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Title: Analysis of protein oxidation in serum of fetal and newborn piglets and the influence of iron dextran on induction of protein carbonyls.

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

Submitted to: American Society of Animal Science
Publication Type: Abstract Only
Publication Acceptance Date: 4/27/2009
Publication Date: 7/12/2009
Citation: Caperna, T.J., Shannon, A.E., Ramsay, T.G., Blomberg, L., Garrett, W.M. 2009. Analysis of protein oxidation in serum of fetal and newborn piglets and the influence of iron dextran on induction of protein carbonyls. American Society of Animal Science, Abstract #33536.

Interpretive Summary: N/A

Technical Abstract: Methods were employed to evaluate serum biomarkers associated with protein oxidative stress and damage, to determine potential sources 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 (1:1) and quantified by spectrophotometry. DNP-labeled proteins were then separated by 2D PAGE, electro-blotted onto PVDF membranes and visualized by western immunoblot analysis using a polyclonal anti-DNP reagent. Proteins which reacted positively were extracted from a duplicate 2D gel, digested with trypsin and identified by MALDI-TOF mass spectrometry (MS). At birth, significant amounts of oxidized proteins were readily determined in piglet serum (~1 nmole/mg protein). Fetuses at 50 and 110 d of gestation were also evaluated and found to have potentially higher levels of protein carbonyls, than newborns. We then determined that the standard iron dextran treatment used to prevent iron deficiency anemia (100 mg iron given intramuscularly), was associated with a two-fold increase (P<.05) in oxidized proteins by 48 hrs, if given on the first day of life. Oxidized proteins which were identified by MS included; albumin, transferrin, alpha fetoprotein, immunoglobulins and fetuin. Postponement of iron treatment until the third day of life also resulted in an increase in protein carbonyls, but at attenuated levels (P<.05), compared to earlier-treated piglets. These results indicate that it may be possible to reduce the impact of iron dextran as a source of oxidative stress in newborn pigs by adjusting the timing of treatment. In addition, because of the sensitivity and simplicity of the analysis which was routinely performed using one mg of serum protein, evaluation of protein carbonyls may be a rapid and useful tool to monitor oxidative stress associated with neonatal mortality in the pig.