L-arginine catabolism is driven mainly towards nitric oxide synthesis in the erythrocytes of patients with type 2 diabetes at first clinical onset

Abstract

Background

We investigated the l-arginine (l-Arg)–nitric oxide (NO) metabolic pathway in the erythrocytes (RBCs) and plasma of subjects with type 2 diabetes at first clinical onset.

Methods

RBCs and plasma were collected from 26 patients with type 2 diabetes at first clinical onset and 19 age-matched non-diabetes subjects as controls. l-Arg content was assayed by capillary electrophoresis. We measured arginase activity and nitrate/nitrite concentrations by spectrophotometry, and glycosylated haemoglobin (HbA1c) by standardized immunoturbidimetry.

Results

We found that, when compared with controls, l-Arg content was similar in RBCs while decreased in the plasma of patients with type 2 diabetes. Interestingly, arginase activity was lower in RBCs and increased in plasma of patients with diabetes. NO production was higher in RBCs in patients with type 2 diabetes, while no difference was found in the plasma of our subjects.

Conclusions

l-Arg catabolism is driven mainly towards NO synthesis in RBCs of patients with type 2 diabetes at first clinical onset. The decreased RBC arginase activity could be considered a potential mechanism of increased RBC NO production in early diabetes. Therefore, the RBC pool would represent a potentially compensatory intravascular compartment for endothelial dysfunction in diabetes.

Keywords

nitric oxide type 2 diabetes

Background

l-Arginine (l-Arg) exerts its important cardiovascular role mainly via nitric oxide (NO)-dependent processes.¹ In adults, endogenous synthesis of l-Arg involves the intestinal–renal axis.¹ Namely, citrulline is synthesized from glutamine, glutamate and proline in the mitochondria of enterocytes, released from the small intestine and taken up primarily by the kidneys for l-Arg production. The hepatic urea cycle does not lead to a net synthesis of l-Arg because the liver contains an exceedingly high arginase activity to hydrolyse arginine into urea plus ornithine.¹ l-Arg degradation produces NO, ornithine, urea, polyamines, proline, glutamate, creatine and/or agmatine and involves several pathways.¹ These pathways are initiated by arginases, three isoforms of nitric oxide synthase (NOS), l-Arg:glycine amidinotransferase and l-Arg decarboxylase.

In mammals, the arginase pathway is quantitatively most important for l-Arg catabolism.¹ Two arginase isoforms are expressed in mammals. While type II arginase is synthesized in all mitochondria-containing extrahepatic cells,² type I arginase is produced mainly in hepatocytes,³ and, to a limited extent, in extrahepatic cells,⁴ including red blood cells (RBCs).⁵

NO plays a critical role in vascular homeostasis.⁶ NO can be enzymatically synthesized in a reaction catalysed by NOS.⁷ Three isoforms of NOS are described: neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2) and endothelial NOS (eNOS or NOS3). A mitochondrial NOS isoform (mtNOS) is still under debate. Despite controversy,⁸ recent data⁹ have shown that NOS3 is expressed in RBCs and is potentially used as vascular source of NO. The non-enzymatic NO synthesis is activated when NO production from NOS is impaired.¹⁰ This alternative route involves nitrite-derived production of NO particularly under ischaemia conditions.¹¹ Enzyme-independent NO formation has been described both in different tissues¹⁰ and erythrocytes (RBCs).¹²

Endothelial dysfunction is accompanied by decreased plasma concentrations of l-arginine (l-Arg) in patients with diabetes.^13,14 However, data based on clinical studies using l-Arg supplementation in subjects with diabetes are still conflicting,^13,14 Despite evidence of RBC implication in NO-dependent l-Arg metabolic pathways,¹⁵ to our knowledge, there is a lack of information related to the involvement of RBCs in arginine metabolic pathways in type 2 diabetes at first clinical onset.

In the current study, we investigated the l-Arg–NO metabolic pathway in RBCs and plasma of subjects with type 2 diabetes at first clinical onset, with no previous medical history of diabetes.

Methods

Subjects

We enrolled 26 patients (10 women, 16 men) with type 2 diabetes at first clinical onset in the study. All the patients had no previous medical history of diabetes. Among the patients with diabetes, 13 (50%) had no chronic complications (5 women, 8 men), and hypertension was initially found in 13 (50%) cases (5 women, 8 men). All patients received no previous treatment for diabetes or high blood pressure. Nineteen age-matched volunteers (12 women, 7 men) were recruited as controls from general practitioner files. The study was approved by the ethics committee of the National Institute of Diabetes, Nutrition and Metabolic Diseases ‘N. C. Paulescu’ (reference number 199/2009). Written informed consent was obtained from all participants in the study. Subject characteristics are presented in Table 1.

Table 1.

Subjects characteristics.

	Controls	Type 2 DM (n = 26)		P
	Controls	No complications	HBP	P
n (%)	19 (100)	13 (50)	13 (50)
Gender	12 women, 7 men	5 women, 8 men	5 women, 8 men
Age (years)	59.7 ± 2.9	54.9 ± 2.9	52.8 ± 4.0	ns
BMI (kg/m²)	27.3 ± 0.4	26.3 ± 0.9	28.7 ± 0.7	ns
HbA1c (%)	5.5 ± 0.05	8.3 ± 0.5	8.5 ± 0.5	<0.0001
SBP (mmHg)	133.3 ± 1.2	130.5 ± 2.8	171.9 ± 2.6	<0.0001
DBP (mmHg)	77.7 ± 1.1	82.4 ± 2.1	91.2 ± 2.6	<0.0001

Values are mean ± SEM, ANOVA one-way Tukey test. P = ns for SBP and DBP between controls and type 2 diabetes mellitus group.

DM: diabetes mellitus; BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; HBP: high blood pressure.

Sample processing

Following overnight fasting, blood samples (5 mL) were collected into tubes containing lithium-heparin. After centrifugation (2000 g, 15 min, room temperature [RT]), the plasma was retained on ice, and the remaining RBCs were washed three times with 0.9% NaCl and lysed (1:1) in ultrapure water.¹⁶ The samples were stored at −80℃ until further analysis. l-Arg content, arginase activity and nitrate and nitrite analysis were performed in plasma and RBCs. Glycosylated haemoglobin (HbA1c) was measured in whole blood.

Arginine detection

A stock solution of internal standard (I.S.) homoarginine (1 mmol/L dissolved in 10 mmol/L of HCl) was prepared and stored at −80℃.¹⁷ Work 100 µM homoarginine standards were used for reactions. Plasma (100 µL) was mixed with 50 µL (100 µmol/L) of I.S. homoarginine, and 300 µL of acetonitrile/ammonia (90/10) were then added to precipitate the proteins. After centrifugation (3000 g, 5 min), the clear supernatant was kept.¹⁷ The RBC lysates were mixed with 50 µL (100 µmol/L) of I.S. homoarginine, and the proteins were precipitated with a mixture of chloroform/ethanol (vol/vol, 33.33/66.66).¹⁶ The upper, clear aqueous layer was collected. Both supernatants were then evaporated under a nitrogen stream, and the residues were re-dissolved with 200 µL of ultrapure water and injected into the system.¹⁷ l-Arg detection was performed in all samples by an MDQ capillary electrophoresis system equipped with a diode array detector (Beckman Coulter, CA, USA).¹⁷ The system was fitted with a 30 kV power supply with a current limit of 300 µA. The analysis was performed in an uncoated fused-silica capillary, 75 µm ID and 60.2 cm length (50 cm to the detection window), injecting 10 mm of water plug (0.5 pounds per square inch [psi] × 11 s) followed by electrokinetic injection of 10 kV for 40 s and a buffer plug of 4.5 mm (0.5 psi × 5 s). The separation was carried out in a 50 mmol/L Tris buffer containing potassium chloride 0.3 M¹⁸ titrated with 1 mmol/L phosphoric acid to the pH 2.30, 15℃ at normal polarity 12 kV (68 µA). The use of K⁺ concentrations of 0.3 M in the operating buffer is a certified method for minimizing the adsorption of proteins on fused-silica capillaries. The increased ionic strength results in a competition between K⁺ and proteins for cation-exchange sites on the silica surface.¹⁸ At the beginning of every working day, the capillary was treated with HCl 0.1 M (20 psi × 5 min), NaOH 0.1 M (20 psi × 20 min), H₂O (20 psi × 10 min) and Tris buffer (20 psi × 15 min).¹⁷ After each run, the capillary was rinsed with 0.1 mmol/L HCl 0.1 M (20 psi × 2 min), NaOH 0.1 M (20 psi × 5 min) and H₂O (20 psi × 2 min) and equilibrated with the Tris buffer for 7 min at 20 psi.¹⁷ Arginine was detected at 200 nm.¹⁷

Assay for arginase activity

This was performed based on urea production in the sample.¹⁹ Briefly, RBC lysates or plasma samples (50 µL) were first placed in a mixture containing 0.1 M glycine buffer of pH 9.5, 0.4 M MnCl₂ and 1 mM 2-mercaptoethanol. The mixture was then activated by incubation – for 1 h at 37℃. Next, 0.9 M arginine solution, pH 9.5, was added, and the mixture was incubated for 10 min. The reaction was stopped by adding 0.33 mmol/L perchloric acid. The mixture was centrifugated for 15 min at 3500 rotations per minute (rpm). To evaluate the basal urea concentration, a blank solution was prepared by dissolving each sample in a mixture containing 0.1 M glycine buffer of pH 9.5 and perchloric acid 0.33 mmol/L followed by centrifugation – 15 min at 3500 rpm. The urea concentration in each sample supernatant was measured spectrophotometrically by an automatic analyser (Cobas Mira Plus; Roche Diagnostics, Switzerland) at 578 nm by the urease method according to the manufacturer’s recommendations (Dialab, Austria). One unit represents the activity that produces 1 µmol urea for 1 min at 37℃.

Nitrate and nitrite analysis

This was performed in all samples after protein precipitation. Plasma samples (100 µL) were mixed with ethanol (500 µL), and the proteins were precipitated after centrifugation (10,000 g, 15 min, RT).²⁰ RBC proteins were precipitated with a mixture of ethanol/chloroform, as previously described.¹⁶ Nitrate and nitrite analysis was conducted spectrophotometrically by an assay that combined reduction of nitrate with vanadium (III) and measurement of nitrite in a single step.²⁰ Reactions were carried out at 37℃, bearing in mind that, at temperatures below 80℃, nitrate reduction by vanadium (III) is suspended following nitrite formation. Griess reagents [N-1-Naphthylethylenediamine dihydrochloride (NEDD) and sulphanilamide (SULF)] (Promega, WI, USA) were used as trapping agents for the simultaneous detection of nitrite and nitrate. Briefly, for standard curve, a nitrate standard solution (50 µL) was serially diluted (from 100 to 5 µM) in duplicate in a 96-well flat-bottomed, polystyrene microtiter plate (Corning, NY, USA). After loading the plate with 50 µL samples, addition of a saturated solution of VCl₃ (40 mg in 5 mL HCl 1 M) to each well (50 µL) was immediately followed by addition of SULF 2% in HCl 1 M (25 µL) and NEDD 0.1% in dH₂O (25 µL). The samples were then incubated for 40 min at 37℃, and the total nitrite and nitrate (NOx) content was measured spectrophotometrically at 540 nm. The VCl₃ solution and the Griess reagents were freshly prepared immediately prior to application to the plate. Sample blank values were obtained by substituting diluting medium for Griess reagent. Nitrite was similarly measured, except that samples and nitrite standards were only exposed to Griess reagents.

HbA1c

HbA1c was assayed by standardized immunoturbidimetry (Cobas Integra; Roche, Switzerland) according to the manufacturer’s recommendations.²¹

Statistical analysis

Data analysis was performed using GraphPad InStat software package (GraphPad Software Inc., CA, USA). Differences between groups were computed using ANOVA parametric (Tukey test) or non-parametric tests (Kruskal–Wallis test). A P < 0.05 was considered statistically significant.

Results

We first evaluated the l-Arg pool in RBCs and the plasma of our subjects by capillary electrophoresis. This method is well recognized as a sensitive approach for quantification of ionic charged trace analytes.²² Compared with non-diabetic controls, l-Arg content was similar in RBCs (Figure 1(a)) while decreased in the plasma (Figure 1(b)) of all patients with type 2 diabetes, even when hypertension was documented at first clinical onset. Bearing in mind that, in mammals, the arginase pathway is quantitatively most important for l-Arg catabolism,¹ we further analysed RBC and plasma arginase activity in all subjects. Interestingly, arginase activity was lower in RBCs (Figure 2(a)) and increased in the plasma (Figure 2(b)) of all patients with early type 2 diabetes compared with controls. l-Arg degradation generates NO.¹ Measurement of nitrate and nitrite concentrations in plasma and blood is used as an index of NO production.²³ Moreover, it has been reported that haemoglobin (Hb) is recognized as a powerful catalyst of NO oxidation.²⁴ Therefore, we further investigated the l-Arg–NO metabolic pathway in the RBCs and plasma of our subjects. We observed significantly higher concentrations of NO production in RBCs from all early type 2 diabetes patients when compared with non-diabetic subjects (Figure 3). In contrast, we did not find any statistically significant difference in NO production in the plasma of our subjects (Figure 4).

Figure 1.

l-Arginine pool is unchanged in RBCs (a) and decreased in the plasma (b) of patients with diabetes. Erythrocytes (RBC) and plasma from patients with type 2 diabetes at first clinical onset and from non-diabetic age-matched subjects were assayed for l-arginine content. Values are mean ± SEM. P = 0.045 vs. controls (b), ANOVA Kruskal–Wallis test.

Figure 2.

Arginase activity decreased in RBCs and increased in the plasma of patients with diabetes. RBCs and plasma from patients with type 2 diabetes at first clinical onset and from non-diabetic age-matched individuals were evaluated for arginase activity. Values are mean ± SEM. P = 0.001 vs. controls (a and b), ANOVA Kruskal–Wallis test.

Figure 3.

RBC NO production increased in patients with diabetes. Nitrite (a) and nitrite plus nitrate (Nox) (b) were measured in RBCs from early type 2 diabetes patients and from non-diabetic age-matched subjects. Values are mean ± SEM. P < 0.001 vs. controls (a and b), ANOVA one-way Tukey test.

Figure 4.

Plasma NO production remains unmodified in patients with diabetes. Nitrite (a) and nitrite plus nitrate (Nox) (b) were assayed in plasma from patients with type 2 diabetes at first clinical onset and from age-matched non-diabetic subjects.

Discussion

In this paper, we investigated, for the first time, l-Arg catabolism in the RBCs of patients with type 2 diabetes at first clinical onset. To our knowledge, this is the first report to measure the l-Arg content in RBCs from patients with no previous medical history of diabetes. We found no significant differences when compared with non-diabetic controls (Figure 1(a)). In the RBCs of subjects with type 2 diabetes at different stages of the disease, Ramirez-Zamora et al.²⁵ found lower l-Arg when compared with healthy individuals. The authors suggested that l-Arg pool is released from RBCs in order to maintain within reference range the serum concentration of l-Arg in patients with diabetes. Our data show that the lack of difference maintained even when hypertension at first clinical onset was documented in patients with diabetes (Figure 1(a)). A decreased bioavailability of l-Arg from RBCs was previously reported by Moss et al.²⁶ in patients with known medical history of systemic arterial hypertension. The authors concluded that the diminished rate of l-Arg transport in human RBCs in hypertension is mediated selectively by a downregulation of the specific transport system y+L activity.²⁶ The transport of l-Arg in RBCs is mediated via the cationic amino acid transport (CAT) systems y+ and y+L.²⁷ However, other chronic medical conditions characterized by systemic high blood pressure, i.e. uraemia and heart failure, induce adaptative increases in the activity of the CAT system y+, elevating the transport of l-Arg in red blood cells.^28,29 Thus, our result suggests that l-Arg pool from RBCs is not yet depleted in early stages of diabetes, even when hypertension at first clinical onset was documented.

Arginase activity was decreased in the RBCs of all patients with early type 2 diabetes (Figure 2(a)). Despite previous data³⁰ showing increased arginase activity in RBC membranes, to our knowledge, there is no direct comparison of the enzymatic activities in both the membrane and cytoplasm of RBCs in diabetes.³¹ It could be speculated that different activities between the two cellular compartments are generated by arginase–flotillin interactions in RBC membrane.³¹ It is notable that decreased arginase activity was maintained in RBCs of patients with diabetes and high blood pressure at first clinical onset (Figure 2(a)). This could represent an adaptative response of RBCs against endothelial dysfunction. That is, arginase inhibits the NO production through several mechanisms, including competition with NOS for the substrate l-Arg.³² Therefore, upregulation of arginase in endothelial cells inhibits eNOS-derived NO production and may contribute to endothelial damage in different conditions such as hypertension and diabetes.³² Furthermore, it has been shown that intraplatelet arginase activity in patients with systemic arterial hypertension is not affected,³³ suggesting that other blood cells may exhibit similar adaptative behavior.

As previously described,¹⁴ we also observed that l-Arg content was decreased (Figure 1(b)) and arginase activity increased (Figure 2(b)) in the plasma of patients with early diabetes. The same tendency was maintained in patients with hypertension and diabetes at first clinical onset (Figures 1(b) and 2(b)). These results are in line with previous data¹⁴ suggesting an increased arginine catabolism in the plasma of patients with type 2 diabetes. Plasma concentrations of l-Arg are increased in patients with systemic arterial hypertension²⁶ and decreased in medical conditions characterized by hypertension, such as chronic renal or cardiac failure.³⁴. Low concentrations of l-Arg in plasma are described instead in normotensive patients with type 2 diabetes.¹⁴ The most important mechanisms behind these variations include alterations of l-Arg transport or arginase activity and increased plasma concentrations of l-Arg analogues, i.e. asymmetric dimethyl-l-arginine (ADMA) and symmetric dimethyl-l-arginine (SDMA) complex modulation of plasma l-Arg in patients with diabetes and hypertension at first clinical onset.

It is also important to note that l-Arg may be found either in plasma or RBC (i.e. RBC/plasma l-Arg ratio). One therefore could suppose that the reduced plasma l-Arg (Figure 1(b)) could compromise its uptake by RBC (Figure 1(a)). Although it cannot be excluded, this alternative is questionable since the ‘arginine paradox’ is taken into consideration, that is the intracellular concentrations of l-Arg are usually well above the Km for NO synthesis, and the transport of l-Arg is essential for NO production.³⁵ In other words, the observation has been primarily described in endothelial cells and refers to the discrepancy that, at physiological l-Arg concentrations, eNOS should be well saturated with substrate, and the addition of exogenous l-Arg should not affect the enzyme’s activity.³⁶ There have been several explanations postulated, although none of them can fully explain the phenomenon.²⁶

We here reported for the first time that NO production in the RBCs of patients with early type 2 diabetes was significantly increased when compared with non-diabetic subjects (Figure 3). Our data are in line with very recent observation showing increased nitrites concentration in RBCs from patients with type 2 diabetes at different stages of the disease.²⁵ Palmerini et al.³⁷ observed similar results on patients with metabolic syndrome and erectile dysfunction. We obtained the same result in RBCs of patients with diabetes and hypertension at first clinical onset (Figure 3). This observation suggests that RBCs could maintain their production of NO in spite of the reduced plasma concentrations of l-Arg (Figure 1(b)). A possible explanation could be activation of the l-Arg transport, already described in uraemic platelets and mononuclear cells, which may provide a protective mechanism against increased aggregation and accelerated atherosclerosis characteristic of renal failure.^34,38

NO bioavailability varies in the serum of patients with diabetes. NO metabolites are either increased,^25,39 decreased^40,41 or unchanged⁴² in patients with early type 2 diabetes mellitus. The same conflicting results are also reported in the presence of chronic complications of diabetes.⁴³ We here reported no differences in NO metabolites in the plasma of all patients with type 2 diabetes, in spite of documented hypertension at first clinical onset when compared with non-diabetic controls (Figure 4).

Our data suggest a putative increase of RBC NO availability. It is well known that RBCs are the major intravascular storage sites of nitrite in human blood⁴⁴ and also that they have important roles in NO bioavailability.^8,24 Production of intracellular nitrite in RBCs involves several pathways.⁴⁴ However, recent data⁴⁵ provide evidences for the transport of NO via Hb within RBCs. Nitrite has the potential to be a major intravascular NO storage molecule in humans that is capable of transducing NO bioactivity distal to its site of formation.⁴⁶ The significance of our observation may rely in the fact that RBCs are essential in mediating NO bioavailability under specific conditions characterizing endothelial dysfunction. Hence, there are data suggesting that RBC nitrite reservoir can be enzymatically reduced to NO by Hb under hypoxic conditions.⁴⁶ These results suggest that RBC Hb acts as a nitrite reductase, potentially contributing to hypoxic vasodilation in tissues with low oxygen tension. In medical conditions characterized by chronic hypoxia, such as diabetes mellitus, these mechanisms may represent a potential adaptative response against endothelial damage.

Taking together, our data show that l-Arg catabolism is driven mainly towards NO synthesis in the RBCs of patients with type 2 diabetes, even when hypertension was documented at first clinical onset. Despite previous data stating that production of intracellular nitrite in RBCs involves several pathways,⁴⁴ our observation – showing a decrease in RBCs arginase activity – could be considered a potential mechanism of increased RBC NO production in early diabetes. Hence, the RBC pool would represent a potentially compensatory intravascular compartment for endothelial dysfunction in diabetes.

Footnotes

Acknowledgements

The group of subjects was recruited at courtesy of Ioana Veronica Grajdeanu, Senior MD, Senior Lecturer, Department of Internal Medicine, University of Medicine and Pharmacy, Bucharest, Romania.

Declaration of conflicting interests

None declared.

Funding

Raluca Papacocea was supported by grants from Sectoral Operational Programme Human Resources Development, financed from the European Social Fund and by the Romanian Government under contract number POSDRU/89/1.5/S/64109.

Ethical approval

The study was approved by the ethics committee of the National Institute of Diabetes, Nutrition and Metabolic Diseases ‘N. C. Paulescu’ (reference number 199/2009).

Guarantor

IS.

Contributorship

OS, LI and IS performed research; OS, IS, LI, OMB, RP and CS contributed to data acquisition, new reagents/analytic tools; OS, IS, LI, OMB, RP and CS analysed data; OS and IS wrote the paper; OS and IS designated the research; OS, IS, OMB, RP and CS reviewed the paper.

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