Alcoholic proteomics: Ethanol effects on proteins in rat liver and serum

Alcohol abuse

Proteomics has been used in the past to try and elucidate the changes that occur to the proteins in the body following chronic alcohol abuse, in order to locate biomarkers and to try and figure out the widespread alterations that take place. Liver damage is one serious outcome of long-term alcohol exposure and this organ is generally targeted by researchers in the field.

A team of scientists in Japan has taken a broader approach, preferring to examine both liver tissue and serum. Fumio Nomura and colleagues from Chiba University, Chiba University Hospital and the National Institute of Biomedical Innovation, Osaka, hoped that this combined would give a more comprehensive view of the proteomic alterations and provide greater insight into the disease. In their studies on rats fed for eight weeks on a liquid diet containing ethanol as 36% of the total energy, they decided on two unconventional methods to extract the proteins.

Protein extraction

Proteins in liver homogenate and the liver cytosol fractions were extracted using two-dimensional gel electrophoresis in which the first step was agarose gel isoelectric focusing, rather than immobilized pH gradient (IPG) gels. The advantages of this switch are two-fold, allowing more proteins to be loaded to the gel and resolving proteins with molecular masses larger than 150 kDa.

Serum samples were processed by a three-step method developed by the team themselves in which preliminary immunodepletion of the most abundant proteins was followed by RP-HPLC then one-dimensional SDS-PAGE. This simple and highly reproducible procedure helps to uncover the less abundant proteins and has been used with some success to find low-abundance biomarkers for sepsis and breast cancer.

The separated proteins on the final gels from both systems were located by staining and identified by tandem mass spectrometry following digestion with trypsin. Many protein spots were observed with molecular masses ranging from 21.5 to 200 kDa. When compared with control samples from rats fed a normal diet, 46 proteins from the liver were found to be either up- or down-regulated and 77 were altered in serum.

Biomarkers in liver and serum

Only eight of the proteins from the liver with altered expressions could be identified by mass spectrometry but they included one which had been reduced a huge amount by the effects of alcohol. The abundance of carbonic anhydrase III was reduced by 75% in the liver homogenate and 70% in the liver cytosol fraction.

Carbonic anhydrases are zinc-containing proteins whose main function is to convert carbon dioxide and water into bicarbonate, thereby regulating pH and fluid balance in different parts of the body. Carbonic anhydrase III has also been reported to have antioxidant properties. Nomura speculated that its decline in the liver after sustained alcohol exposure is linked to oxidative stress.

An earlier report on alcohol exposure in minipigs found that carbonic anhydrase III was increased in the liver, so there are some interspecies differences. The observation of reduced levels in the rat does not automatically mean that it will also be reduced in humans.

In serum, carbonic anhydrase was not detected by the three-step procedure used to isolate the proteins. One notable finding was the up-regulation of betaine homocysteine methyltransferase (BHMT) in the alcohol-exposed rats. In fact, this was the only protein found to be higher in both the liver and serum, although different proteomics approaches might detect others. The biological significance of increased levels of BHMT is unclear at present but it has been associated with alcoholic fatty liver in previous studies.

For the other proteins with altered expressions, “in-depth analyses of how these proteins are involved in alcohol-induced liver injury pathways are needed,” Nomura declared. “Moreover, the biological relevance of some of the changes in protein expression we observed remains to be clarified.”

A number of those proteins have antioxidant properties, so the researchers are planning to carry out another study that will look at oxidised proteins in particular.