Long-Term N-Acetylcysteine and L-Arginine Administration Reduces Endothelial Activation and Systolic Blood Pressure in Hypertensive Patients with Type 2 Diabetes

Emerging Treatments and Technologies ORIGINAL ARTICLE

Long-Term N-Acetylcysteine and L-Arginine Administration Reduces Endothelial Activation and Systolic Blood Pressure in Hypertensive Patients With Type 2 Diabetes

1 1 VALENTINO MARTINA, MD PAOLA MASSARENTI, MD ardiovascular complications repre- 1 1 ANDI MASHA, MD FABIO SETTANNI, PHD sent 80% of the causes of death in 1 2 VALENTINA RAMELLA GIGLIARDI, MD LARA DELLA CASA, PHD 1 patients with type 2 diabetes. Several OREDANA ROCATO MD 2 C L B , STEFANIA BERGAMINI, PHD causes may explain this mortality excess. 3 2 ENZO MANZATO, MD, PHD NNA ANNONE MD, PHD 1 A I , Among these, the decreased availability of ARRIGO BERCHIO, MD nitric oxide (NO) has increasingly gained credit. In fact, reduced NO availability has been demonstrated not only in diabe- OBJECTIVE — Reactive oxygen and nitric oxide (NO) have recently been considered to be tes (1) but also in other diseases, such as involved in the cardiovascular complications of patients with type 2 diabetes, as NO is thought atherosclerosis and hypertension, known to lose its beneficial physiological effects in the presence of oxygen radicals. For this reason, we to be associated with increased mortality tested the effects of L-arginine (ARG) and N-acetylcysteine (NAC) administration in increasing due to cardiovascular causes (2,3). NO is NO bioavailability by reducing free radical formation. crucial for regulating the vascular tone RESEARCH DESIGN AND METHODS — A double-blind study was performed on 24 and maintaining the intrinsic thrombore- male patients with type 2 diabetes and hypertension divided into two groups of 12 patients that sistant and atheroprotective properties of randomly received either an oral supplementation of placebo or NAC ϩ ARG for 6 months. the vascular wall (4). NO production by constitutive NO RESULTS — The NAC ϩ ARG treatment caused a reduction of both systolic (P Ͻ 0.05) and synthase (cNOS) is mainly dependent on diastolic (P Ͻ 0.05) mean arterial blood pressure, total cholesterol (P Ͻ 0.01), LDL cholesterol the availability of L-arginine (ARG) (5). (P Ͻ 0.005), oxidized LDL (P Ͻ 0.05), high-sensitive C-reactive protein (P Ͻ 0.05), intracellular Several studies have demonstrated that adhesion molecule (P Ͻ 0.05), vascular cell adhesion molecule (P Ͻ 0.01), nitrotyrosine (P Ͻ ARG infusion is able to improve endothe- 0.01), fibrinogen (P Ͻ 0.01), and plasminogen activator inhibitor-1 (P Ͻ 0.05), and an improve- lial function in normal subjects and pa- ment of the intima-media thickness during endothelial postischemic vasodilation (P Ͻ 0.02). tients with coronary heart disease and Ͻ HDL cholesterol increased (P 0.05). No changes in other parameters studied were observed. hypertension (6), but the results, although encouraging, are not conclusive, CONCLUSIONS ϩ — NAC ARG administration seems to be a potential well-tolerated an- probably because of the short-term effects tiatherogenic therapy because it improves endothelial function in hypertensive patients with of ARG intravenous administration. Oral type 2 diabetes by improving NO bioavailability via reduction of oxidative stress and increase of NO production. Our study’s results give prominence to its potential use in primary and second- ARG has a longer half-life and longer- ary cardiovascular prevention in these patients. term effects than ARG given intra- arterially or intravenously so that in long- Diabetes Care 31:940–944, 2008 term health maintenance or symptoms management the oral route would be pre- ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ferred. Unfortunately, studies in diabetic patients are not so widely positive, since From the 1Department of Internal Medicine, University of Torino, Torino, Italy; the 2Department of Bio- medical Sciences, University of Modena and Reggio Emilia, Modena, Italy; and the 3Department of Medical oral ARG does not improve endothelial and Surgical Sciences, University of Padova, Padova, Italy. function (7). Corresponding author: Valentino Martina, MD, Department of Internal Medicine, University of Torino, It is well known that thiols are active Corso Dogliotti 14, I-10121 Torino, Italy. E-mail: [email protected]. intermediates in the NO pathway (8). Ex- Received for publication 7 December 2007 and accepted in revised form 2 February 2008. Published ahead of print at http://care.diabetesjournals.org on 11 February 2008. DOI: 10.2337/dc07- isting data document an increased free 2251. radical production in diabetes and hyper- Abbreviations: ABP, arterial blood pressure; ABPM, ambulatory blood pressure monitoring; ADMA, tension with a consequent decrease in asymmetrical dimethyl-arginine; ARG, L-arginine; hs-CRP, high-sensitive C-reactive protein; sGC, soluble thiol levels. In this situation, NO reacts Ј Ј guanylyl cyclase; cGMP, intracellular guanosine 3 ,5 -cyclic monophosphate; GSH, glutathione; GSSG, with the superoxide anion and to a lesser oxidized glutathione; HPLC, high-performance liquid chromatography; ICAM, intercellular adhesion molecule; IL-6, interleukin-6; IMT, intima-media thickness; ox-LDL, oxidized LDL; NAC, N-acetylcysteine; extent with thiols so that the beneficial cNOS, constitutive NO synthase; PAI-1, plasminogen activator inhibitor-1; ROS, reactive oxygen species; effects of NO are lost. We might suppose SDMA, symmetrical dimethyl-arginine; TNF-␣, tumor necrosis factor-␣; VCAM, vascular cell adhesion that in patients with diabetes, diminished molecule. NO availability and the failure to amelio- © 2008 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby rate NO availability after oral ARG sup- marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. plementation could be attributed to this

940 DIABETES CARE, VOLUME 31, NUMBER 5, MAY 2008 Martina and Associates mechanism. Accordingly, we previously overlapping in terms of mean age (62.5 SDMA were determined using an HPLC demonstrated that, in type 2 diabetic pa- [95% CI 59.3–74.5] vs. 67.0 years [51.0– assay (Waters, Milford, MA). Normal sub- tients, a short-term intramuscular admin- 69.7]) and BMI (27.6 [23.6–38.9] vs. jects evaluated in a previous study istration of glutathione (GSH) is able to 28.5 kg/m2 [25.1–38.4]). Group A pa- showed the following mean values for increase NO availability (9). tients received placebo (compounds ARG, ADMA, and SDMA: 105 Ϯ 6.1, Based on these observations, we car- identical in appearance to ARG and NAC) 1.41 Ϯ 0.2, and 0.68 Ϯ 0.07 ␮mol/l, re- ried out a study where ARG and N-acetyl- for 6 months, while group B patients respectively (10). cysteine (NAC) were administered ceived NAC (600 mg Acetilcisteina, one Nitrites/nitrates were measured si- together in patients with type 2 diabetes tablet twice a day) plus ARG (1,200 mg multaneously: nitrates were first con- and hypertension: ARG was administered Zentrum, one vial a day) per os. Compli- verted to nitrites by means of enzymatic to enhance NO production while NAC ance was checked by pill and vial counts. conversion, then total nitrites were mea- was administered to ameliorate antioxi- No adverse effects were noted during the sured in the sample using the Griess re- dant defense and increase intracellular ni- treatment. There was one dropout in each agent. The concentration of nitrites in trosothiol concentration, thus increasing group, both not for medical reasons. samples was calculated by comparison NO availability. In all subjects, before and after 6 with a standard curve of sodium nitrite months of treatment/placebo, blood sam- (Sigma-Aldrich, Milan, Italy). Normal RESEARCH DESIGN AND ples were obtained in order to evaluate value Ϯ SEM was 20.1 Ϯ 4.3 mmol/l. METHODS — The study was ap- A1C, total, HDL, and LDL cholesterol, ox- GSH and GSSG in erythrocytes were proved by the ethics committee at our in- idized LDL (ox-LDL), triglycerides, ratio determined by HPLC after a derivatisa- stitution, and written informed consent was of reduced GSH to oxidized GSH (GSSG) tion of hemolysed samples using a fluo- obtained from all recruited subjects. It in erythrocytes, nitrites/nitrates, asym- rescence detector (Varian 9070). GSSG wasarandomized,double-blind,placebo- metrical and symmetrical dimethyl- was calculated as the difference between controlled small-scale study of 6 months. arginine (ADMA and SDMA, respec- total and reduced GSH. Male patients (n ϭ 24, median age 64 tively), nitrotyrosine, arginine, homocys- Plasma levels of nitrotyrosine were as- years [95% CI 51–74 ]) with type 2 dia- teine, high-sensitive C-reactive protein sessed by ELISA (HyCult Biotechnology); betes and hypertension treated with oral (hs-CRP), interleukin-6 (IL-6), tumor ne- the normal range was 0–120 ␮mol/l. hypoglycemic and antihypertensive crosis factor-␣ (TNF-␣), intercellular and Plasma levels of homocysteine were de- drugs were enrolled. All the subjects were vascular cell adhesion molecules (ICAM termined by EIA (Axis-Shield Diagnos- recruited from the outpatient clinic of the and VCAM, respectively), plasminogen ac- tics, Dundee, U.K.); the normal range was Diabetic Centre of the Division of Endo- tivator inhibitor-1 (PAI-1), and fibrinogen. 3–37 ␮mol/l. crinology of the University of Turin, Italy. Moreover, before and after 6 months of Serum levels of hs-CRP (ng/ml) were Only male subjects were selected to treatment, the patients underwent an ABPM measured with a high-sensitive ELISA avoid interferences on NO due to the ac- for 24 h and an ultrasound assessment of (Bender MedSystems); the normal range tion of estrogens. All the subjects were intima-media thickness (IMT) after endo- was 136–800 ng/ml. Serum levels of cy- nonsmokers, had no secondary hyperten- thelium-dependent flow-mediated vasodi- tokines (IL-6 and TNF-␣) and adhesion sion, cancer, or systemic, hepatic, pulmo- lation of the brachial artery. molecules (ICAM-1 and VCAM-1) were nary, cardiovascular, or renal diseases or The different variables were mea- measured with ELISA (R&D Systems); psychiatric disorders, and were not being sured in the laboratories of the Endocri- the normal range was 0.43–8.87 pg/ml treated with nitrates, NAC, or ARG; fur- nology Division at the University of for IL-6, 0–4.22 pg/ml for TNF-␣, 115– thermore, they were not hypersensitive to Torino, the Department of Biomedical 306 ng/ml for ICAM-1, and 341–897 NAC and had no contraindications to Sciences at the University of Modena and ng/ml for VCAM. Fibrinogen was mea- ␤-blocker treatment. Subjects were all Reggio Emilia, and the Department of sured with ELISA (GENTAUR). Normal following a diet for diabetic patients with- Medical and Surgical Sciences at the Uni- plasma concentration was 2.26–3.30 mg/ out changing their food habits for the versity of Padova. ml. Human PAI-1 Ag (endothelial type) whole duration of the study. Serum levels of A1C were measured was measured with ELISA (GENTAUR). The eligible patients underwent an using a high-performance liquid chroma- Normal poor-platelet plasma concentra- antihypertensive treatment washout for tography (HPLC) autoanalyzer (Auto- tion was 5–40 ng/ml. 15 days. The subjects were enrolled only Alc-HA-8121; Menarini, Florence, Italy). The ABPM was performed using an in- if, at the end of this period, they presented Total cholesterol and triglycerides in the strument of AND INTERMED s.r.l. (TM- a systolic mean arterial blood pressure plasma were measured with enzymatic 2430). ABP was measured every 15 min (ABP) between 135 and 180 mmHg and a methods using the Cobas Mira Plus auto- between 7 A.M. and 11 P.M. and every 20 min diastolic mean ABP between 85 and 110 analyzer (ABX Diagnostic Italia, Rome, between 11 P.M. and 7 P.M.; mean systolic mmHg, measured with ambulatory blood Italy). HDL cholesterol was measured af- and diastolic ABP values were calculated as pressure monitoring (ABPM) for 24 h. ter precipitation of lipoproteins contain- the media of the 24-h registrations. During the washout time, and for the ing apolipoprotein B (Lipid Research The ultrasound assessment of the en- whole duration of the study, all patients Clinics Program, 1982). LDL cholesterol dothelial-dependent flow-mediated vaso- received atenolol (50 mg per os daily) for was calculated according to the Friede- dilation of the brachial artery was used to precautionary reasons. wald equation. Plasma levels of ox-LDL measure the IMT and performed using B- Patients were randomly assigned to were measured with a competitive ELISA mode ultrasonography with a 7-MHz lin- either the placebo group (n ϭ 12, group (Alpco Diagnostics). Normal values were ear transducer (ATL-APOGEE 800 PLUS) A) or the NAC ϩ ARG group (n ϭ 12, 37–1,200 units/l. as follows. The subject was positioned su- group B). Subjects in the two groups were Plasma levels of arginine, ADMA, and pine with the arm in a comfortable posi-

DIABETES CARE, VOLUME 31, NUMBER 5, MAY 2008 941 NAC and ARG administration in diabetic patients

Table 1—Baseline characteristics of the study groups reduced concentration of nitrosothiols. This could represent the first step of the Group A Group B P (A vs. B) endothelial damage, since it causes a reduced production of cGMP. In the mean- n 12 12 time, superoxide anions react with NO, Age (years) 67.0 (51.0–69.7) 62.5 (59.3–74.5) NS producing a strong pro-oxidant repre- Height (cm) 165.0 (160.5–175.5) 170.0 (161.3–179.5) NS sented by peroxynitrite. In brief, when Weight (kg) 77.0 (70.8–103.4) 84.0 (65.5–107.7) NS cellular conditions are favorable (i.e., dur- 2 BMI (kg/m ) 28.5 (25.1–38.4) 27.6 (23.6–38.9) NS ing the reductive state), NO produces its Mean systolic ABP (mmHg) 140.5 (136.0–170.7) 148.5 (137.3–175.2) NS antiatherosclerotic effects; whereas when Mean diastolic ABP (mmHg) 87.0 (86.0–94.7) 87.5 (86.0–92.6) NS NO is synthesized under unfavorable Data are median (95% CI). conditions (i.e., during oxidative stress), it can be harmful. Recently, it has been tion for imaging the brachial artery. After cholesterol remained unchanged, as did demonstrated that the metabolism of ev- acquiring a baseline rest image, a sphyg- the other evaluated parameters (Table 2). ery molecule of glucose yields a superox- momanometric cuff was placed above the ide anion, so that chronic hyperglycemia antecubital fossa to create a flow stimulus. CONCLUSIONS — NO has several leads to oxidative stress (13). Arterial occlusion was created by cuff effects realized through different path- This study was designed to induce inflation to at least 50 mmHg above sys- ways; the most important physiological NO antiatherosclerotic effects in diabetic tolic pressure for 5 min, causing ischemia. one being its interaction with soluble gua- patients by increasing NO production, in- Subsequent cuff deflation induced a brief nylyl cyclase (sGC) leading to an accumu- duced by ARG supplementation, in the high-flow state (reactive hyperemia); the re- lation of intracellular guanosine 3Ј,5Ј- presence of a reductive state obtained sulting increase in shear stress caused the cyclic monophosphate (cGMP) (12), through supplementation of NAC, which brachial artery to dilate (endothelial post- which mediates its antiatherosclerotic ef- acts as an antioxidant and GSH precursor ischemic vasodilation) and, subsequently, fect. NO has a half-life of only 5–15 s as it (Fig. 1). As estrogens increase NO pro- the intima to become thinner (11). rapidly reacts with other molecules de- duction, in the young female individuals pending on the redox state of the cell. In NO changes during the cycle represent a Data analysis the presence of a reduced intracellular confounding factor. Therefore, only male All data in text and figures are provided as state, NO reacts with thiols, producing s- subjects were chosen for this study, and medians and 95% CIs. Statistical signifi- nitrosothiols, which are relatively long- consequently the results can only be ap- cance was tested using Wilcoxon’s lived reservoirs of NO that transport it on plied to the male sex. The study was car- signed-rank test or the Mann-Whitney the heme group of the sGC, inducing ried out in a 6-month period, and as that rank-sum test when appropriate; the level cGMP synthesis (8). On the other hand, time was not enough for adequate evalu- selected for statistical significance was set when large amounts of reactive oxygen ation of hard end points, the study as- at P Ͻ 0.05. The analysis was performed species (ROS) are generated, burning the sesses only intermediate end points. using Statistica ‘98 edition software (Stat- antioxidant defenses (i.e., oxidative As in vivo NO measurement is very Soft, Tulsa, OK). stress), the s-thiols decrease, leading to a difficult, its production is usually esti-

RESULTS — The baseline characteristics of the two groups are outlined in Ta- ble 1; no differences existed in the measured parameters. Basal values were superimposable in the two groups except for HDL cholesterol and fibrinogen (Table 2). After 6 months of NAC ϩ ARG administration, in comparison with baseline, the following results were obtained (Table 2): 1) reduction of systolic and diastolic me- dium ABP, total cholesterol, LDL cholesterol, ox-LDL, hs-CRP, ICAM, VCAM, nitrotyrosine, fibrinogen, PAI-1, and IMT; 2) increase of HDL cholesterol and nitrites/nitrates; and 3) no change in A1C, triglycerides, IL-6, TNF-␣, GSH-to-GSSG ratio, arginine, ADMA, SDMA, and homocysteine levels. Figure 1—NO pathways involved in NAC ϩ ARG supplementation. Arginine improves NO genera- After 6 months of placebo administration, leading to nitrosothiol formation through the increased thiol (SH) groups availability, furnished by tion, PAI-1, total cholesterol, LDL choles- NAC supplementation; nitrosothiols transport NO to the sGC, inducing cGMP synthesis. NAC inhibits terol, and ox-LDL levels were significantly superoxide formation and, as a consequence, peroxynitrite, nitrogen dioxide, and nitrotyrosine forma- • • ؊ ؊ • higher than those observed at the base- tion. NO, nitric oxide; NO2 , nitrite; NO3 , nitrate; O2 , superoxide; ONOO-, peroxynitrite; NO2 , line; however, the ratio of ox-LDL to LDL nitrogen dioxide; RS-NO, nitrosothiol; SH-NAC, thiol group of NAC.

942 DIABETES CARE, VOLUME 31, NUMBER 5, MAY 2008 Martina and Associates aaaemda 9%CI). (95% median are Data 2— Table enssoi B mH)100(3.–6.)110(2.–7.)190(3.–7.)140(2.–6.)NS (128.3–161.5) 144.0 (138.5–175.5) 149.0 (128.0–173.0) 141.0 (136.0–168.3) 140.0 (mmHg) ABP systolic Mean endatlcAP(mg 70(609.)8. 7.–88 80(609.)8. 7.–63 NS (78.0–96.3) 83.0 (86.0–92.8) 88.0 (76.5–98.8) 89.0 (86.0–94.8) 87.0 (mmHg) ABP diastolic Mean M ( IMT irtrsn ( Nitrotyrosine irtsadntae mo/)2. 2.–72 83(786.)2. 1.–57 82(837.)NS (18.3–70.9) 38.2 (13.9–65.7) 24.8 (17.8–64.5) 28.3 (23.1–47.2) 27.3 (mmol/l) nitrates and Nitrites A- n/l 64(209.)4. 3.–90 82(067.)3. (20.6–62.0) 30.5 (20.6–70.7) 38.2 (35.9–89.0) 47.4 (32.0–90.5) 46.4 (ng/ml) PAI-1 ( SDMA ( ADMA ( Arginine CM(gm)13(.–.)13(.–.)13(.–.)11(.–.)NS (0.5–1.2) 1.1 (0.7–1.9) 1.3 (0.8–1.6) 1.3 (0.8–1.7) 1.3 (ng/ml) ICAM CM( VCAM rgyeie mo/)09(.–.)12(.–.)13(.–.)15(.–.)N SNS NS NS (0.6–3.8) (3.1–4.9) 1.5 4.5 (0.8–2.7) (3.5–5.8) 1.3 4.5 (0.7–2.2) (3.8–6.5) 1.2 4.8 (0.6–2.1) (3.9–5.7) 0.9 4.4 (mmol/l) cholesterol Total (mmol/l) Triglycerides D hlseo mo/)11(.–.)12(.–.)09(.–.)10(.–.)NS (0.6–1.4) 1.0 (0.6–1.2) 0.9 (1.0–1.5) 1.2 (0.9–1.5) 1.1 (mmol/l) cholesterol HDL D hlseo mo/)29(.–.)32(.–.)28(.–.)27(1.5–3.3) 2.7 (27.2–58.9) 47.7 (32.4–61.9) 50.0 (2.1–4.4) 2.8 (34.3–66.2) 55.6 (2.2–4.6) 3.2 (35.2–58.1) 49.0 (2.3–4.0) 2.9 (units/l) ox-LDL (mmol/l) cholesterol LDL sCP(gm)155(0.–5.)155(72122 4. 6.–2.)197(61184 NS (36.1–168.4) 119.7 (69.0–224.5) 141.3 (67.2–172.2) 135.5 (102.1–152.8) 135.5 (ng/ml) hs-CRP TNF- 1 % . 5998 . 6096 . 611.)74(.–23 SN NS NS NS NS NS NS NS NS (5.8–12.3) 7.4 NS (3.8–7.2) 5.4 (6.1–10.1) 6.8 (5.9–33.1) 10.0 (3.2–6.4) 4.5 (6.0–9.6) 7.5 (5.6–19.4) 8.7 (3.0–10.5) 5.7 (5.5–12.3) (5.9–9.8) 6.4 7.2 (3.0–8.6) 5.7 (3.7–13.2) 5.6 ( Homocysteine (%) A1C ratio GSH-to-GSSG ( Fibrinogen (pg/ml) IL-6 mated by measuring nitrites and nitrates,

␣ which represent stable end products of its ⌬ m .5( 0.05 mm) p/l . 1789 . 1183 . 2169 . 2051 SN NS NS NS (2.0–5.1) 3.4 (2.1–6.9) 3.4 (1.1–8.3) 2.9 (1.7–8.9) 3.2 (pg/ml) ␮ ␮ ␮ metabolism. In group B, the observed in- o/)11(.–.)09(.–.)08(.–.)09(.–.)N SNS NS NS (0.1–1.6) 0.9 (0.5–1.9) 0.8 (0.8–1.1) 0.9 (0.2–1.6) 1.1 mol/l) /l . 1318 . 1318 . 0719 . 0613 NS (0.6–1.3) 1.0 (0.7–1.9) 1.3 (1.3–1.8) 1.6 (1.3–1.8) 1.6 g/ml) o/)18(.–.)18(.–.)18(.–.)17(.–.)N SNS NS NS (1.3–2.8) 1.7 (1.5–2.8) 1.8 (1.5–2.1) 1.8 (1.5–3.0) 1.8 mol/l) ␮ vlae parameters Evaluated

o/)164(20130 55(47164 94(66125 0. 4.–5.)N SNS NS NS (47.5–157.2) 105.8 (56.6–192.5) 99.4 (84.7–146.4) 95.5 (82.0–143.0) 126.4 mol/l) crease of plasma nitrites and nitrates and ␮ o/)1. 511.)1. 611.)1. 1.–90 33(.–64 NS (5.8–16.4) 13.3 (14.5–19.0) 16.8 (6.1–17.6) 14.7 (5.1–17.0) 15.3 mol/l)

␮ the improvement of IMT, a good index ␮ o/)17(.–.)16(.–.)12(.–.)10(.–.)NS (0.3–2.3) 1.0 (0.4–2.6) 1.2 (1.2–2.2) 1.6 (1.2–2.2) 1.7 mol/l)

o/)2. 2.–54 31(233.)2. 2.–35 59(123.)N SNS NS NS (21.2–34.3) 25.9 (20.6–33.5) 26.8 (12.3–37.8) 23.1 (22.9–35.4) 25.2 mol/l) that directly correlates with NO bioavailability, suggest a gain in NO production, with the decrease of ABP representing the clinical effect of these changes. Nitrotyrosine, synthesized by the interaction of tyrosine residues and peroxynitrite, is considered indirect evidence of oxidative stress (14). Accordingly, the Ϫ 1 aa 2 otsB:BslB:6mnh 2v.A 2v.B 1v.A1 vs. B1 B1 vs. B2 A1 vs. A2 months 6 B2: Basal B1: months 6 A2: Basal A1: diminished nitrotyrosine levels found .5t 0.23) to 0.05 demonstrate an improvement of the redox state of the cells (i.e., reduction of oxidative stress). Indeed, being a thiol, NAC both in its own form and by conver-

ru ru B Group A Group sion in glutathione has a potent antioxidant effect that opposes the activity of

Ϫ ROS by neutralizing them. In a wider .2( 0.02 view, the reduction of oxidative stress led to increased NO bioavailability. Although Ϫ

.9t .2 .6(0.00–0.11) 0.06 0.12) to 0.09 an increased GSH-to-GSSG ratio after 6 months of NAC administration would be expected, no statistical change in glutathione was observed. Though in contrast with most data in the literature, this ﬁnd- ing agrees with that of Patriarca et al. (15), who also showed no change in GSH content after NAC administration. The failure to ﬁnd an increased GSH-to-GSSG ratio after thiol administration could be ex- plained by the antioxidant properties of NAC that probably, in oxidized states of the cell, more easily act by directly neutralizing ROS than by participating in GSH production (16). Besides the vasodilator effect, NO ex- Ϫ

.5( 0.05 erts additional antiatherosclerotic effects by inhibiting several signaling pathways

Ϫ including vascular smooth muscle cell .1t .2 NS 0.12) to 0.11 migration and monocyte activation, che- motaxis, adhesion, and migration. Such effects are mediated via inhibition of nuclear factor-␬B, which plays an important role in the regulation of redox-sensitive inﬂammation-related genes (17) respon- sible for the expression of several pro- Ͻ Ͻ Ͻ Ͻ 0.05 0.05 0.05 0.05 teins, including adhesion molecules (ICAM-1 and VCAM-1) and PAI-1. On the other hand, nuclear factor-␬B is acti- vated by oxygen free radicals, promoting

Ͻ endothelial dysfunction. Therefore, the Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ .0 NS 0.005 .5NS 0.05 .5NS 0.05 .2NS 0.02 .5NS 0.05 .5NS 0.05 .5NS 0.05 .1NS 0.01 .1NS 0.01 0.02 .5NS 0.05 .5NS 0.05 0.01 .1NS 0.01

P observed decrease of both adhesion molecules and PAI-1 might be the result of the same mechanisms, an increase in bioac- tive NO, and/or a reduction of ROS. It has been demonstrated that high Ͻ Ͻ 0.02 0.05 levels of CRP are a risk factor for cardiovascular morbidity and death. High levels of CRP have been found in diabetes (18)

DIABETES CARE, VOLUME 31, NUMBER 5, MAY 2008 943 NAC and ARG administration in diabetic patients and essential hypertension (19) and, fur- lar prevention in type 2 diabetic patients. oxide-sensitive guanylyl cyclase. Circ Res thermore, have been shown to signifi- Further clinical studies on a larger scale are 93:96–105, 2003 cantly correlate with both oxidative stress needed to support our experimental data. 13. Brownlee M: Biochemistry and molecular and endothelial activation (20). NAC ϩ cell biology of diabetic complications. Na- ARG treatment has been able to decrease Acknowledgments— This work was sup- ture 414:813–820, 2001 ported by a grant from MURST. 14. Ceriello A: Nitrotyrosine: new findings as CRP, likely due to both an antioxidant a marker of postprandial oxidative stress. effect and an increased NO availability, We thank Marin Raffaella for her valuable contribution to this study. Int J Clin Pract Suppl 129:51–58, 2002 which downregulates CRP expression in 15. Patriarca S, Furfaro AL, Domenicotti C, vivo (21), supporting the hypothesis that References Odetti P, Cottalasso D, Marinari UM, strategies meant to lower plasma CRP 1. Martina V, Bruno GA, Trucco F, Zumpano Pronzato MA, Traverso N: Supplementa- could include the increase of both NO E, Tagliabue M, Di Bisceglie C, Pescarmona tion with N-acetylcysteine and taurine production and availability. G: Platelet cNOS activity is reduced in pa- failed to restore glutathione content in A growing number of studies has fo- tients with IDDM and NIDDM. Thromb liver of streptozotocin-induced diabetics cused attention on fibrinogen in diabetes, Haemost 79:520–522, 1998 rats but protected from oxidative stress. suggesting its role as a marker of cardio- 2. Napoli C, Ignarro LJ: Nitric oxide and ath- Biochim Biophys Acta 1741:48–54, 2005 erosclerosis. Nitric Oxide 5:88–97, 2001 16. Tredger JM: N-acetylcysteine: not simply vascular risk in this disease. In fact, an a glutathione precursor. Transplantation association between diabetes and fibrino- 3. Taddei S, Virdis A, Ghiadoni L, Sudano I, Salvetti A: Endothelial dysfunction in hy- 69:703–704, 2000 gen levels has been demonstrated (22), pertension. J Cardiovasc Pharmacol 38 17. D’Acquisto F, Maiuri MC, de Cristofaro and fibrinogen seems to be a major inde- (Suppl. 2):S11–S14, 2001 F, Carnuccio R: Nitric oxide prevents in- pendent predictor of cardiovascular mor- 4. Moncada S, Radomski MW, Palmer RM: ducible cyclooxygenase expression by tality in type 2 diabetes. On the other Endothelium-derived relaxing factor: inhibiting nuclear factor-␬B and nuclear hand, different evidence (23) indicates identification as nitric oxide and role in factor-interleukin-6 activation. Naunyn that a short-term inhibition of NOS in- the control of vascular tone and platelet Schmiedebergs Arch Pharmacol 364:157– creases fibrinogen levels. The findings of function. Biochem Pharmacol 37:2495– 165, 2001 the present study are indirectly in accor- 2501, 1988 18. Yuan G, Zhou L, Tang J, Yang Y, Gu W, Li dance with that assertion. 5. Palmer RM, Moncada S: A novel citrul- F, Hong J, Gu Y, Li X, Ning G, Chen M: Serum CRP levels are equally elevated in No changes in IL-6 and TNF-␣ levels line-forming enzyme implicated in the formation of nitric oxide by vascular en- newly diagnosed type 2 diabetes and im- were found in our patients during the paired glucose tolerance and related to ϩ dothelial cells. Biochem Biophys Res Com- ARG NAC treatment. This is probably mun 158:348–352, 1989 adiponectin levels and insulin sensitivity. due to the fact that the treatment was 6. Panza JA, Casino PR, Badar DM, Quyyumi Diabetes Res Clin Pract 72:244–250, 2006 meant to increase NO production and AA: Effect of increased availability of 19. Lakoski SG, Cushman M, Palmas W, lower the oxidative state but not to de- endothelium-derived nitric oxide precur- Blumenthal R, D’Agostino RB, Jr, Her- crease an inflammatory state. sor on endothelium-dependent vascular rington DM: The relationship between We also evaluated ADMA concentra- relaxation in normal subjects and in blood pressure and C-reactive protein in tion, which has been considered by some patients with essential hypertension. Cir- the Multi-Ethnic Study of Atherosclerosis authors as a novel cardiovascular risk fac- culation 87:1475–481, 1993 (MESA). J Am Coll Cardiol 46:1869– 1874, 2005 tor (24). The structural homology be- 7. Mullen MJ, Wright D, Donald AE, Thorne S, Thomson H, Deanfield JE: Atorvastatin 20. Cottone S, Mule G, Nardi E, Vadala A, tween ARG and ADMA suggests that this but not L-arginine improves endothelial Guarneri M, Briolotta C, Arsena R, Pal- compound might regulate NO synthesis function in type I diabetes mellitus: a dou- ermo A, Riccobene R, Cerasola G: Rela- through a competition for the active site ble-blind study. J Am Coll Cardiol 36: tion of C-reactive protein to oxidative of NOS. In patients supplemented with 410–416, 2000 stress and to endothelial activation in es- ARG ϩ NAC, we did not observe any vari- 8. Ignarro LJ, Edwards JC, Gruetter DY, sential hypertension. Am J Hypertens 19: ation in ADMA levels or in its analog Barry BK, Gruetter CA: Possible involve- 313–318, 2006 SDMA levels. More importantly, we found ment of S-nitrosothiols in the activation of 21. McCarty MF: AMPK activation may sup- that the observed levels of this methylargi- guanylate cyclase by nitroso compounds. press hepatic production of C-reactive nine in both groups of patients were lower FEBS Lett 110:275–278, 1980 protein by stimulating nitric oxide synthase. Med Hypotheses 63:328–333, 2004 than the concentration range of 3–15 9. Martina V, Bruno GA, Zumpano E, Origlia ␮ C, Quaranta L, Pescarmona GP: Adminis- 22. Le Devehat C, Khodabandehlou T, Vimeux mol/l which has been demonstrated to be tration of glutathione in patients with type M: Diabetes mellitus and fibrinogen: hem- necessary to significantly inhibit NO forma- 2 diabetes mellitus increases the platelet orrheological and microcirculatory conse- tion (25). From these data, we cannot hy- constitutive nitric oxide synthase activity quences. J Mal Vasc 25:53–57, 2000 pothesize an involvement of ADMA in the and reduces PAI-1. J Endocrinol Invest 24: 23. Kawabata A: Evidence that endogenous dysregulation of the NO pathway. 37–41, 2001 nitric oxide modulates plasma fibrinogen In conclusion, combined NAC and 10. Bergamini S, Vandelli L, Bellei E, Rota C, levels in the rat. Br J Pharmacol 117:236– ARG administration seems to be a success- Manfredini P, Tomasi A, Albertazzi A, 237, 1996 ful and well-tolerated antiatherogenic ther- Iannone A: Relationship of asymmetric 24. Miyazaki H, Matsuoka H, Cooke JP, Usui apy, capable of improving endothelial dimethylarginine to haemodialysis hypo- M, Ueda S, Okuda S, Imaizumi T: Endog- function in hypertensive diabetic male pa- tension. Nitric Oxide 11:273–278, 2004 enous nitric oxide synthase inhibitor: a 11. Hashimoto M, Miyamoto Y, Matsuda Y, novel marker of atherosclerosis. Circula- tients because it reduces oxidative stress Akita H: New methods to evaluate endo- tion 99:1141–1146, 1999 and, at the same time, promotes NO anti- thelial function: non-invasive method of 25. Kurose I, Wolf R, Grisham MB, Granger DN: atherosclerotic effects. Our study’s results, evaluating endothelial function in hu- Effects of an endogenous inhibitor of nitric therefore, give prominence to its potential mans. J Pharmacol Sci 93:405–408, 2003 oxide synthesis on postcapillary venules. use in primary and secondary cardiovascu- 12. Friebe A, Koesling D: Regulation of nitric Am J Physiol 268:H2224–H2231, 1995

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