L-Arginine infusion has no effect on systemic haemodynamics in

Br J clin Pharmac 1993; 36: 45-49 L-Arginine infusion has no effect on systemic haemodynamics in normal volunteers, or systemic and pulmonary haemodynamics in patients with elevated pulmonary vascular resistance S. V. BAUDOUIN', P. BATH2, J. F. MARTIN2, R. DU BOIS' & T. W. EVANS1 'The Intensive Care Unit and Department of Thoracic Medicine, The National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, 2The Department of Medicine, King's College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ and 3The Department of Rheumatology, Royal Free Hospital, Pond Street, London NW3 2QG 1 The evidence that the infusion of L-arginine, the precursor of endothelium-derived relaxing factor (EDRF)/nitric oxide (NO), may reduce systemic blood pressure, via the generation of intracellular cyclic guanosine-3,5-monophosphate(cgmp), in normotensive volunteers is controversial. In the first part of the study we investigated the effect of an L-arginine infusion on systemic blood pressure and plasma cgmp in healthy volunteers. 2 Patients with systemic sclerosis have widespread endothelial damage which, by reducing the release of NO, could contribute to the raised pulmonary vascular resistance (PVR) often found in this condition. We hypothesised that if there were a failure of NO synthesis this might be overcome by infusing L-arginine into the pulmonary artery, thereby lowering PVR. In the second part of the study we investigated the effect of L-arginine infusion on systemic and pulmonary haemodynamics, and on plasma cgmp levels in patients with pulmonary hypertension and systemic sclerosis. 3 L-arginine (500 mg kg-1) was infused over 30 min into five normotensive volunteers and five patients with systemic sclerosis and pulmonary hypertension. Blood pressure, heart rate and skin temperature were measured non-invasively in the volunteers and systemic and pulmonary haemodynamics recorded via radial artery cannulae and balloon-tipped, flow directed, pulmonary artery catheters in the patients with systemic sclerosis. 4 L-arginine had no significant effect on blood pressure, heart rate or skin temperature in the normotensive volunteers nor on systemic or pulmonary haemodynamics in the systemic sclerotic group. Cyclic-GMP levels did not significantly change in either group. 5 In both normotensive volunteers and patients with pulmonary hypertension and systemic sclerosis, EDRF/NO release is unlikely to be substrate-limited by the availability of L-arginine. Keywords L-arginine vasodilation endothelium-derived relaxing factor pulmonary hypertension systemic sclerosis systemic blood pressure haemodynamics Introduction Nitric oxide (NO), which accounts for the biological essential amino acid, L-arginine [3] and relaxes smooth activity of endothelium-derived relaxing factor (EDRF) muscle by stimulating soluble guanylate cyclase and [1], is released by endothelial cells lining blood vessels thereby raising intracellular cyclic guanosine-3,5- and causes relaxation of vascular smooth muscle cells monophosphate (cgmp) concentrations [4]. [2]. NO is synthesised by NO synthase from the semi- Evidence now suggests that NO contributes to the Correspondence: Dr T. W. Evans, The Royal Brompton National Heart and Lung Hospital, Sydney Street, London SW3 6LY 45

46 S. V. Baudouin et al. regulation of basal blood pressure and flow through its inhibitory effect on vascular smooth muscle cell tone. NO synthesis may be inhibited using the competitive antagonist of L-arginine, NG-monomethyl-L-arginine (L-NMMA), which increases blood pressure in anaesthetized rabbits [5]. Similarly, L-NMMA infused locally into human forearm arteries [6] results in vasoconstriction and reduced blood flow. NO-dependent dilatation is known to be reduced in systemic and pulmonary hypertension [7-10], hypercholesterolaemia [11, 12] and atherosclerosis [12, 13], both in experimental animals and man suggesting that attenuated EDRF/NO function may be a general finding in vascular disease. Intravenous infusions of L-arginine have been reported to lower blood pressure acutely in man [14, 15] and in some laboratory studies [16], but not in others [5, 17]. Similarly, the endothelial dysfunction present in the coronary micro-circulation of hypercholesterolaemic patients may be corrected with the intra-coronary infusion of L-arginine [18]. Evidence that these effects are mediated by the conversion of L-arginine to NO, and thence the stimulation of vascular soluble guanylate cyclase and intracellular cgmp concentrations, is suggested by a small rise in both plasma cgmp and L-citrulline levels (a by-product of the conversion of L-arginine to EDRF/NO) [15, 19] and urinary nitrite/ nitrate concentrations [15]. However, others have challenged this interpretation suggesting that L-arginine does not lower blood pressure by this mechanism since D-arginine (which does not form EDRF/NO) also modulated blood flow [20]. Widespread endothelial damage occurs in patients with systemic sclerosis [21-23], a condition frequently complicated by increased pulmonary vascular resistance (PVR) [24]. We hypothesised that EDRF/NO synthesis and release might be impaired in such patients and that L-arginine, by increasing substrate availability, might increase EDRF release and lower vascular resistance. To investigate the effect of L-arginine on systemic blood pressure we performed a study infusing L-arginine intravenously into normal volunteers. Secondly, L-arginine was infused into the pulmonary artery of patients with systemic sclerosis and pulmonary hypertension and its effects on haemodynamics measured. In both studies the effects of L-arginine on plasma cgmp concentrations was also assessed. Methods Normal volunteers Five healthy male volunteers (age range 29-37 years) who were not taking any medication were studied in the Intensive Care Unit. Peripheral, venous cannulae (Abbocath 20G, Abbott Ireland Ltd, Sligo, Ireland) were inserted under sterile conditions into both antecubital fossae. Supine blood pressure was measured non-invasively and automatically (Hewlett Packard Careview 9000, Hewlett Packard CA, USA) and heart rate, electrocardiography (lead II), and skin temperature were recorded. At 4 min intervals throughout the study systolic, diastolic and mean arterial pressures, heart rate and skin temperature were recorded. L-arginine monochloride (Guy's Hospital, London, UK) was infused into the left antecubital vein to give a total dose of 500 mg kg-' [19] over a 30 min period. Venous blood samples were taken 15 min before the start of the infusion and then at 20 min into the infusion period. These samples were assayed for cyclic 3'- 5 '-guanosine monophosphate levels (see below). Measurements were made for 30 min following the end of the infusion. Patients with systemic sclerosis and pulmonary hypertension Five patients with systemic sclerosis (all meeting the American Rheumatology Association criteria for diagnosis) who were undergoing right heart catheterization for the diagnosis or management of pulmonary hypertension were studied. Systemic supine blood pressure was monitored by a radial arterial line (Abbocath 20G, Abbott Ireland Ltd, Sligo, Ireland). A balloontipped, flow-directed, thermodilution pulmonary artery catheter (Arrow Thermodilution catheter, AH-05050- H, Arrow International, Pennsylvania, USA) was introduced by an internal jugular venous approach. Systemic and pulmonary artery pressure, pulmonary capillary wedge pressure, cardiac output (Hewlett Packard 66S, Hewlett Packard CA, USA) and arterial and mixed venous oxygen saturations were measured at 10 min intervals for 30 min before the start of the L-arginine infusion and at 10 min intervals throughout the study. Cardiac output was measured by thermodilution. L-arginine monochloride (Guy's Hospital, London, UK) was infused into the pulmonary artery at a total body dose of 500 mg kg-' over a 30 min period. Cardiac index, systemic and pulmonary vascular resistances were calculated. Venous blood samples were taken 15 min prior to the start of the infusion and at 20 min into the infusion period for the assay of 3'- 5'-guanosine monophosphate concentrations. Measurements continued to be made for 30 min following the end of the infusion. The study protocols were approved by the Ethics Committee of the Royal Brompton National Heart and Lung Hospital and informed, written consent was obtained from all subjects and patients studied. Cyclic GMP assay Venous blood was taken onto edetic acid and centrifuged at 400 C. The separated plasma aliquots were frozen at -70 C prior to assay. cgmp concentrations were measured by radioimmunoassay (Amersham, Aylesbury, UK) according to the manufacturer's instructions; plasma samples were acetylated before analysis [25]. Statistics Mean (s.d.) values are presented for each set of data. Two-way analysis of variance (ANOVA) was performed for the haemodynamic data for both volunteers and patients for the five time points studied; multiple comparisons were not undertaken since the ANOVA did not reveal any significant differences at any time-point.

L-arginine and haemodynamics 47 Baseline and 20 min cgmp concentrations were compared by paired t-test. Probability values of P < 0.05 were considered significant. Results Normal volunteers The mean values for all the measured haemodynamic parameters are shown in Table 1. There were no significant changes recorded in pulse rate (F = 0.99, P = 0.44), mean systemic systolic (F = 0.67, P = 0.62) or diastolic blood pressure (F = 2.12, P = 0.13) or skin temperature (F = 0.17, P = 0.95) during the infusion of L-arginine. Similarly, basal and peri-infusion (20 min) cyclic GMP concentrations were not significantly different (Table 2, P = 0.99). Systemic sclerosis Haemodynamic data are shown in Table 3. Pulmonary artery pressure and vascular resistance were raised in all patients. There were no significant changes in any of the parameters measured including heart rate (F = 0.31, P = 0.87), mean systemic systolic (F = 1.21, P = 0.38) or diastolic arterial pressure (F = 2.41, P = 0.13), mean pulmonary systolic (F = 1.17, P = 0.39) or diastolic artery pressure (F = 2.95, P = 0.09), cardiac output (F = 0.05, P = 0.99), systemic vascular resistance (F = 0.08, P 0.99), pulmonary vascular resistance (F = 0.05, P = 1.00) or mixed venous oxygen saturation (F = 0.17, P = 0.95). Plasma cgmp concentrations did Table 1 Mean (s.d.) pulse rate, systolic and diastolic arterial pressure and skin temperature during L-arginine infusion in the five volunteers. Comparison by two-way analysis of variance Systolic Diastolic Skin Time Pulse rate pressure pressure temperature (min) (beats min-') (mm Hg) (mm Hg) (OC) Pre-infusion 71 (6) 121 (9) 63 (2) 30.5 (1.6) 0 72 (7) 120 (6) 61 (6) 29.9 (1.5) 20 70 (6) 119 (7) 58 (6) 30.0 (1.8) 30 77 (5) 120 (7) 59 (1) 30.0 (1.2) Post-infusion 75 (10) 119 (6) 61 (4) 29.8 (1.1) F value 0.99 0.67 2.12 0.17 P value 0.44 0.62 0.13 0.95 Table 2 Mean (s.d.) cyclic GMP concentrations (nm) in plasma before, and during L-arginine infusion, in the control subjects and patients with systemic sclerosis. Comparison by paired t-test Controls Patients Plasma 2.30 2.10 4.59 3.23 [cgmp] (0.33) (0.10) (1.51) (0.81) P value 0.99 0.63 not change during the infusion of L-arginine (Table 2, P = 0.63). Discussion Infusion of L-arginine into healthy volunteers did not produce any significant change in systemic haemodynamics as assessed by non-invasive methods. This finding contradicts previous reports that L-arginine infusion reduces blood pressure in healthy normotensive subjects [14, 15], but is similar to studies suggesting that L-arginine has no effect on blood pressure in some animal species [5, 17]. L-arginine is a precursor of EDRF/NO and could increase the synthesis and release of NO if this reaction is substrate-limited at physiological concentrations of L-arginine. Intra-arterial infusions into the forearm of both L-arginine and D-arginine only increase blood flow at high concentrations [9, 20] and since D-arginine is not a substrate for EDRF/NO synthesis it is likely that vasodilation was not mediated by increased release of EDRF/NO. In addition, results from both animal and in vitro studies suggest that the supply of L-arginine is not normally the rate-limiting step for EDRF/NO synthesis in endothelial cells [26, 27]. Abnormalities of EDRF/NO release have been reported in secondary pulmonary hypertension in both experimental studies and in pulmonary vascular rings from patients undergoing lung transplantation [10]. The loss of intrinsic vasodilation could then contribute to the rise in pulmonary vascular resistance. Pulmonary hypertension is relatively common in patients with systemic sclerosis [24]. Endothelial damage occurs in both the systemic and pulmonary circulations in systemic sclerosis [21, 23] and we hypothesised that this could also lead to the loss of EDRF/NO release. Although the synthesis of NO is unlikely to be limited by lack of L-arginine in normal endothelial cells, a relative lack of substrate could reduce NO production in damaged cells. However, infusion of L-arginine did not significantly alter either pulmonary or systemic haemodynamics in our patients. Equally important, cgmp levels did not change suggesting that intracellular second messenger activity is not inducible by L-arginine. Limitation of L-arginine is therefore unlikely to contribute to the pulmonary hypertension of systemic sclerosis. The discrepancies between the previously published results showing that L-arginine lowers blood pressure in man [14, 15] and our present findings are difficult to explain since similar protocols (including quantity and form of L-arginine) were followed. Firstly, it could be argued that our results do not exclude an effect of L-arginine on blood pressure, and perhaps NO synthesis, in view of the relatively small number of subjects studied (a type II statistical error). However, our sample size is similar to that used in the studies that originally showed the hypotensive effect of infused L-arginine [14, 15]. Secondly, despite the identical infusion rates of L-arginine used in both studies it is possible that the blood levels of L-arginine achieved were higher in the volunteers in Nakaki's study and produced vasodilation by non-specific mechanisms, as reported by Calver in the forearm [9, 20] and others in isolated vessels in vitro [27].

48 S. V. Baudouin et al. Table 3 Mean (s.d.) heart rate (HR), mean systolic (MSAP) and diastolic (MDAP) systemic arterial pressures, mean systolic (MSPAP) and diastolic (MDPAP) pulmonary artery (MPAP) pressures, cardiac output (CO), systemic (SVR) and pulmonary (PVR) vascular resistance and mixed venous oxygen saturations (MvSaO2) during L-arginine infusion in the five patients with systemic sclerosis. Comparison by two-way analysis of variance HR SVR PVR Time (beats MSAP MDAP MSPAP MDPAP (dyn s- (dyn s- MvSaO2 (min) min-) (mmhg) (mmhg) (mmhg) (mmhg) CO(lmin-) cm-5) cm-5) (%) Preinfusion 91 (3) 139 (20) 72 (6) 58 (28) 22 (13) 4.7 (0.6) 1427 (50) 530 (341) 72 (1) 10 93 (2) 136 (12) 66 (5) 59 (30) 22 (12) 5.1 (1.3) 1366 (182) 470 (318) 20 92 (4) 134 (18) 66 (7) 60 (32) 23 (13) 5.1 (1.6) 1379 (285) 518 (363) 30 91 (3) 136 (7) 64 (4) 56 (30) 25 (14) 5.1 (1.8) 1404 513 (305) (404) Postinfusion 88 (5) 145 (10) 66 (5) 58 (32) 24 (14) 5.2 (1.6) 1297 (257) 472 (317) 72 (1) F value 0.31 1.21 2.41 1.17 2.95 0.05 0.08 0.05 0.17 P value 0.87 0.38 0.13 0.39 0.09 0.99 0.99 1.00 0.95 Finally, it is noteworthy that the positive and negative studies were performed in Japanese and Caucasian subjects respectively. This raises the possibility that racial differences exist in blood pressure sensitivity to L-arginine, perhaps related to genetic differences in NO synthase [28]. Dr P. Bath was supported by a Medical Research Council grant, Professor J. F. Martin is British Heart Foundation Professor of Cardiovascular Science. The authors gratefully acknowledge the technical assistance of Dr E. Nava. References 1 Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987; 327: 524-526. 2 Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980; 288: 373-376. 3 Palmer RMJ, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 1988; 333: 664-666. 4 Rapoport RM, Draznin MB, Murad F. Endotheliumdependent relaxation in rat aorta may be mediated through cyclic GMP-dependent protein phosphorylation. Nature 1983; 306: 174-176. 5 Rees DD, Palmer RMJ, Moncada S. Role of endotheliumderived nitric oxide in the regulation of blood pressure. Proc Natl Acad Sci 1989; 86: 3375-3378. 6 Vallance P, Collier J, Moncada S. Effects of endotheliumderived nitric oxide on peripheral arteriolar tone in man. Lancet 1989; ii: 997-1000. 7 Linder L, Kiowski W, Buhler FR, Luscher TF. Indirect evidence for release of endothelium-derived relaxing factor in human forearm circulation in vivo. Blunted response in essential hypertension. Circulation 1990; 81: 1762-1767. 8 Panza JA, Quyyumi AA, Brush JE, Epstein SE. Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. New Engl J Med 1990; 323: 22-27. 9 Calver A, Collier J, Vallance P. Dilator actions of arginine in human peripheral vasculature. Clin Sci 1991; 81: 695-700. 10 Cremona G, Dinh-Xuan AT, Higenbottam TW. Endothelium-derived relaxing factor and the pulmonary circulation. Lung 1991; 169: 185-202. 11 Creager MA, Cooke JP, Mendelsohn ME et al. Impaired vasodilation of forearm resistance vessels in hypercholesterolemic humans. J clin Invest 1990; 86: 228-234. 12 Shimokawa H, Vanhoutte PM. Impaired endotheliumdependent relaxation to aggregating platelets and related vasoactive substances in porcine coronary arteries in hypercholesterolemia and atherosclerosis. Circ Res 1989; 64: 900-914. 13 Jayakody L, Kappagoda T, Senaratne MPJ, Thomson ABR. Impairment of endothelium-dependent relaxation: an early marker for atherosclerosis in the rabbit. Br J Pharmac 1988; 94: 335-346. 14 Nakaki T, Hishikawa K, Suzuki H, Saruta T, Kato R. L-arginine-induced hypotension. Lancet 1990; 336: 696. 15 Hishikawa K, Nakaki T, Tsudu M et al. Effect of systemic L-arginine administration on hemodynamics and nitric oxide release in man. Jap Heart J 1992; 33: 41-48. 16 Cernadas MR, Riesco A, Gallego MJ et al. L-arginineinduced hypotension. Lancet 1990; 336: 1017. 17 Murakami M, Suzuki H, Ichihara A, Naitoh M, Nakamoto H, Saruta T. Effects of L-arginine on systemic and renal haemodynamics in conscious dogs. Clin Sci 1990; 81: 727-732. 18 Drexler H, Zeiher AM, Meinzer K, Just H. Correction of endothelial dysfunction in coronary microcirculation of hypercholesterolaemic patients by L-arginine. Lancet 1991; 338: 1546-1550. 19 Hishikawa K, Nakaki T, Suzuki H, Saruta T, Kato R. L-arginine-induced hypotension. Lancet 1991; 337: 683-684. 20 Calver A, Collier J, Vallance P. L-arginine-induced hypotension. Lancet 1990; 336: 1016-1017. 21 Harrison NK, Myers AR, Corrin B et al. Structural features

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