Nitric oxide synthetic pathway in red blood cells is impaired in coronary artery disease

Abstract

Background: All the enzymatic factors/cofactors involved in nitric oxide (NO) metabolism have been recently found in red blood cells. Increased oxidative stress impairs NO bioavailability and has been described in plasma of coronary artery disease (CAD) patients. The aim of the study was to highlight a potential dysfunction of the metabolic profile of NO in red blood cells and in plasma from CAD patients compared with healthy controls.

Methods: We determined L-arginine/NO pathway by liquid-chromatography tandem mass spectrometry and high performance liquid chromatography methods. The ratio of oxidized and reduced forms of glutathione, as index of oxidative stress, was measured by liquid-chromatography tandem mass spectrometry method. NO synthase expression and activity were evaluated by immunofluorescence staining and ex-vivo experiments of L-[(15)N2]arginine conversion to L-[(15)N]citrulline respectively.

Results: Increased amounts of asymmetric and symmetric dimethylarginines were found both in red blood cells and in plasma of CAD patients in respect to controls. Interestingly NO synthase expression and activity were reduced in CAD red blood cells. In contrast, oxidized/reduced glutathione ratio was increased in CAD and was associated to arginase activity.

Conclusion: Our study analyzed for the first time the whole metabolic pathway of L-arginine/NO, both in red blood cells and in plasma, highlighting an impairment of NO pathway in erythrocytes from CAD patients, associated with decreased NO synthase expression/activity and increased oxidative stress.

Figure 1. Plasma levels of analytes involved in Arginine/NO pathway.

(A) L-arginine (Arg), L-citrulline (Cit), L-ornithine (Orn) were simultaneously determined by LC-MS/MS. (B) Arg bioavailability and the relative activity of arginase and NOS enzymes are expressed as Arg/(Orn+Cit) and Orn/Cit ratios, respectively. (C) The endogenous inhibitors asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) were determined by LC-MS/MS. (D) Tetrahydrobiopterin (BH₄) and oxidized biopterins (Box) were detected by HPLC after selective oxidation with iodine. Data are presented as age and sex adjusted geometric means and 95% C.I. Comparisons between groups (CAD, n = 22; Ctrl, n = 20) were performed by covariance analysis, adjusting for age and sex.

Figure 2. RBC levels of analytes involved in Arginine/NO pathway.

(A) L-arginine (Arg), L-citrulline (Cit), L-ornithine (Orn) were simultaneously determined by LC-MS/MS. (B) Arg bioavailability and the relative activity of arginase and RBC-NOS enzymes are expressed as Arg/(Orn+Cit) and Orn/Cit ratios, respectively. (C) The endogenous inhibitors asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) were determined by LC-MS/MS. (D) Tetrahydrobiopterin (BH₄) and oxidized biopterins (Box) were detected by HPLC after selective oxidation with iodine. Data are presented as age and sex adjusted geometric means and 95% C.I. Comparisons between groups (CAD, n = 22; Ctrl, n = 20) were performed by covariance analysis, adjusting for age and sex.

Figure 3. Immunostaining of NO synthase (NOS) protein in human red blood cells (RBCs).

NOS was detected in RBCs from CAD patients (A) and from Ctrl (B). RBCs were incubated with a monoclonal anti eNOS antibody (2.5 µg/ml) and with an anti-mouse conjugated secondary antibody (40 µg/ml; Alexa Fluor488). The samples were mounted with fluorescent mounting medium and examined by laser scanning confocal microscope (LSM710, Carl Zeiss) using a 63×/1.3 oil immersion objective lens. Fluorescent images were captured with a digital camera using the image processor Zen (Carl Zeiss). (C) Fluorescence intensity (densitometric sum of grey) was quantified. Data are expressed as the log median of total fluorescence intensity per field ± interquartile range subtracted of the negative control value. Means derive from n = 10 CAD and n = 10 Ctrl.

Figure 4. NO synthase (NOS) activity in lysed human red blood cells (RBCs).

RBCs were washed and lysed with H₂O containing protease inhibitors. RBC-NOS activity was measured after the addition of L-[¹⁵N₂]arginine in the presence of arginase inhibitor nor-NOHA (50 µM). The levels of L-[¹⁵N]citrulline and L-[¹⁵N₂]arginine were determined by LC-MS/MS. The RBC-NOS activity was evaluated by the ratio between L-[¹⁵N]citrulline formed and L-[¹⁵N₂]arginine residue. Individual data are represented as geometric means for CAD (n = 8) and Ctrl (n = 8).