Determination of enzymatic source of alanine aminotransferase activity in serum from dogs with liver injury.
BACKGROUND
Increase of serum alanine aminotransferase (ALT) activity is widely used as a surrogate marker for tissue damage. Two ALT isoforms, ALT1 and ALT2, have been cloned recently in mammals. The study investigated the source of elevated ALT activity in serum of dogs treated with a hepatotoxic compound.


METHODS
ALT activity was measured by enzyme assay. Immunoblot analysis was performed using generated specific peptide antibodies against dog ALTs. LC-MS/MS-based proteomics analysis was conducted to independently identify dog ALT peptides. Serum samples immunodepleted of major serum components by Seppro IgY-D11 microbead spin column were evaluated by the immunoblot analysis, and compared with those of the ALT activity.


RESULTS
Involvement of ALT enzyme(s) is consistent with the following observations: 1) all the substrates (L-alanine and alpha-ketoglutarate) were required for serum ALT activity as purified porcine ALT1 needed for activity, 2) serum ALT activity was inhibited by L-cycloserine, a known ALT inhibitor, and 3) apparent Km value for the ALT reaction catalyzed by the serum, liver, and skeletal muscle was roughly similar. Immunoblot analysis showed that ALT1 was detected in liver and both ALTs were detected in the skeletal muscle. The relative expression level of ALTs was -: liver ALT1>skeletal muscle ALT1>skeletal muscle ALT2. LC-MS/MS-based proteomics analysis gave similar results. Immunoblot analysis of the depleted serum samples revealed the presence of ALT1 in compound-treated dogs. Intensity of the ALT1 band detected in the sera correlated well with the ALT activity measured by the enzyme assay.


CONCLUSIONS
Based on these findings, we conclude that the elevation of serum ALT activity in dogs with liver injury is attributed to elevation of ALT1 protein level in serum. The methodology to directly detect ALT proteins in serum could be a tool to facilitate our understanding of biological and toxicological significance of the ALT isoenzymes.
Miyazaki
Masafumi
M
ActivX Biosciences Inc., 11025 North Torrey Pines Road, La Jolla, California 92037, USA. masafumi.miyazaki@mb.kyorin-pharm.co.jp
Rosenblum
Jonathan S
JS
Kasahara
Yasushi
Y
Nakagawa
Ippei
I
Patricelli
Matthew P
MP
D000410:Alanine Transaminase / Q000097:blood* / Q000145:classification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers / Q000097:blood / Q000378:metabolism; D004195:Disease Models, Animal; D004285:Dogs; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000097:blood / Q000378:metabolism; D008107:Liver Diseases / Q000097:blood* / Q000201:enzymology* / Q000473:pathology; D008297:Male; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D015427:Reperfusion Injury / Q000097:blood* / Q000201:enzymology* / Q000473:pathology; D013379:Substrate Specificity / Q000502:physiology; D013552:Swine
D015415:Biomarkers; D007527:Isoenzymes; D000410:Alanine Transaminase