Abundance of plasma antioxidant proteins confers tolerance to acute hypobaric hypoxia exposure

Abstract

Systematic identification of molecular signatures for hypobaric hypoxia can aid in better understanding of human adaptation to high altitude. In an attempt to identify proteins promoting hypoxia tolerance during acute exposure to high altitude, we screened and identified hypoxia tolerant and susceptible rats based on hyperventilation time to a simulated altitude of 32,000 ft (9754 m). The hypoxia tolerance was further validated by estimating 8-isoprotane levels and protein carbonyls, which revealed that hypoxia tolerant rats possessed significant lower plasma levels as compared to susceptible rats. We used a comparative plasma proteome profiling approach using 2-dimensional gel electrophoresis (2-DGE) combined with MALDI TOF/TOF for both groups, along with an hypoxic control group. This resulted in the identification of 19 differentially expressed proteins. Seven proteins (TTR, GPx-3, PON1, Rab-3D, CLC11, CRP, and Hp) were upregulated in hypoxia tolerant rats, while apolipoprotein A-I (APOA1) was upregulated in hypoxia susceptible rats. We further confirmed the consistent higher expression levels of three antioxidant proteins (PON1, TTR, and GPx-3) in hypoxia-tolerant animals using ELISA and immunoblotting. Collectively, these proteomics-based results highlight the role of antioxidant enzymes in conferring hypoxia tolerance during acute hypobaric hypoxia. The expression of these antioxidant enzymes could be used as putative biomarkers for screening altitude adaptation as well as aiding in better management of altered oxygen pathophysiologies.

FIG. 1.

Hyperventilation time of Sprague Dawley rats on exposure to hypobaric hypoxia at 9754 m, 32°C. The hyperventilation time is expressed in Mean±SEM for each group of animals.

FIG. 2.

Determination of 8-isoprostane and protein carbonyls in hypoxia tolerant and susceptible rats. (A) Plasma 8-isoprostane levels were lower in tolerant while susceptible have higher levels and were measured in pg/mL. (B) The amount of protein carbonyls were observed to be higher in susceptible animals and expressed as nmol/mL. All the results are expressed as Mean±SEM. (* represents p<0.05 vs. Control, and $ indicates p<0.05 vs. hypoxia tolerant according to Newman–Keuls multiple comparison tests).

FIG. 3.

A representative 2-D gel image of hypobaric hypoxia exposed rat plasma following albumin and IgG depletion. Proteins (500 μg) were separated by IEF using pH 4–7, 18 cm IPG strips (GE Healthcare, Sweden) and 12% SDS PAGE. The gels were silver stained and analyzed by Progenesis Same spots 2D gel image analysis software (Nonlinear Dynamics). Spot numbers represent differentially expressed proteins (±1.5-fold, p<0.05) among tolerant and susceptible compared with control.

FIG. 4.

Zoom-in images of 2-DGE of differentially expressed spots in control, Susceptible, Tolerant rat plasma. Spots were identified by MS/MS. C, Control, S, Susceptible, T, Tolerant.

FIG. 5.

Estimation of PON 1 activity in rat blood plasma. Results are given in Mean±SEM. (** indicates p<0.001 vs. Control). The concentration of plasma PON 1activity were higher in tolerant rats was 0.31±0.01 U/μL as compared to hypoxia susceptible animals (0.03±0.01 U/μL, p<0.001).

FIG. 6.

(A) Validation of differentially expressed protein (PON 1, TTR, GPx-3) by immunoblot analysis. Total plasma protein (30 μg/lane) were separated by 10% SDS-PAGE and probed with primary antibody for PON 1, TTR, and GPx-3. (B) Graphical representation of the optical density of PON 1, TTR, GPx-3 normalized to β-tubulin indicating the expression levels. Data represent Mean±S.E.M of three independent experiments. (* represents p<0.05 vs. Control according to Newman–Keuls multiple comparison tests).

FIG. 7.

Estimation of nitric oxide levels in rat blood plasma. Results are given in Mean±SEM. (* indicates p<0.05 vs. Control). The concentration of plasma nitric oxide was higher in tolerant rats (10.64±0.87 μmol/L, p<0.005) as compared to the hypoxia susceptible animals (6.4±0.48 μmol/L).