Hyperhomocysteinemia and Low Folate and Vitamin B12 Are Associated with Vascular Dysfunction and Impaired Nitric Oxide Sensitivity in Morbidly Obese Patients

Abstract

There is a high prevalence of hyperhomocysteinemia that has been linked to high cardiovascular risk in obese individuals and could be attributed to poor nutritional status of folate and vitamin B12. We sought to examine the association between blood homocysteine (Hcy) folate, and vitamin B12 levels and vascular dysfunction in morbidly obese adults using novel ex vivo flow-induced dilation (FID) measurements of isolated adipose tissue arterioles. Brachial artery flow-mediated dilation (FMD) was also measured. Subcutaneous and visceral adipose tissue biopsies were obtained from morbidly obese individuals and non-obese controls. Resistance arterioles were isolated in which FID, acetylcholine-induced dilation (AChID), and nitric oxide (NO) production were measured in the absence or presence of the NO synthase inhibitor, L-NAME, Hcy, or the superoxide dismutase mimetic, TEMPOL. Our results demonstrated that plasma Hcy concentrations were significantly higher, while folate, vitamin B12, and NO were significantly lower in obese subjects compared to controls. Hcy concentrations correlated positively with BMI, fat %, and insulin levels but not with folate or vitamin B12. Brachial and arteriolar vasodilation were lower in obese subjects, positively correlated with folate and vitamin B12, and inversely correlated with Hcy. Arteriolar NO measurements and sensitivity to L-NAME were lower in obese subjects compared to controls. Finally, Hcy incubation reduced arteriolar FID and NO sensitivity, an effect that was abolished by TEMPOL. In conclusion, these data suggest that high concentrations of plasma Hcy and low concentrations of folate and vitamin B12 could be independent predictors of vascular dysfunction in morbidly obese individuals.

Keywords: bariatric surgery; folate; homocysteine; nitric oxide; obesity; vascular dysfunction; vitamin B12.

Figure 1

Duplex B-mode/pulsed wave Doppler (PWD) ultrasound of brachial artery flow-mediated dilation (FMD). This figure shows a long axis scan of brachial artery with simultaneous blood velocity profile by pulsed wave Doppler before (A) and after cuff deflation (B). For offline image analyses, a representative section of brachial artery is selected for an automated measurement of diameter. Baseline diameter (BSL) was averaged from a serial of recorded frames before cuff deflation and the maximum diameter was measured after cuff deflation during reactive hyperemia (RH).

Figure 2

Plasma Hcy, folate, vitamin B12, and NO concentrations and brachial artery FMD measurements. Plasma from obese subjects (n = 40) and non-obese controls (n = 40) were analyzed for Hcy (A), folate (B), and vitamin B12 (C) using specific ELISA assays and for nitrates + nitrites (NO metabolites) using the Griess chemical reaction assay (D). Percentage of brachial artery FMD was calculated by subtracting the mean baseline diameter from the largest mean values obtained after cuff deflation in obese subjects (n = 40) and non-obese controls (n = 40) (E). All measurements are presented as means ± standard error (SE). * (p < 0.05) for comparing obese subjects with controls.

Figure 3

FID and acetylcholine-induced dilation (AchID) in SAT isolated resistance arterioles. FID measurements in SAT arterioles isolated from obese (n = 40) and non-obese (n = 40) subjects corresponding to increasing intraluminal pressure gradients of 10–100 cmH₂O (A). AchID measurements in SAT arterioles corresponding to increasing concentrations of Ach (10⁻⁹ to 10⁻⁴ M) (B). Absolute reduction in FID in response to eNOS inhibition via L-NAME (10⁻⁴ M) (C). Absolute reduction in AchID in response to eNOS inhibition via L-NAME (D). All measurements are presented as means ± standard error (SE). * (p < 0.05) for comparing obese subjects with controls.

Figure 4

FID and AChID in VAT isolated resistance arterioles. FID measurements in VAT arterioles isolated from obese (n = 40) and non-obese (n = 40) subjects corresponding to increasing intraluminal pressure gradients of 10–100 cmH₂O (A). AchID measurements in VAT arterioles corresponding to increasing concentrations of Ach (10⁻⁹ to 10⁻⁴ M) (B). Absolute reduction in FID in response to eNOS inhibition via L-NAME (10⁻⁴ M) (C). Absolute reduction in AchID in response to eNOS inhibition via L-NAME (D). All measurements are presented as means ± standard error (SE). * (p < 0.05) for comparing obese subjects with controls.

Figure 5

Effect of Hcy incubation on baseline FID and L-NAME-mediated reduction in FID. SAT (A,B) and VAT (C,D) isolated arterioles from obese subjects (n = 10) and non-obese controls (n = 10) were incubated in100 µM of Hcy for 180 min followed by measuring FID with and without eNOS inhibition via L-NAME (10⁻⁴ M). Charts (A,C) present absolute reductions in FID in Hcy preconditioned arterioles compared to corresponding unconditioned arterioles. Charts (B,D) present absolute reduction in FID caused by L-NAME in Hcy preconditioned arterioles compared with baseline FID after Hcy incubation. All measurements are presented as means ± standard error (SE). * (p < 0.05) for comparing obese subjects with controls.

Figure 6

Effect of TEMPOL on restoring FID in Hcy preconditioned arterioles. Isolated arterioles from obese subjects (n = 10) and non-obese controls (n = 10) were incubated in100 µM of Hcy and the superoxide dismutase mimetic, TEMPOL (10⁻⁵ M) for 180 min followed by measuring the FID. Charts (A) and (B) present the absolute increase in FID in response to combined incubation with Hcy and TEMPOL relative to Hcy alone in SAT and VAT arterioles, respectively. All measurements are presented as means ± standard error (SE). * (p < 0.05) for comparing obese subjects with controls.

Figure 7

Endothelium-independent vasodilation in SAT and VAT isolated arterioles. The intraluminal diameter of SAT (A) and VAT (B) isolated arterioles was measured in response to increasing concentrations of SNP (10⁻⁹−10⁻⁴ M) in obese subjects (n = 40) and non-obese controls (n = 40). The absolute difference in SNP-induced vasodilation between Hcy-preconditioned and unconditioned SAT (C) and VAT (D) arterioles in obese subjects (n = 10) and non-obese controls (n = 10). All measurements are presented as means ± standard error (SE).

Figure 8

NO and Reactive Oxygen Species (ROS) production in isolated adipose tissue arterioles. (A): Representative images by fluorescence microscopy of NO (red fluorescence) and ROS (green fluorescence) generation at baseline conditions and after incubation with L-NAME in adipose tissue arterioles collected obese subjects (n = 40) and non-obese controls (n = 40). The charts present NO (B) and ROS (C) fluorescent signals that were measured and expressed in arbitrary units using NIH Image J software. All measures are represented as means± SE. * (p < 0.05) for comparing L-NAME to baseline in each group, † (p < 0.05) for comparing obese subjects with controls, and ₸ (p < 0.05) for comparing SAT and VAT arterioles in each treatment condition.

Figure 9

NO and ROS changes in response to homocysteine (Hcy) and TEMPOL. (A): Representative images by fluorescence microscopy of NO (red fluorescence) and ROS (green fluorescence) generation in response to Hcy and Hcy + TEMPOL treatment conditions in adipose tissue arterioles collected obese subjects (n = 10) and non-obese controls (n = 10). The charts present NO (B) and ROS (C) fluorescent signals that were measured and expressed in arbitrary units using NIH Image J software. All measures are represented as means± SE. * (p < 0.05) for comparing L-NAME to baseline in each group, and † (p < 0.05) for comparing obese subjects with controls.