age 45. Sequence analysis of her LCAT gene showed a novel homozygous TG-deletion at residues 138-139 that resulted in a frameshift causing the generation of a stop codon and premature termination of the LCAT protein at amino acid residue 144. Western blotting of the patient's plasma using a polyclonal IgY primary antibody against human LCAT failed to demonstrate the presence of a truncated LCAT protein. A 53 bp mismatched PCR primer was designed to generate an Fsp 1 restriction site in the wild type sequence of exon 4 where the mutation occurred. The 155 bp PCR product from the wild type allele produced a 103 bp and 52 bp fragment with Fsp 1 and no cleavage products with the mutant allele thus permitting rapid screening for this novel mutation.

Adolescent↗
PubMed1999

High plasma cholesterol in drug-induced cholestasis is associated with enhanced hepatic cholesterol synthesis.

In alpha-naphthylisothiocyanate-treated mice, plasma phospholipid (PL) levels were elevated 10- and 13-fold at 48 and 168 h, respectively, whereas free cholesterol (FC) levels increased between 48 h (17-fold) and 168 h (39-fold). Nearly all of these lipids were localized to lipoprotein X-like particles in the low-density lipoprotein density range. The PL fatty acyl composition was indicative of biliary origin. Liver cholesterol and PL content were near normal at all time points. Hepatic hydroxymethylglutaryl CoA reductase activity was increased sixfold at 48 h, and cholesterol 7alpha-hydroxylase activity was decreased by approximately 70% between 24 and 72 h. These findings suggest a metabolic basis for the appearance of abnormal plasma lipoproteins during cholestasis. Initially, PL and bile acids appear in plasma where they serve to promote the efflux of cholesterol from hepatic cell membranes. Hepatic cholesterol synthesis is then likely stimulated in the response to the depletion of hepatic cell membranes of cholesterol. We speculate that the enhanced synthesis of cholesterol and impaired conversion to bile acids, particularly during the early phase of drug response, contribute to the accumulation of FC in the plasma.

1-Naphthylisothiocyanate↗
PubMed1999

Characterization of C-terminal histidine-tagged human recombinant lecithin:cholesterol acyltransferase.

Lecithin:cholesterol acyltransferase (LCAT) is the plasma enzyme that catalyzes esterification of the sn-2 fatty acid of phospholipid to cholesterol. To facilitate the isolation of large quantities of LCAT and to assist in future structure;-function studies, LCAT containing a carboxy-terminal histidine-tag (H6) was expressed in Chinese hamster ovary cells (CHO). A high level of CHO-hLCATH6 expression ( approximately 15 mg L(-1)) was achieved over a 72-h period using 10 mm sodium butyrate to enhance transcription and PFX-CHO protein-free medium. The pure enzyme ( approximately 96%) was isolated by cobalt metal affinity chromatography with an activity yield of 82 +/- 26%. CHO-hLCATH6 and CHO-hLCAT species had identical specific activities (26 +/- 6 and 26 +/- 3 nmol CE formed microg(-1) h(-1), respectively). The enzymatic activity of CHO-hLCATH6 was stable at 4 degrees C in excess of 60 days. Substrate saturation studies, using rHDL composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and apolipoprotein A-I (80:5:1) indicated that the appK(m) for CHO-hLCATH6, CHO-hLCAT, and purified plasma LCAT were nearly identical at approximately 2 microm substrate cholesterol. We conclude that carboxy-terminal histidine-tagged LCAT is a suitable replacement for both plasma LCAT and CHO-hLCAT.

Animals↗
PubMed1996

Abnormal lipoproteins in the ANIT-treated rat: a transient and reversible animal model of intrahepatic cholestasis.

The alpha-naphthylisothiocyanate (ANIT)-treated rat was evaluated as a model for lipoprotein metabolism in cholestatic liver disease. Alterations in lipoprotein composition over a period of 120 h after ANIT treatment (100 mg/kg) were studied. Eighteen hours after treatment, plasma bilirubin and bile acid levels began to rise, together with significant increases in free cholesterol. C-18/16, C-18/18, and C-18/20 phospholipid molecular species. By 48 h, plasma lipid levels were maximal, free cholesterol was 935%, cholesteryl ester 294%, phospholipid 611%, and triacylglycerols 176% of controls, and the cholesteryl ester to free cholesterol ratio began to recover with a modest shift from cholesteryl esters containing C-20 fatty acids to those containing C-18 fatty acids. Lecithin: cholesterol acyltransferase activity was near normal, lipoprotein lipase activity was increased, and hepatic triacylglycerol lipase activity was decreased. Density gradient ultracentrifugation of rat plasma demonstrated a marked shift in lipoprotein density towards the low density lipoprotein range, with the increased lipid being associated with apolipoproteins A-I and E. The presence of large 300-400 A particles and the high surface to core lipid ratio in this density range was consistent with the presence of lipoprotein-X-like vesicles. Apolipoprotein B-48 accumulation was observed in the high density fractions (d15 > 1.063 g/ml) suggesting that these rats have impaired lipoprotein remnant removal. All of these increased levels returned to near normal by 120 h. This study demonstrates that ANIT-treatment induces a transient, fully reversible, non-surgical intrahepatic cholestasis that results in many of the plasma lipoprotein abnormalities associated with human hepatic cholestasis and the bile duct-ligated rat.

1-Naphthylisothiocyanate↗