Decreased Whole Body Endogenous Nitric Oxide Production in Patients with Primary Pulmonary HypertensionDemoncheaux E.A.G.a · Higenbottam T.W.c · Kiely D.G.b · Wong J.-M.a · Wharton S.a · Varcoe R.a · Siddons T.a · Spivey A.C.d · Hall K.e · Gize A.P.e
aDivision of Clinical Sciences (South), University of Sheffield, Royal Hallamshire Hospital, and bRespiratory Function Unit, Sheffield Teaching Hospital NHS Trust, Sheffield, cAstraZeneca R&D Charnwood, Loughborough, dDepartment of Chemistry, South Kensington Campus, Imperial College, London, and eManchester Medical Mass Spectrometry Facility, Unit A Millbrook Business Centre, Manchester, UK Corresponding Author
Impaired pulmonary release of nitric oxide (NO) is one of the characteristic phenotypic changes of vascular cells in pulmonary hypertension. The aim of this study was to determine nitric oxide synthase (NOS)-dependent whole body NO production in patients with primary pulmonary hypertension. NOS-dependent whole body NO production was assessed by giving an intravenous infusion of L-[15N]2-arginine (50 μmol/min for 30 min) and measuring isotopic urinary enrichment of 15N-nitrite and 15N-nitrate. Four female patients with no signs of infection were recruited and compared with 6 age-matched control subjects. Mean 12-hour excretion of 15N-nitrite and 15N-nitrate in the total urine over 36 h was smaller in patients than in control subjects (57.2 ± 27.6 vs. 229.1 ± 65.2 nmol/mmol creatinine, p< 0.01, Mann-Whitney U test, respectively). Neither mean 12-hour excretion of 14N-nitrite and 14N-nitrate (51.6 ± 10.0 vs. 72.4 ± 10.0 μmol/mmol creatinine, p = 0.3) nor glomerular filtration rates (84.5 ± 15.8 vs. 129.7 ± 16.0 ml/min, p = 0.1) were different between patients and control subjects. Our results suggest that either basal NOS-dependent whole body NO production is impaired or excess NO metabolism occurs in patients with primary pulmonary hypertension.
Copyright © 2005 S. Karger AG, Basel
The extensive phenotypic changes of pulmonary vascular cells cause the characteristic structural and physiological abnormalities in pulmonary hypertension . While basal rate of endothelial nitric oxide (NO) production modulates pulmonary vascular tone  the role of NO in the pathogenesis of pulmonary hypertension remains controversial.
While decreased endothelial nitric oxide synthase (eNOS) expression has been reported , almost all studies to date have used exhaled breath NO and biochemical products of NO to infer the involvement of NOS in pulmonary hypertension [4,5,6]. However, basal levels vary according to diet, lifestyle and kidney function. Many of the problems associated with using point estimates of NO, nitrite and nitrate concentrations can be overcome by using the conversion of a subsystemic dose of non-radioactively labelled L- [15N] 2-arginine to 15N-nitrite and 15N-nitrate in man .
The aim of this study was therefore to determine if NOS-dependent whole body NO production is affected in patients with primary pulmonary hypertension. We measured the formation of 15N-nitrite and 15N-nitrate in urine after an intravenous dose of L- [15N] 2-arginine as indicator of whole body endogenous NO production in vivo and compared patients with age- and sex-matched control subjects.
Patients and Methods
Four female patients (43 ± 3 years old) referred to the Sheffield Pulmonary Hypertension Service were included in the study. None of the patients were on non-steroidal anti-inflammatory drugs in the week before the study. Six healthy, non-smoking female volunteers (36 ± 4 years old) were used as a control group. Subjects abstained from eating nitrite- and nitrate-containing foods, such as green vegetables and preserved red meat. All provided written informed consent and the study had received approval of the South Sheffield Ethics Committee.
Venous blood samples were taken for liver function tests, basal nitrite and nitrate measurements, and determination of background isotope ratios of urine nitrite and nitrate concentrations. L- [15N] 2-arginine (purity > 98%, Mass Trace, USA) was dissolved in physiological saline (0.9%; Baxter Healthcare Ltd., Thetford, UK). Pharmaceutical grade L- [15N] 2-arginine was infused at a sub-systemic dose of 50 μmol/min for 30 min. Total dose of arginine infused was therefore chosen so that normal circulating levels would not be affected . Total urine collection was collected over 48 h, from 12 h before until 36 h after infusion. Serum and urine samples were frozen and stored at –20°C until analysis.
The ratio of urinary 15N-nitrate to 14N-nitrate was determined in 100-μl aliquots of urine using a method adapted from Tsikas et al.  which involved reduction to 15N-nitrite by pre-treatment with cadmium. Spiking a sample of a known amount of 14N-nitrate with 15N-nitrate (98 atom %, Aldrich, UK) demonstrated that our technique standard curve was linear over the range of empirical enrichments from 0 to 5% (r2 = 0.996). The coefficient of variation, within assay precision, was 0.5% on replicate measures. The Griess assay was used to measure the total amount of urinary nitrate in each of the 12-hour samples. Nitrate was reduced to nitrite by pre-treatment with cadmium. This assay was linear up to 500 μmol/l with a coefficient of correlation of 0.998 and a limit of detection of 1.9 μmol/l. Both our recoveries were greater than 80%. Urinary and blood creatinine concentrations were measured by the Clinical Chemistry Department of the Royal Hallamshire Hospital.
Data are given as means ± SEM or as a box plot. Median values are presented as a line inside the box. 25th and 75th percentiles are given in each box. Top and bottom of each bar are the 90th and 10th percentiles, respectively. Non-parametric analysis was applied and differences between groups were analysed by the Mann-Whitney U test (SPSS for Windows, version 12.0, SPSS, Chicago, Ill., USA). Values of p < 0.05 were considered significant.
The patients had primary pulmonary hypertension, with a mean value for the group’s mean pulmonary artery pressure of 51.5 ± 8.8 mm Hg (table 1). No patient or normal subject had systemic hypertension (systolic blood pressures < 140 mm Hg and diastolic blood pressures < 90 mm Hg). All patients were receiving oral anticoagulants to achieve an international normalised ratio >2.2 and were also taking diuretics and calcium channel blockers (diltiazem, n = 2, amlodipine, n = 1, or nifedipine, n = 1).
Table 1. Hemodynamic parameters and demographic data for the patients with primary pulmonary hypertension
Mean 12-hour excretion of 15N-nitrite and 15N-nitrate in the total urine over 36 h was smaller in patients than in controls, i.e. 57.2 ± 27.6 vs. 229.1 ± 65.2 nmol/mmol creatinine (p< 0.01, Mann-Whitney U test), respectively (fig. 1). Mean 12-hour excretion of 14N-nitrite and 14N-nitrate was not different between patients and control subjects (51.6 ± 10.0 vs. 72.4 ± 10.0 μmol/mmol creatinine, p = 0.3). There was no correlation between hemodynamic parameters and whole body nitric oxide synthesis in this small cohort of subjects (table 1). Glomerular filtration rates were similar between patients and control subjects (84.5 ± 15.8 vs. 129.7 ± 16.0 ml/min, respectively, p = 0.1).
Fig. 1. Urinary excretion of 15N-nitrate in female control subjects (n = 6) and patients with primary pulmonary hypertension (PPH, n = 4) after an intravenous injection of L- [15N] 2-arginine (50 μmol/min for 30 min). Medians, and 75th and 25th percentiles are given in each box. Top and bottom of each bar are the 90th and 10th percentiles, respectively (Mann-Whitney U test).
In the present study, we tested the hypothesis that NOS-dependent whole body NO production was affected in patients with primary pulmonary hypertension. We have demonstrated that it was decreased in this patient population indicating an abnormal balance between NO enzymatic synthesis and its rate of consumption. An important strength of this study is that our results are independent from exogenous sources of NO, nitrite and nitrate.
The inability of point estimates of concentration of NO in breath or its biochemical products in serum to detect changes in rates of NO production is supported by earlier work. Breath NO may be both elevated  or decreased [3, 4] in pulmonary hypertension. Serum NO products do not appear to be affected when subjects with pulmonary hypertension are not controlled for dietary intake of nitrite and nitrate [4, 5].
We saw no relationship between age and endogenous NO production. Neither reduced renal function nor nitrate excretion rates accounted for the differences we observed. Treatment with dihydropyridine calcium antagonists, dialtizem, amlodipine and nifedipine, which can cause NO release from endothelium [9, 10] did not elevate the rates of NO production to normal levels in our patients.
Our results can be explained by both decreased NOS activity or increased NO, nitrite and nitrate rate of consumption. In support of the former hypothesis, decreased NOS expression has been reported in patients with pulmonary hypertension . Furthermore, recent advances from Pearson et al.  suggest that functional polymorphism in carbamoyl-phosphate synthetase, a rate-limiting enzyme in arginine synthesis, might be associated with the development of pulmonary hypertension and that the resulting low serum arginine levels may decrease NOS activity. Finally, elevated serum concentration of asymmetric dimethylarginine, an endogenous inhibitor of NOS, has been reported and may inhibit NO production in pulmonary hypertension .
Impairment in the NO metabolism may also be involved in the pathogenesis of pulmonary hypertension . Indeed, Bowers et al.  have recently reported formation of nitrotyrosine in the lungs of patients with severe pulmonary hypertension. While over 70% NO is oxidised to nitrite and nitrate and excreted in the urine over 48 h , formation of nitrotyrosine or other nitrated proteins might shunt NO consumption away from terminal oxidation to nitrite, which acts as source of NO in the vasculature [16,17,18].
In conclusion, the measurement of the rate of NO production using conversion of [15N] 2-arginine to 15N-nitrate and 15N-nitrite offers a specific measure of NOS-dependent whole body endogenous NO production. The need for further investigations is emphasised by our findings to explain how whole body endogenous NO production is decreased in patients with primary pulmonary hypertension.
We acknowledge the advice and constructive criticism of Drs. C. Emery, D. Bee, R. Patel G. Barer (University of Sheffield, UK), D. Crowther (Sheffield Hallam University, UK), and A. P. L. Smith (Scripps Research Institute, USA). A National Institute of Health grant (HL-60753) supported this work.
Dr. D.G. Kiely
Respiratory Function Unit, Sheffield Teaching Hospital NHS Trust
Sheffield S10 2JF (UK)
Tel. +44 114 2712132, Fax +44 114 2711718, E-Mail email@example.com
Received: August 5, 2004
Accepted after revision: November 22, 2004
Published online: January 21, 2005
Number of Print Pages : 4
Number of Figures : 1, Number of Tables : 1, Number of References : 18
Journal of Vascular Research (Incorporating 'International Journal of Microcirculation')
Vol. 42, No. 2, Year 2005 (Cover Date: March-April 2005)
Journal Editor: Pohl, U. (Munich)
ISSN: 1018–1172 (print), 1423–0135 (Online)
For additional information: http://www.karger.com/jvr