Effect of increasing maternal body mass index on oxidative and nitrative stress in the human placenta

Abstract

Maternal obesity is an increasing problem in obstetrics associated with adverse pregnancy outcomes and delivery complications. As an inflammatory state, where elevated levels of pro-inflammatory cytokines are found, obesity can lead to the increased incidence of oxidative and nitrative stress. These stresses may result in protein oxidation and protein nitration respectively, which are post- translational covalent modifications that can modify the structure and subsequently alter the function of a protein. The objective of this study was to examine whether placental oxidative and nitrative stress increase with increasing maternal body mass index. Placental tissue was collected from three groups of patients categorized as lean, overweight and obese. The presence of nitrotyrosine residues, a marker of nitrative stress, and antioxidant enzymes, as markers of oxidative stress, were assessed by immunohistochemistry, Western blot and ELISA. Protein carbonyl formation, a specific measure of protein oxidation, was measured by OxyBlot kit. Nitrotyrosine residues were increased in obese compared to lean and overweight groups although localization was unaltered across the three groups. Superoxide dismutase enzyme expression, localization and activity was unaltered between the groups. Protein carbonyl formation was greater in the lean compared to the overweight individuals. This study demonstrates that with increasing maternal body mass index there is an increase in placental nitrative stress. There does not appear to be a corresponding increase in oxidative stress and indeed we demonstrate some evidence of a decrease in oxidative effects in these placenta samples. Potentially the formation of peroxynitrite may be consuming reactive oxygen species and reducing oxidative stress. There may be a shift in the balance between nitrative and oxidative stress, which may be a protective mechanism for the placenta.

Figure 1

Immunolocalization of nitrotyrosine residues (A-C), MnSOD (E-G), CuZnSOD (I-K) and ECSOD (M-O) in placental sections from lean (LN; A, E, I, M), overweight (OV; B, F, J, N) and obese (OB; C, G, K, O) patients. Negative control sections where primary antibody was omitted (D, H, L, P). Original magnification 200×; scale bar = 50μm. Positive staining is indicated by the arrow heads (▶).

Figure 2

Representative Western blots demonstrating A (i); nitrated protein expression, B (i); MnSOD monomer expression, C (i) CuZnSOD and D (i); ECSOD expression in lean (LN), overweight (OV) and obese (OB) patient placental samples. A-D (ii); Corresponding blots from A-D (i) stripped and re-probed with β-actin as a protein loading control. Molecular weight markers are indicated (kDa).

Figure 3

ELISA data showing A; total nitrotyrosine residue levels, B; total superoxide dismutase (SOD) enzyme activity and C; glutathione peroxidase (Gpx) enzyme activity in lean (LN), overweight (OV) and obese (OB) patient placental homogenate samples. *p<0.001 one way ANOVA with Tukey post hoc test.

Figure 4

A; Representative Oxyblot showing derivatized (+) and paired non-derivatized controls (-) for three lean and three obese patients. Molecular weight markers are indicated (M) and sizes reported (kDa). Three bands were selected for densitometric analysis (labeled 1-3). B; Graphs showing densitometry data for bands 1, 2 and 3, * p<0.05 Mann Whitney U test. C; Graph showing the combined total densitometry data for bands 1-3, * p<0.05 Mann Whitney U test.