Pradeep Ghosh

Emerging Biomarkers : New directions and clinical applications

This article summarizes content proceedings of a symposium held at the 2004 Research Society on Alcoholism Scientific Annual Meeting in Vancouver, Canada. The chairs were Friedrich M. Wurst and Raye Litten. The presentations were (1) Introduction, by Raye Litten; (2) Direct Ethanol Metabolites--On the Threshold From Science to Routine Use, by Friedrich M. Wurst; (3) Sialic Acid Index of Plasma Apolipoprotein J (SIJ) as a Viable Marker for Chronic Alcohol Consumption, by Philippe Marmillot; (4) The Emergence of Ethyl Glucuronide (EtG) Testing as a Tool in Monitoring Healthcare Professionals, by Gregory E. Skipper; (5) Application of Biomarkers for Alcohol Use Disorders in Clinical Practice, by Tim Neumann; (6) Utility of Biomarkers in Assessing the Efficacy of Medications for Treating Alcoholism, by Marty Javors; and (7) Discussion, by Raye Litten.

Long-term ethanol exposure impairs glycosylation of both N- and O-glycosylated proteins in rat liver

Carbohydrate residues of glycoproteins play important roles in their functions. We have previously shown that long-term ethanol treatment in rats alters the normal glycosylation pattern of plasma transferrin and apolipoprotein (apo) E. Glycosylation of proteins is a posttranslational process that is regulated by both glycosyltransferases and glycosidases, the resident enzymes of hepatic subcellular organelles. In this investigation using rat transferrin and apo E as model N- and O-glycosylated proteins, respectively, we have explored the effects of long-term ethanol treatment on the (1) incorporation of various labeled sugar precursors into these specific glycoproteins, (2) activities of mannosyltransferase, galactosyltransferase, and sialytransferases, and (3) hepatic synthetic rate of N-acetyl glucosamine (GlcNAc) alpha 2,6-sialyltransferase (2,6-ST). The relative ratio of labeled sugar to leucine incorporation (glycosylation index) showed a 43% (P < .01) decrease for relative mannosylation of transferrin molecule at both the microsomal and Golgi level in the ethanol group (AN) versus the control group (CN). For apo E, relative mannosylation was reduced by 48.9% (P < .01) and 46.9% (P < .01), respectively, at the microsomal and Golgi level in the AN versus CN. More importantly, relative sialation of transferrin was reduced by 86% (P < .001) in AN as compared with CN. Relative sialation of apo E was reduced by 35% (P < .01) in AN as compared with CN.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma sialic-acid index of apolipoprotein J (SIJ): a new alcohol intake marker

Although plasma carbohydrate-deficient transferrin (CDT) is considered a viable biochemical marker for chronic alcohol consumption, it is valid only when an individuals daily alcohol consumption exceeds 60 g. In addition, it is less sensitive in women drinkers than in men drinkers. We have established that chronic alcohol consumption impairs the hepatic sialylation of a number of glycoproteins by specifically down-regulating Gal-beta-1,4GlcNAc alpha2,6-sialyltransferase mRNA. Significantly, we found that chronic ethanol consumption markedly inhibits hepatic sialylation of apolipoprotein J (Apo J), a 70-kDa N-glycosylated protein of plasma HDL. Because the sialic-acid index of Apo J (SIJ; moles of sialic acid per mole of Apo J protein) is approximately seven times more than that for transferrin (28 vs. 4), we have evaluated whether plasma SIJ would be an even more sensitive marker for chronic ethanol consumption than CDT in both rats and human subjects. The method involves immunoaffinity purification of plasma HDL-Apo J, followed by its sialic acid determination. We have found that chronic ethanol feeding resulted in loss of sialic acid residues of plasma HDL-Apo J in rats. This loss of sialic acid was positively correlated with both amount and duration of ethanol treatment. In human subjects, an intake of about 60 g of alcohol for 30 days led to almost 50% (P <.01) depletion of sialic acid from plasma HDL-Apo J. Further, we established that there was a positive correlation of alteration in SIJ with alcohol consumption, detoxification, abstinence, and relapse in human alcohol-dependent patients (sensitivity, 90%-92%). In addition, plasma SIJ was decreased by 50%-57% (P <.01) in both male and female alcohol-dependent subjects. We suggest that plasma SIJ can be used as a viable marker for early detection of chronic alcohol consumption in human beings.

Purification and Partial Characterization of a Cellular Carotenoid-binding Protein from Ferret Liver

A cellular carotenoid-binding protein was purified to homogeneity from β-carotene-fed ferret liver utilizing the following steps: ammonium sulfate precipitation, ion exchange, gel filtration, and affinity chromatography. The final purification was 607-fold. [14C]β-Carotene co-purified with the binding protein throughout the purification procedures. SDS-PAGE of the purified protein showed a single band with an apparent molecular mass of 67 kDa. Scatchard analysis of the specific binding of the purified protein to β-carotene showed two classes of binding sites, a high affinity site with an apparent K d of 56 × 10−9 m and a low affinity site with aK d of 32 × 10−6 m. The B max for β-carotene binding to the high affinity site was 1 mol/mol, while that for the low affinity site was 145 mol/mol. The absorption spectrum of the complex showed a 32-nm bathochromic shift in λmax with minor peaks at 460 and 516 nm. Except for α-carotene and cryptoxanthin, none of the model carotenoids or retinol competed with β-carotene binding to the protein. Thus, a specific carotenoid-binding protein of 67 kDa has been characterized in mammalian liver with a high degree of specificity for binding only carotenoids with at least one unsubstituted β-ionone ring.

Regulatory enzymes of lipid metabolism in LA/N-cp rats.

Hepatic activities of rate limiting enzymes in fatty acid and cholesterol synthesis and cholesterol degradation were determined in lean and obese LA/N-cp rats. The hepatic activities of acetyl-CoA carboxylase and fatty acid synthetase, the key enzymes of fatty acid synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase (the rate limiting enzyme in cholesterol synthesis), were increased 2-fold in the obese rats as compared with their lean littermates. In contrast, the activity of cholesterol 7alpha-hydroxylase, the rate limiting enzyme of cholesterol degradation to bile acids, was significantly decreased by 28% in the obese group as compared with the control group. Significantly, compared with the control group, the obese animals exhibited similar magnitudes of differences in the activities of the above enzymes even when they were pair-fed with the control animals. Thus these differences in the obese group are not due to hyperphagia but possibly to hypersecretion of the lipogenic hormone, insulin in this strain. These results indicate that the LA/N-cp obese rat has twice the capacity to synthesize body fat and cholesterol but has a reduced capacity to degrade the cholesterol, leading to increased accumulation of cholesterol and fat.

Ethanol, Lipoprotein Metabolism, and Fatty Liver

Persistent excessive intake of ethanol can lead to liver cell injury and eventually to cirrhosis of the liver after prolonged abuse over many years. There are individual variations in susceptibility to alcohol toxicities. Hepatocellular necrosis results in a wide variety of clinical symptoms ranging from a relatively asymptomatic enlargement of the liver to massive fatty infiltration that ultimately leads to hepatic failure.1,2As the process of alcohol-mediated injury progresses, liver fibrosis and eventually cirrhosis ensue, resulting in total failure of the liver functions. The pathogenesis of alcohol-induced toxicity generally begins with specific episodes of hepatocellular injury accompanied by varying degrees of fatty liver.3As the alcohol abuse continues, this degenerative process manifests itself into liver cell dysfunction, chronic inflammation, and structural distortion leading to the proliferation of fibrous tissue, which ultimately culminates into the cirrhosis of the liver and death.