Åke Wennmalm

Metabolism and excretion of nitric oxide in humans. An experimental and clinical study.

Despite the increasing insight in the clinical importance of nitric oxide (NO), formerly known as endothelium-derived relaxing factor (EDRF), there is limited information about the metabolism and elimination of this mediator in humans. We studied the degradation of NO in healthy subjects inhaling 25 ppm for 60 minutes and in patients with severe heart failure inhaling 20, 40, and 80 ppm in consecutive 10-minute periods. In other healthy subjects, the renal clearance of NO metabolite was measured. The metabolism ex vivo was evaluated by direct incubation of nitrite, the NO oxidation product, in blood from healthy humans. During inhalation of NO, the plasma levels of nitrate increased progressively, both in the healthy subjects (from 26 to 38 mumol/L, P < .001) and in the patients (from 72 to 90 mumol/L, P < .001). Methemoglobin (MetHb) also increased in the healthy subjects (from 7 to 13 mumol/L, P < .001) as well as in the patients (from 19 to 42 mumol/L, P < .01). No change in nitrosohemoglobin (HbNO) was detected, either in the healthy subjects or in the patients. In arterialized blood (O2 saturation, 94% to 99%), incubated nitrite was semiquantitatively converted to nitrate and MetHb. In venous blood (O2 saturation, 36% to 85%) moderate amounts of HbNO were also formed. Plasma and urinary clearance of nitrate in healthy subjects averaged 20 mL/min. We conclude that uptake into the red blood cells with subsequent conversion to nitrate and MetHb is a major metabolic pathway for endogenously formed NO. Nitrate may then enter the plasma to be eliminated via the kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)

Dependence of the metabolism of nitric oxide (NO) in healthy human whole blood on the oxygenation of its red cell haemoglobin.

Plasma or whole venous or arterialized blood from healthy human donors was incubated with NO (50–300 μm), and the resulting formation of methaemoglobin (MetHb), nitrosyl haemoglobin (HbNO), and plasma nitrite and nitrate were measured. In plasma, NO was converted to nitrite and nitrate in a ratio of 5:1. In arterial blood (O2 sat. 94–99%) NO was almost quantitatively converted to nitrate and MetHb. No nitrite was detected and HbNO formation was low. In venous blood (O2 sat. 36–85%) more HbNO and less nitrate was formed, in comparison to arterialized blood. We propose that NO liberated from endothelium of conductance and resistance vessels is taken up by red blood cells and inactivated by HbO2 via stoichiometric conversion to MetHb and nitrate.

IMPORTANCE OF A NOVEL OXIDATIVE MECHANISM FOR ELIMINATION OF BRAIN CHOLESTEROL : TURNOVER OF CHOLESTEROL AND 24(S)-HYDROXYCHOLESTEROL IN RAT BRAIN AS MEASURED WITH 18O2 TECHNIQUES IN VIVO AND IN VITRO

The brain is the most cholesterol-rich organ in the body. Brain cholesterol is characterized by a very low turnover with very little exchange with lipoproteins in the circulation. Very recently we showed that there is a continuous age-dependent flux of 24(S)-hydroxycholesterol from the human brain into the circulation (Lütjohann, D., Breuer, O., Ahlborg, G., Nennesmo, I., Sidén, Å., Diczfalusy, U., and Björkhem, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9799–9804). Here we measured the rate of synthesis of cholesterol as well as the conversion of cholesterol into 24(S)-hydroxycholesterol in rat brain in vivo with use of an18O2 inhalation technique and mass isotopomer distribution analysis. Cholesterol synthesis was found to correspond to 0.03 ± 0.01% of the pool per h. Conversion of cholesterol into 24(S)-hydroxycholesterol was of a similar magnitude, about 0.02% of the pool per h. Brain microsomes converted endogenous cholesterol into 24(S)-hydroxycholesterol at a similar rate when incubated in the presence of NADPH. When incubated with whole homogenate and subcellular fractions of rat brain, there was no significant conversion of tritium-labeled 24-hydroxycholesterol into more polar products. Plasma from 18O2-exposed rats contained 24(S)-hydroxycholesterol with an enrichment of 18O similar to that in 24(S)-hydroxycholesterol in the brain. The results suggest that the present 24(S)-hydroxylase mediated mechanism is most important for elimination of cholesterol from the brain of rats. There is a slow conversion of brain cholesterol into 24(S)-hydroxycholesterol with a rapid turnover of the small pool of the latter oxysterol due to leakage to the circulation (half-life of brain 24(S)-hydroxycholesterol is about 0.5 days as compared with 2–4 months for brain cholesterol). It is evident that the 24(S)-hydroxylation greatly facilitates transfer of cholesterol over the blood-brain barrier and that this hydroxylation may be critical for cholesterol homeostasis in the brain.

Detection of endothelial-derived relaxing factor in human plasma in the basal state and following ischemia using electron paramagnetic resonance spectrometry

The endothelial-derived relaxing factor is a vasodilator agent that is formed in the vascular endothelium in response to various stimuli. It has been identified as nitric oxide (NO). Due to its short half-life the endothelial-derived relaxing factor offers certain analytical problems. We present here a method for quantitative analysis of nitrite, the oxidation product of NO, in human plasma. NO binds strongly to hemoglobin. If the resulting NO-hemoglobin (Hb) complex is subjected to a magnetic field and microwave radiation, a characteristic electron paramagnetic resonance spectrum is obtained. This spectrum is highly specific and its amplitude can be used for quantitative determination of NO in the nanomolar range. Columns of bovine Hb covalently bound to agarose were prepared, and an excess amount of dithionite was used to ensure that the Hb was reduced to a ferrous, nonoxygenated state. Samples of human plasma were treated with dithionite to convert nitrite to nitric oxide. They were then passed over the columns, which were subsequently analyzed at 77 degrees K in an electron paramagnetic resonance spectrometer. As an external standard nitrite was used. The amplitude of the spectrum was linear in the range 1-100 nmol. In healthy subjects the venous plasma level of nitrite ranged from 0 to 0.6 microM. Following forearm or leg ischemia the plasma level of nitrite increased substantially. These data are the first to demonstrate circulating levels of an index of the endothelial-derived relaxing factor in human plasma.

Conversion of inhaled nitric oxide to nitrate in man

1 Nitric oxide (NO) is potentially useful as a selective vasodilator drug in infants and adults with pulmonary hypertension. In vitro and in vivo observations demonstrate that NO may be converted to nitrate in the blood, to be further excreted into the urine. The aim of the present study was to assess quantitatively the importance of this pathway for inhaled NO in human subjects. 2 Healthy subjects inhaled 15NO (25 p.p.m.) for 1 h. The plasma and urine levels of 15NO3‐ were followed for 2 and 48 h, respectively. 3 The measured retention of 15NO in the lungs was 224 ± 13 μmol, corresponding to 90 ± 2% of the inhaled amount. Plasma 15NO3‐ increased during the inhalation of 15NO, to about 15μmoll_1, and fell when inhalation of 15NO was terminated. 4 Urinary excretion of 15NO3‐ during the first 24 h after inhalation was 154 ± 12μmol. During the following 24 h another 8 ± 2 μmol of 15NO3 appeared in the urine. 5 We conclude that conversion of inhaled NO to nitrate is a major metabolic pathway in man, covering more than 70% of its inactivation. The metabolic fate of the remaining NO inhaled requires further study.

Tobacco use and urinary excretion of thromboxane A2 and prostacyclin metabolites in women stratified by age.

BackgroundActivated platelets have been implicated in both acute thrombus formation and atherogenesis. Because smoking is a risk factor for cardiovascular disease in men and women and male smokers have biochemical evidence of increased platelet activation, we found it of interest to study whether smoking augments platelet activity in women as well. Methods and ResultsData on smoking habits and a urinary sample were obtained from 125 healthy female nonsmokers and an equal number of smokers, stratified by age in five groups from 18 to 59 years old. Urinary samples were analyzed with gas chromatography/mass spectrometry for the 2,3-dinormetabolites of thromboxane A2 (Tx-M), reflecting platelet activity, and prostacyclin (PGI-M), representing platelet/vessel wall interaction. Urinary Tx-M in smokers was higher than in nonsmokers (p<0.001), increasing with the number of cigarettes smoked per day and with age. In nonsmokers, there was no difference in Tx-M between the age groups. Urinary PGI-M in smokers was higher than that in nonsmokers (p<0.001) and decreased with age in nonsmokers but not in smokers. There was no difference in Tx-M between previous smokers and lifelong nonsmokers. ConclusionsThe elevated Tx-M in women who smoke cigarettes indicates an increased platelet activity that is dependent on smoking intensity. In parallel, PGI-M is augmented, suggesting that platelet/vessel wall interaction is stimulated. Quitting smoking is an effective means to restore platelet function. We propose that the observed increase in platelet activity in women who smoke cigarettes may be related to subsequent development of cardiovascular disease and that quitting smoking should be considered a high-priority medical target also in this sex.

Endothelium-dependent and -independent vasodilation and reactive hyperemia in healthy smokers

Summary: Although cigarette smoking elicits a transient increase in peripheral vascular resistance in humans, the basal blood pressure (BP) is usually slightly lower in smokers. We hypothesized that this may be due to a compensatory increase in sensitivity in smokers to endogenous vasodilators. To test this, we studied endotheliumdependent and ‐independent vasodilator responses, and reactive hyperemia (RH) in the forearm of 25 healthy subjects [11 smokers (S) and 14 nonsmokers (NS)]. Endothelium‐dependent vasodilation was induced by infusion into a brachial artery of stepwise increasing dosages of acetylcholine (ACh, 10‐60 μg/kg/min) and endotheliumindependent vasodilation by a similar infusion of nitroprusside (N, 1‐6 μg/kg/min). RH was induced by release of a 5‐min upper arm arterial occlusion. Forearm blood flow (FBF) was recorded by plethysmography. Endothelium‐dependent vasodilation was more pronounced in S than in NS (p < 0.01), the maximal FBF during infusion of ACh being 20 ± 6 and 14 ± 4 ml/100 ml/min, respectively. Endothelium‐independent vasodilation was also larger in S than in NS (p < 0.001), the maximal FBF during infusion of N being 14 ± 3 and 12 ± 2 ml/100 ml/min, respectively. ACh‐induced vasodilator responses in S and NS were completely blocked by atropine. They were not decreased by trimethaphan, a nicotinic receptor inhibitor. RH was slightly more pronounced in S than in NS (p < 0.02), the postocclusive FBF 15 s after release of the occlusion being 25 ± 4 and 21 ± 3 ml/100 ml/min, respectively We propose that cigarette smoking increases the sensitivity of the vascular smooth muscle to vasodilator stimuli.

Acute supplementation with the nitric oxide precursor l-arginine does not improve cardiovascular performance in patients with hypercholesterolemia

Endothelial dysfunction based on lack of nitric oxide (NO) may contribute to several settings of cardiovascular disorder. Chronic oral supplementation with the NO precursor L-arginine counteracts the development of aortic atherosclerosis in cholesterol-fed rabbits, and i.v. infusion of L-arginine may acutely improve endothelium-dependent coronary epicardial vasodilation in patients with hypercholesterolemia (HC). To clarify whether excess NO precursor may also improve general cardiovascular performance in HC, we measured working capacity indices of myocardial ischemia, and basal and post-occlusive forearm and skin blood flow in nine patients with elevated plasma cholesterol (9.1 +/- 0.2 mumol/l) following random double-blinded administration of L-arginine (16 g i.v.) or placebo. Infusion of L-arginine raised the plasma concentration of this amino acid from 85 +/- 12 to 2460 +/- 230 mumol/l but did not change the plasma level of the major NO metabolite nitrate. Maximal working capacity, indices of myocardial ischemia, and basal and post-occlusive blood flow in the skin or forearm did not differ between the treatments. The lack of positive effect of L-arginine compared to placebo indicates that excess NO precursor did not improve microvascular endothelial function in the patients, or alternatively, that the indices measured in the present study were not dependent on endothelial microvessel function. Thus, in patients with HC, deficiency of precursor for NO formation does not seem to impair either maximal exercise capacity myocardial perfusion during maximal exercise, or maximal vasodilator capacity in skeletal muscle or skin.

Effect of Nitric Oxide Gas on Platelets During Open Heart Operations

BACKGROUND The increased bleeding tendency observed after cardiopulmonary bypass is caused in part by thrombocytopenia and impaired platelet function induced by the procedure. Previous in vitro studies have shown that nitric oxide (NO) added to the oxygenator sweep gas reduces platelet activation during experimental perfusion. We evaluated the effect of 40 ppm of NO, added to the oxygenator sweep gas, on platelet consumption and activation in patients undergoing cardiopulmonary bypass. METHODS Twenty patients scheduled to undergo cardiopulmonary bypass were randomized to either the control or the NO arm of the study. Their platelet count, plasma beta-thromboglobulin level, platelet membrane glycoprotein Ib and IIb/IIIa levels, adenosine diphosphate-induced platelet aggregation, plasma nitrate level, and plasma hemoglobin were assayed before, during, and after cardiopulmonary bypass. RESULTS After operation, slightly higher platelet counts were observed in the NO-treated patients than in the control patients, which might indicate a lower degree of platelet adhesion to the artificial surfaces of the extracorporeal circuit. However, this difference did not reach statistical significance. In addition, a difference in platelet membrane expression of glycoprotein Ib was seen between the NO and control groups after operation; the platelets of the control patients had significantly higher glycoprotein Ib expression than those of the NO-treated patients. The results of platelet aggregometry indicated preserved platelet function in both the NO-treated and control patients. The blood methemoglobin levels also were low in both groups. CONCLUSIONS Nitric oxide might reduce the platelet consumption encountered during cardiopulmonary bypass without having any adverse effect on platelet function, as reflected by the preserved aggregation response seen in our patients. However, the best route of NO administration and the optimum dose remain to be established.

NO‐dependent and ‐independent elevation of plasma levels of insulin and glucose in rats by L‐arginine

1 L‐Arginine elevates plasma insulin in man. Recent in vitro data indicate that this is based on stimulation of endogenous nitric oxide (NO) with subsequent pancreatic release of insulin by L‐arginine. L‐Arginine also raises plasma glucose. 2 We studied plasma levels of insulin, glucose and NO metabolites, as well as systemic blood pressure, in anaesthetized rats during i.v. infusion of L‐arginine (25–200 mg kg‐1 min‐1) or glucose (55 mg kg‐1 min‐1), before and after administration of the NO synthesis inhibitor, Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 50 mg kg‐1). 3 Before L‐NAME, L‐arginine elevated plasma insulin from about 15 to 65 u 1‐1 and glucose from 5.2 to 6.7 mmol 1‐1. These effects of L‐arginine were not dose‐related. 4 L‐NAME alone had no effect on plasma insulin and glucose levels, but diminished the effects of a low dose (25 mg kg‐1 min‐1) of L‐arginine on plasma insulin by about 40%, and that on plasma glucose by more than 90%. In contrast, the effects of a high dose (200 mg kg‐1 min‐1) of L‐arginine on plasma insulin and glucose levels were not affected by L‐NAME. 5 L‐NAME elevated systemic blood pressure by about 35 mmHg. L‐Arginine (25–100 mg kg‐1 min‐1) had no effect on systemic blood pressure, either before or after L‐NAME. L‐Arginine (200 mg kg‐1 min‐1) lowered systemic blood pressure, both before and after L‐NAME. 6 Glucose infusion elevated plasma glucose from about 5.5 to 6.8 mmol 1‐1, and plasma insulin from about 18 to 26 u 1‐1. 7 The basal plasma levels of the NO metabolite nitrate (18 ± 4 μMol 1‐1) were not affected by L‐arginine (200 mg kg‐1 min‐1). Plasma nitrosohaemoglobin was likewise unaffected by L‐arginine (200 mg kg‐1 min‐1). 8 We conclude that L‐arginine separately elevates plasma insulin and glucose levels, both by NO‐dependent and ‐independent mechanisms.