Gerald S. Marks

Does carbon monoxide have a physiological function

Recently endothelium-derived relaxing factor (EDRF) has been identified as nitric oxide. The source of the nitric oxide is L-arginine, and the L-arginine-nitric oxide pathway has been proposed to function as a widespread transduction mechanism for the regulation of cell function and communication. Gerald Marks and colleagues suggest that carbon monoxide, which is formed endogenously from heme catabolism and which shares some of the chemical and biological properties of nitric oxide, may play a similar role. This would be achieved by carbon monoxide binding to the iron atom of the heme moiety of soluble guanylyl cyclase and to the iron-sulfur centers of macrophage enzymes.

Pharmacokinetic-hemodynamic studies of intravenous nitroglycerin in congestive cardiac failure.

SUMMARYThe expanding role of intravenous nitroglycerin (GTN) in the management of critically ill hospitalized patients demands a clear knowledge of its pharmacodynamics and kinetics in both normal and diseased states. Accordingly, we studied 16 patients with congestive cardiac failure to establish the relationship between blood levels of GTN and its physiologic effects during and after an i.v. infusion. The end point of this study was either a greater than 25% fall in pulmonary capillary wedge presure or more than a tenfold increment over the initial GTN infusion rate. Infusion rate of GTN and blood concentration correlated well (r = 0.75, p < 0.001). Patients were divided into two groups based on their blood GTN concentration. Group I patients (n = 8) achieved blood GTN concentrations of 1.2-11.1 ng/ml and all reached the hemodynamic end point. The minimum effective blood GTN concentration was 1.2 ng/ml at an infusion rate of 15 μg/min. Group 2 patients (n = 8) had blood levels greater than 11.1 ng/ml and only three achieved the hemodynamic end point. Group 2 had greater systemic venous congestion than group 1 (right atrial pressure 19 ± 4 mm Hg (SD) vs 10 ± 4 mm Hg [p < 0.0011). In addition, group 2 had lower total body clearance of GTN (3.6 ± 1.8 1/min) than group 1 (13.8 ± 5.8 1/min) (p < 0.005). The low clearance of GTN in group 2 patients may be explained in part by impaired hepatic metabolism secondary to severe systemic venous congestion. Complete blood GTN data were available on five patients after cessation of the GTN infusion and revealed a short half-life of 1.9 minutes. Some patients failed to reach the hemodynamic end point with high infusion rates of GTN (220-4 μg/min), and blood levels of 42.2-481.3 ng/ml. There was no evidence of toxicity despite these high GTN blood levels.

Blood levels after sublingual nitroglycerin.

Pharmacokinetic analysis of nitroglycerin (GTN) has been hampered by the lack of a sensitive and specific method for measuring GTN in blood. Therefore, we examined the appearance of GTN in blood after administering 0.6 mg sublingually in 10 studies of normal volunteers. We used a gas-liquid chromatographic method with electron-capture detection and isosorbide dinitrate as the internal standard. GTN appeared in blood at 0.5 minutes, reached a peak of 2.3 ± 0.36 ng/ml at 2 minutes, fell to 50% of peak value at 7.5 minutes and was barely detectable at 20 minutes. These blood levels paralleled the changes in heart rate and systolic blood pressure. These data show rapid appearance and disappearance of GTN from blood after sublingual administration, a large volume of distribution, and a rapid rate of total body clearance that precludes the liver from being the sole elimination site. This method for analysis of GTN and isosorbide dinitrate should be helpful in defining the role of chronic nitrate therapy.

Exposure to Toxic Agents: The Heme Biosynthetic Pathway and Hemoproteins as Indicator

The heme biosynthetic pathway is closely controlled by levels of the end product of the pathway, namely, heme, and porphyrins are normally formed in only trace amounts. When control mechanisms are disturbed by xenobiotics, porphyrins accumulate and serve as a signal of the interaction between a xenobiotic and the heme biosynthetic pathway. For example, an increase in erythrocyte protoporphyrin is a useful measurement for early detection of exposure to lead and porphyrinuria was an early manifestation of a hexachlorobenzene-induced porphyria in Turkey. In recent years a variety of additional xenobiotics has been shown to interact with the heme biosynthetic pathway, namely, halogenated aromatic hydrocarbons, pesticides, sulfides, and a variety of metals. Moreover, different xenobiotics (e.g., dihydropyridines and compounds containing unsaturated carbon-carbon bonds) interact with the prosthetic heme of cytochrome P-450 forming novel N-alkylporphyrins.

Carbon monoxide formation in the ductus arteriosus in the lamb: implications for the regulation of muscle tone.

1 We have previously shown that carbon monoxide (CO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450‐based mono‐oxygenase reaction controlling the formation of endothelin‐1 (ET‐1). In the present study, we have examined whether CO is formed naturally in the vessel. 2 The CO‐forming enzyme, haem oxygenase (HO), was identified in ductal tissue in its constitutive (HO‐2) and inducible (HO‐1) isoforms by Western immunoblotting and immunological staining procedures (both light and electron microscopy). HO‐1 was localized to endothelial and muscle cells, while HO‐2 was found only in muscle cells. Inside the muscle cells, HO‐1 and HO‐2 immunoreactivity was limited to the perinuclear region, and the Golgi apparatus in particular. However, upon exposure to endotoxin, HO‐1 became more abundant, and both HO isoforms migrated towards the outer region of the cytoplasm close to the sarcolemma. 3 CO was formed enzymatically from added substrate (hemin, 50 μm) in the 10,000 g supernatant of the ductus and its formation was inhibited by zinc protoporphyrin IX (ZnPP, 200 μM). 4 ZnPP (10 μM) had no effect on the tone of the ductus under normal conditions (2.5 to 95% O2), but it contracted the endotoxin‐treated ductus (at 2.5% O2). At the same concentration, ZnPP also tended to contract the hypoxic vessel (zero O2). 5 ZnPP (10 μm) curtailed the relaxant response of the oxygen (30%)/indomethacin (2.8 μM)‐contracted ductus to bradykinin (35 nM), while it left the sodium nitroprusside (35 nM) relaxation unchanged. 6 We conclude that CO is formed in the ductus and may exert a relaxing influence when its synthesis is upregulated by an appropriate stimulus.

DISTRIBUTION OF α‐ AND β‐ADRENOCEPTORS IN HUMAN URINARY BLADDER

1 The distribution of α‐ and β‐adrenoceptors in isolated preparations of human bladder neck and detrusor muscle has been studied. 2 Adrenaline caused contraction of the bladder neck which was blocked by phenoxybenzamine but unaltered by propranolol. 3 Isoprenaline caused relaxation of the bladder neck which was blocked by propranolol. High concentrations caused contraction which was enhanced by propranolol but blocked by phenoxybenzamine. 4 Detrusor muscle was relaxed by isoprenaline and this effect was blocked by propranolol. Phenylephrine caused relaxation of detrusor which was unaffected by phenoxybenzamine; in some cases contraction was produced in the presence of propranolol. 5 It is concluded that the bladder neck contains mainly α‐receptors and the detrusor mainly β‐receptors but both regions possess both types of adrenoceptor.

Pharmacokinetic-hemodynamic studies of nitroglycerin ointment in congestive heart failure

Abstract The aim of this study was to determine the bioavailability of nitroglycerin after application of nitroglycerin ointment in patients with heart failure and to examine whether a correlation exists between plasma nitroglycerin and its hemodynamic effects. The dose of nitroglycerin ointment selected was based on the prior hemodynamic response of individual patients to an intravenous infusion of nitroglycerin. Nine patients received 1 to 2 inches of nitroglycerin ointment on a single skin site (small dose group) and five patients received 4 inches (2 inches to two separate skin sites, large dose group). There was good correlation between the dose of nitroglycerin ointment and bioavailability (area under the plasma concentration time curve from 0 to 240 minutes) (r = 0.81, p mm Hg and in right atrial pressure from 14 ±7 to 11 ±6 mm Hg. There was a greater decrease in pulmonary capillary wedge pressure in the small dose group (31 percent) compared with the large dose group (13 percent) (p ng / ml at 60 minutes and remained at that level through 240 minutes. In the large dose group a plasma nitroglycerin level of 8.9 ± 4.0 ng / ml was achieved at 60 minutes and sustained through 240 minutes. Despite this plasma nitroglycerin level there was little decline in pulmonary capillary wedge pressure. This study demonstrates a good relation between the dose of nitroglycerin ointment and nitroglycerin bioavailability; nitroglycerin ointment also provides therapeutic levels of nitroglycerin associated with substantial hemodynamic benefit in selected patients with cardiac failure.

Arterial-venous nitroglycerin gradient during intravenous infusion in man.

We assessed the concentration of nitroglycerin (GTN) measured in plasma at different sampling sites in the circulation during a constant i.v. infusion. Twenty patients with chronic congestive cardiac failure underwent hemodynamic monitoring during i.v. GTN infusion. Our end point was a reduction in pulmonary capillary wedge pressure by at least 25% of its control value or a 10‐fold increment over the initial infusion rate. Nitroglycerin infusion produced no change in heart rate, a reduction in mean arterial pressure from 90 ± 21 (± SD) to 83 ± 17 mm Hg (p< 0.001), a fall in total peripheral resistance from 24.3 ± 10.8 to 20.8 ± 7.0 units (p < 0.02), and a substantial reduction in both pulmonary capillary wedge pressure (from 27 ± 6 to 20 ± 6 mm Hg, p < 0.001) and right atrial pressure (from 12 ± 4 to 8 ± 4mm Hg, p < 0.001). The concentrations of GTN in the pulmonary artery and systemic artery were similar (29.8 ng/ml ± 52.8 and 25.1 ± 48.4 ng/ml, respectively) and considerably higher than the concentration in the peripheral vein (7.3 ± 15.4 ng/ml). There was a 17.4 ± 19.1% extraction of GTN across the pulmonary vascular bed and a 60.8 ± 27.2% extraction across the arterial-venous bed (p < 0.001). There was norelationship between the arterial-venous extraction and the magnitude of arterial plasma GTN concentration, the duration of the infusion of GTN or the total dose administered. These data show that a better understanding of GTN pharmacokinetics is provided by simultaneous arterial and venous samples.

Disruption of hepatic heme biosynthesis after interaction of xenobiotics with cytochrome P-450.

Heme biosynthesis in hepatocytes is controlled by a free heme pool, which regulates δ‐aminolevulinic acid synthase. Porphyrinogenic chemicals deplete the regulatory free heme pool by interacting with cytochrome P‐450 thereby inhibiting heme biosynthesis and/or causing heme breakdown. Recent developments allow us to predict which groups of chemicals are likely to be porphyrinogenic. One group is exemplified by 3,5‐diethoxycarbonyl‐1,4‐dihydro‐2,4,6‐trimethylpyridine. Heterocyclic compounds of this type cause mechanism‐based inactivation of cytochrome P‐450, leading to the formation of N‐alkylporphyrins, with ferrochelatase‐inhibitory activity resulting in lowering the free heme pool. Allylisopropylacetamide exemplifies a second group. Such compounds containing a terminal olefinic or acetylenic group, cause mechanism‐based inactivation of cytochrome P‐450. In the process, the heme moiety of cytochrome P‐450 is destroyed and the free heme pool is lowered. A third group is exemplified by planar polyhalogenated or polycyclic aromatic hydrocarbons. These compounds induce specific cytochrome P‐450 isozymes but are poor substrates. Active oxygen is formed, which interacts with a hepatic substrate to form a uroporphyrinogen decarboxylase inhibitor. Inhibition of this enzyme leads to depletion of the free heme pool.—Marks, G. S.; McCluskey, S. A.; Mackie, J. E.; Riddick, D. S.; James, C. A. Disruption of hepatic heme biosynthesis after interaction of xenobiotics with cytochrome P‐450. FASEB J. 2: 2774‐2783; 1988.

Quantitation of nitric oxide formation from nitrovasodilator drugs by chemiluminescence analysis of headspace gas

A rapid and reliable method has been developed for the quantitation of nitric oxide by chemiluminescence analysis of headspace gas. Aqueous nitric oxide standards are used to calibrate the method. There is a linear relationship between the amount of nitric oxide and the redox chemiluminescence detector response over the range of 52-2445 pmol of nitric oxide in 2.0 mL of deionized water contained in a sealed 6.2-mL flask. The intra-day and inter-day coefficient of variation values of the method do not exceed 4% and 9%, respectively. The lower limit of quantitative sensitivity and the lower limit of qualitative detection are 52 pmol and 26 pmol of nitric oxide, respectively. This method has been used to measure nitric oxide formation during the incubation of glyceryl trinitrate or sodium nitroprusside in the presence of cysteine. This method has two major advantages over the currently available procedures for the quantitation of nitric oxide, viz., no artifactual formation of nitric oxide during sample preparation and decreased instrumental contamination.