Clinical Science (I997) 93,5 19-525 (Printed in Great Britain)

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Impaired nitric oxide-mediated vasodilatation and total body nitric oxide production in healthy old age Declan LYONS, Suzanne ROY, Mahesh PATEL*, Nigel BENJAMIN*and Cameron G. SWIFT Clinical Age Research Unit, King’s College School of Medicine and Dentistry, Bessemer Road, London S€5 9PJ. U.K., and *Department of Clinical Pharmacology, St Bartholomew’s Hospital, West Smithfield, London EClA 7BE. U.K. (Received 25 February/Z9August 1997; accepted 4 September 1997)

1. Basal release of nitric oxide from the vascular endothelium maintains a constant vasodilating tone. Impaired nitric oxide-mediated vasodilatation has been described in hypertension and atheromatous disease. Circulatory diseases account for considerable morbidity and almost half of all deaths in people over the age of 75 years. 2. We have therefore compared nitric oxidedependent vasorelaxation in 12 healthy elderly subjects with 12 young volunteers matched for blood pressure, cholesterol and glucose, using forearm occlusion venous plethysmography combined with brachial artery infusions of the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine(L-NMMA; 1, 2 and 4 pmol/min) with noradrenaline (60, 120 and 240 pmol/min) as a control vasoconstrictor. We also measured urinary nitrate excretion after a controlled 48 h low nitrate diet as an index of total body nitric oxide production and correlated these changes with forearm blood flow responses to L-NMMA and noradrenaline in both groups. 3. The mean age and blood pressure of the elderly subjects was 76 (range 66-82)years and 132/76 (SEM 4/3) mmHg respectively, while in the young these were 27 (20-35) years and 131/72 (4/3) mmHg respectively. L-NMMA and noradrenaline produced dose-dependent reductions in forearm blood flow in both groups. L-NMMA (4 pmollmin) produced less vasoconstriction in the elderly than in the young (-37.7k2.6 versus -48.3 +4.2%; P = 0.017). The mean slope of the L-NMMA dose-response curves in the elderly was significantly less than the younger group ( -35.2 f3.1 versus -63.7 f10.6; P = 0.041). Noradrenaline, 240 pmol/min, also produced less vasoconstriction in the elderly compared with the young (-22.8f2.9 versus -35.3 +5.0%, P = 0.029) although the slopes of the dose-response curves did not differ significantly. 4. Urinary nitrate adjusted for creatinine clearance was also significantly higher in the younger group (460.6 f97.7 versus 205.9 f64.8 pmol/day; P =

0.042) and showed a significant correlation with the percentage change in forearm blood flow in response to the maximum dose of L-NMMA (r = 0.5, P = 0.046). 5. We conclude that nitric oxide-mediated vasodilatation in the forearm vascular bed is diminished in old age and this reflects a more generalized reduction in nitric oxide production (as measured by urinary nitrate) in the circulation of older people. The blunted response to noradrenaline points to a more generalized reduction in vascular reactivity in the elderly. INTRODUCTION

Nitric oxide (NO) is synthesized by both animal and human vascular endothelium [l, 21. It is a potent vasodilator, produced from the semi-essential amino acid L-arginine [3, 41. The synthesis of NO from L-arginine is continuous and provides a background vasodilator influence [5]. It also mediates, at least in part, the vasorelaxant actions of various endogenous hormones including bradykinin, substance P and acetylcholine [ 6 ] . Impaired endothelial function characterizes diseases such as hypertension [7, 81, hypercholesterolaemia and atherosclerosis [9]. Studies in animal models suggest that aging is associated with impaired NO-dependent vasodilatation [lo-121. In man, the relationship between advancing age and NO-dependent vasodilatation was first evaluated in coronary arteries in vivo by intra-arterial infusion of acetylcholine, demonstrating an age-associated blunting of its vasodilating properties [13-151. In addition, when coronary blood flow was measured using the Doppler catheter technique, the vasodilating effect induced by acetylcholine showed an inverse correlation with increasing age [9, 151. Thus, in humans, endothelial function in coronary arteries seems to be decreased with aging. However, blood pressure is not determined by coronary artery tone

Key words: elderly, nitric oxide, noradrenaline, plethysmography. Abbreviations: FABF, forearm blood flow; L-NMMANG-monomethyl-L-arginine;NO, nitric oxide. Correspondence: Dr Declan Lyons, Limerick Rq’onal Hospital, Dooradoyle, Limerick, Ireland.

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but by resistance vessel tone in the systemic circulation. In addition, coronary arteries can be affected by early atherosclerosis, even though angiographically they appear normal. Since atherosclerosis itself increases in old age, this possibility might potentially affect the response to acetylcholine in older patients. Taddei et al. [16] recently demonstrated that the vasodilating response to acetylcholine in the forearm circulation (a vascular bed unaffected by atherosclerosis) declined progressively with increasing age in both normotensive and hypertensive subjects while the response to sodium nitroprusside remained intact, providing indirect evidence for a reduction in NO synthesis as opposed to an endothelial smooth muscle coupling defect. The use of muscarinic agonists such as acetylcholine in the assessment of NO-dependent vasodilatation has certain drawbacks. Muscarinic agonists produce vasodilatation by mechanisms independent of the arginine-NO pathway and responses are dependent on circulating cholinesterase activity, basal blood flow and forearm length [17]. Evidence from animal studies suggests that cholinesterase activity itself is subject to age-related changes making interpretation of muscarinic agonist responsiveness in old age more difficult without knowledge of We believe therefore cholinesterase activity "1. that arterial responsiveness to NO synthase inhibitors [e.g. NG-monomethyl-L-arginine (L-NMMA)] is a better indicator of basal endothelial NO synthesis than responsiveness to muscarinic agonists [7, 17, 191. Aging may be associated with a generalized reduction in responsiveness to vasoactive compounds due to structural changes in the vessel wall. It is therefore important that a vasoconstrictor comparator (e.g. noradrenaline) is used against which responses to L-NMMA may be compared. Although the forearm circulation provides a convenient bed in which to test the integrity of the arginine-NO pathway in man, it is the activity of this pathway in the circulation in general which is of particular interest. We have therefore employed urinary nitrate measurements after a controlled 48 h low nitrate diet as an index of total body NO production and correlated these changes with forearm blood flow (FABF) responses to L-NMMA and noradrenaline in elderly and young groups of healthy volunteers matched for blood pressure. METHODS Patient selection

The study was undertaken in 24 subjects. Twelve healthy elderly (two female) and 12 healthy young male subjects, matched for blood pressure, plasma cholesterol and blood glucose were identified from our unit's database. Exclusion criteria included a history of hypertension, renal impairment (creatinine 2 130 pmol/l), ischaemic heart disease, serious systemic disease or concomitant medication with

drugs likely to affect blood pressure (e.g. steroids, non-steroidal anti-inflammatory drugs). Subjects adhered to their normal sodium intake. Study design

The study design was a cross-sectional, parallel group type (Fig. 1). After an initial screening visit subjects were placed on a strict 48 h low nitrate diet. On the second day of this diet subjects performed a 24 h urine collection for urinary nitrate and creatinine clearance estimation. At the end of this 48 h period subjects returned to the clinical laboratory with the urine collection and for the performance of FABF measurements. Methods of measurement

Forearm occlusion plethysmography. Investigations were performed in a temperature-controlled laboratory (25-27°C) with the subjects lying supine. FABF (ml min-' 100 ml-* forearm) was measured simultaneously in both arms by mercury-in-silastic straingauge plethysmography (Hokanson, EC-4, E20 and AG-101; Bellevue, Washington) 11201. During the recording period the hands were excluded from the circulation by inflation of the wrist cuffs to 200mmHg. The upper arm congesting cuffs were inflated to 40 mmHg for 10 s in each 15 s cycle. The mean of the final five measurements of each recording period was used for analysis. A 27-gauge unmounted steel cannula (Cooper's Needle Works, Birmingham, U.K) was inserted into the left brachial artery using 1 % lignocaine hydrochloride (Pharma Hameln GmbH Germany) to provide local anaesthesia. L-NMMA (CalbiochemNovabiochem, Nottingham, U.K.) and noradrenaline (Sanofi-Winthorp, Surrey, U.K) were dissolved in saline (0.9% NaCl; Baxter Healthcare Ltd, Thetford, Norfolk, U.K) and infused at a constant rate of 1 ml/min throughout the experiment by means of a constant rate infusion pump (Braun Perfusor Ed 2). After insertion of the needle into the brachial artery, 0.9% NaCl was infused at 1 ml/min and baseline measurements of FABF were made for 10-20 min to establish resting control values. During this period measurements were made for 3 min in every 10 min. When resting control values were attained, each patient received three doses of noradrenaline (60, 120 and 240pmol/min, each for 10min with measurements being taken in the final 3min of each) followed by L-NMMA (1, 2 and 4 pmol/min, each for 10 min) to produce cumulative dose-response .curves. Low nitrate diet and urine collection. Cured meats, fruit and green leafy vegetables have a high nitrate content. Knight et al. [21] estimated that the daily intake of nitrate is about 95 mg. Tap water contains nitrates washed from the soil, so subjects were provided with distilled water which was not rationed,

Impaired nitric oxide-mediated vasodilatation in old age

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HYV Control (12)

HEV Control (12) Screening BPx2 IXS

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start 24 hr collection

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minutes Fig. 1. Study design. (a) Study plan; (b) infusion sequences. Abbreviations: investigations; NFD, nitrate-free diet.

HW,healthy young volunteers; HEV, healthy elderly volunteers; BP, blood pressure; Ixs,

and subjects were provided with a low nitrate diet which excluded high-nitrate foodstuffs for 48 h [22]. Day 1 served as a washout period, allowing previously ingested nitrate to be metabolized or excreted in the urine [22]. Endogenous nitrate (the metabolic breakdown product of endogenously produced NO) synthesis could then be measured more accurately as an index of total body NO production in man on day 2 by measurement of urinary nitrate excretion. The urine was collected in 2litre polythene vessels containing a small amount of boric acid to prevent bacterial growth. At the end of this 24 h period the urine volume was measured. Two 10ml aliquots of urine were retained at -20°C for nitrate and creatinine measurement. Urinary nitrate measurements. All urine samples were diluted 1/20 with distilled water and analysed. Measurement of nitrate in urine was automated using a HPLC system adapted from Green et al. [23]. In brief, the nitrate in a sample was reduced to nitrite by a cadmium column. Griess reagent was then added to the system and reacted with the nitrite to produce a purple azo dye. The sample was passed through a heating coil at 71°C to develop colour before being passed into a spectrophotometer (Spectra Physics Spectra SERIES UVlOO) set at absorbance 546 nm. The spectrophotometer was connected to a MacLab/4 chart recorder which relays a trace onto the screen of an Apple MacIntosh computer using the MacLab ‘chart’ application.

Coefficient of variation over the measured concentration range (20-1000 pmol) was ~ 3 % .

Statistics and calculations

FABF is expressed as mlmin-1 100ml-* forearm according to the method of Whitney [20]. The percentage change in FABF after drug administration was calculated as:

where I and NI represent measured FABFs in the infused and non infused arm respectively during periods of drug (d) and vehicle (v) administration. This method is essentially that used by Greenfield and Patterson [24] to minimize the effects of variation in bloodflow caused by minor external factors. Comparison of FAI3F changes was by factorial and repeated measures analysis of variance; P