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Title: Identification of Protein Carbonyls in serum of the fetal and neonatal pig.

Author
item Caperna, Thomas
item Shannon, Amy
item Blomberg, Le Ann
item Garrett, Wesley
item Ramsay, Timothy

Submitted to: Comparative Biochemistry and Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/19/2010
Publication Date: 7/1/2010
Citation: Caperna, T.J., Shannon, A.E., Blomberg, L., Garrett, W.M., Ramsay, T.G. 2010. Identification of protein carbonyls in serum of the fetal and neonatal pig. Comparative Biochemistry and Physiology. 156(3):189-196.

Interpretive Summary: Oxidation of serum proteins leads to carbonyl formation which alters their function and has long been associated with stress-related disease processes. The primary objective of this study was to identify oxidized serum protein biomarkers of metabolic stress in baby pigs and fetuses. Protein carbonyls in serum, were chemically converted to colored compounds so that they could be quantified by spectrophotometry. To identify specific carbonylated proteins, serum protein carbonyls were derivatized to yield biotin derivatives which could separated by electrophoresis and visualized by staining the incorporated biotin with fluorescent stain. To identify individual proteins, stained spots were cut from the two dimensional gels and were digested with trypsin and the resulting peptides were analyzed by mass spectrometry and matched to proteins within public databases. At birth, significant amounts of oxidized proteins were readily determined in piglet serum. Fetuses at 50 and 100 d of gestation were also evaluated and found to have similar levels of protein carbonyls as newborns. The levels of carbonyls were similar for control and runt (< 1 kg at birth) piglets between 1 and 21 days of age, however the distribution of many proteins varied by age and were also influenced by the weight of piglets at birth. Major oxidized proteins identified by mass spectrometry in fetal (f) and newborn (n) pigs included; albumin (f,n), transferrin (f,n), fetuin-A (f,n) alpha fetoprotein (f,n), plasminogen (f,n), fetuin-B (f), alpha 1 antitrypsin (f,) alpha 1-acid glycoprotein (f) immunoglobulins (n) and alpha-antichymotrypsin (n). While, the relative abundance and distribution of individual oxidized proteins changed over time for fetal and neonatal piglets, these changes appear to primarily reflect the relative amounts of those proteins in the serum. In addition, it appears that differences which occur in the physiology of runts compared to normal littermates do not reflect an overall increase in oxidative stress as determined by serum protein carbonyl analysis.

Technical Abstract: Oxidation of serum proteins leads to non-reversible carbonyl formation which alters their function and has long been associated with stress-related disease processes. The primary objective of this study was to identify oxidized serum protein biomarkers of metabolic stress in baby pigs. Protein carbonyls in serum, were converted to dinitrophenyl (DNP) derivatives with DNP hydrazine, precipitated with TCA, extracted in ethyl acetate:ethanol and quantified by spectrophotometry. To identify specific carbonylated proteins, serum protein carbonyls were derivatized with biotin hydrazide, separated by 1D and 2D PAGE and visualized by staining with FITC-avidin and fluorescence imaging. Gels were also stained with Coomassie blue and biotin-labeled proteins were excised from the gels, digested with trypsin and identified by mass spectrometry (MS). At birth, significant amounts of oxidized proteins were readily determined in piglet serum (~600 pmole/mg protein). Fetuses at 50 and 100 d of gestation were also evaluated and found to have similar levels of protein carbonyls as newborns. The levels of carbonyls were similar for control and runt (< 1 kg at birth) piglets between 1 and 21 days of age, however the distribution of many proteins varied by age and were also influenced by the weight of piglets at birth. Major oxidized proteins identified by MS in fetal (f) and newborn (n) pigs included; albumin (f,n), transferrin (f,n), fetuin-A (f,n) alpha fetoprotein (f,n), plasminogen (f,n), fetuin-B (f), alpha 1 antitrypsin (f,) alpha 1-acid glycoprotein (f) immunoglobulins (n) and alpha-antichymotrypsin (n). While, the relative abundance and distribution of individual oxidized proteins changed over time for fetal and neonatal piglets, these changes appear to primarily reflect the relative amounts of those proteins in the serum.