Biotransformation of nitric oxide.

Previous investigations into the health effects of nitrogen oxides (NOx) have mostly been conducted with special reference to nitrogen dioxide (NO2) and its direct effects on the respiratory system, while the study of nitric oxide (NO) has been disregarded. We carried out a study on NO by exposing rats and mice to 15NO or administering 15N-nitrite and 15N-nitrate to these animals by IP injection in order to elucidate the metabolic fate of NO. The results of our study and previous findings led us to assume that the major metabolic path of inhaled NO is as follows: inhaled NO reacts with hemoglobin, forming nitrosyl-hemoglobin (NOHb), and from NOHb, nitrite (NO2-) and nitrate (NO3-) are generated. Major quantities of NO3- are discharged into the urine and a certain amount is discharged into the oral cavity through the salivary glands and transformed to NO2-. Part of this NO2- is converted to N2 gas in the stomach. Nitrate in the intestine is partly reduced to ammonia (NH3) through NO2-, reabsorbed into the body, and converted to urea. Most of the metabolites of inhaled NO are excreted rapidly from the body within 48 hr.


Introduction
In order to clarify the health effects ofnitrogen oxides (NO,), investigation of the direct pathological effects on the respiratory organs and basic studies on the absorption and biotransformation of NO, are important. However, for investigation of the absorption or metabolic transformation of NO,, the half-life of 13N is too short for it to be used as a radioisotope, and in the case of 15N, considerable amounts of the element are contained in the protein of the living body, so little work has been done with "5N (1)(2)(3)(4)(5)(6)(7)(8). In this report, the biotransformation of nitric oxide (NO) and its intermediate metabolites, nitrite (NOr ) and nitrate (NO3 ), are reviewed on the basis of results obtained in our own investigations.

Absorption and Conversion of NO in Blood
In physicochemical comparison with SO2, NO is less readily absorbed in the airway because of its low solubility in water [7.340 cm3/100 mL cold water (9)]. Such observations concerning NO absorption have been made in studies with isolated and perfused lung by Yokoyama and Poslethwait (10) and Mustafa (11). We have also shown that less than 10% of NO is absorbed and oxidized in perfused rabbit lungs (12). However, the results in the case of perfused lung would be expected to differ from those in the case of a living system. With regard *Department of Public Health, School of Medicine, Mie University, Tsu,514,Japan. to NO absorption into the living body, Wagner (13) reported that more than 80% of NO was absorbed in normal breathing and more than 90% was absorbed in deep breathing. We obtained similar results with inhalation of 10 ppm NO (unpublished data). Despite its low solubility in water, the absorption of NO into the body is almost complete. Goldstein et al. (14) showed in experiments with monkeys that in the case of inhalation of 0.3 to 0.9 ppm 13NO, 50 to 60% of the inhaled NO was found in the lung and spread into the other organs through the bloodstream. We found a high 15N content in serum and urine after inhalation of 138 to 880 ppm 15NO, and within 24 hr, about 40% of the inhaled N was excreted into the urine (5).
NO is known to combine strongly with hemoglobin to form nitrosyl-hemoglobin (NOHb) in vitro. According to an investigation by Oda et al. (15), a very small amount of NOHb (0.13% of total hemoglobin) was found in the blood of mice after inhalation of 10 ppm NO. This suggested the possibility ofrapid change ofthe absorbed NO in the blood. In addition, a number of studies have been made on the interaction between NO and blood or hemoglobin (16)(17)(18)(19)(20). Our experimental results with 15NO showed that NO entered the blood, combined with Hb in the first stage, and was oxidized rapidly to 15NOand 15NO (5). Though the amount of NOHb in the blood is very small and in in vivo exposure, NOHb is of important significance as an intermediate in NO metabolism (21). In the reaction process of NO and Hb, degeneration of Hb and damage to erythrocyte membranes are observed (22)(23)(24).
From our results, it is thought that due to its low solubility in water, the major proportion of inhaled NO reaches the deeper portion of the lung, reacts with hemoglobin in erythrocytes to form NOHb, and is converted immediately to NO-and NO-. The major proportion of inhaled NO is excreted in urine in the form of NO3c Metabolism and Excretion of Inhaled NO Biotransformation of NO after conversion to NO-1/ NO is considered to be the same as the metabolism of NO -/NOfrom foodstuffs. Many studies have been devoted to problems related to the fate of ingested nitrate and nitrite in the body (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). It is certain from the literature that increased ingested nitrate or nitrite results in increased urinary excretion of nitrate and nitrite, but the details of the major metabolic pathway are not sufficiently known.
In the case of 1 NO inhalation (145 ppm x 123 min) in rats (5), about 55% of the inhaled 1 N was excreted in urine, 75% of excreted 15N being 15NO3, and 24% 15Nurea (Exp. Al and A2 of Fig. 1). Compared with the case of IP injection of Na15NO2 or K1 NO3, the main metabolites were 15NO3 and '5N-urea. (In the case of "5NO3, a very small quantity of urea was found.) A pattern similar to the case of NO inhalation was found for 15NO-(Exp. Bi and B2 of Fig. 1), but the result for 15NOdiffered. The reason for this is not well understood, but these observations are of some interest. The presence of "5N-urea in urine was confirmed by the urease method and that of '5NO3 was confirmed by gasliquid chromatography/mass spectrometry method using the derivative of 3,4-xylenol (Fig. 2). The peak of M/E 168 in Figure 2B was apparently a result of 15N-6-nitro-3,4-xylenol from inhaled 15NO.
In order to elucidate exactly all the metabolites in the body, Na15NO2 (0.62 mg as 15N per animal) was . @ . . @ K15 NO3 injection 1100 FIGURE 1. Distribution of '5N (excess) in the urine from rats after the exposure to '6NO or IP injection of Na'5NO2 or K15NO3. Rats (2 animals per experiment) were exposed to 145 ppm "5NO for 123 min, or were administered 9.33 mg Na15NO2 or 13.6 mg K`5NO3 (2 mg as 1"N) per anzimal. The urine samples were taken for 48 hr after the exposure or injection. . Mass spectra of 6-nitro-3,4-xylenol (A) and the derivative of 3,4-xylenol from the urine of rats exposed to 1"NO (B). The urine was from the samples described in Fig. 1 injected IP into mice, and the 15N contents of the urine, feces, exhaled gas, and the body were estimated. As shown in Figure 3, 60.7% of the administered '5N was found in the urine, 7.8% in feces, 0.3% in exhaled gas, and 1.6% in the body. The residual 30% was not found. Wang et al. (35) administered '5N-labeled nitrate and nitrite to rats and studied excretion and retention. They found that 60 to 70% of the dose was excreted in the urine, 10 to 20% was eliminated in the feces, and about 10% was retained in the body carcass (in the case of a single dose). The amount of unrecovered 15N that we obtained was high compared to that of Wang et al., although this may have been due to differences in the animal species and administration methods. The unrecovered '5N is assumed to have been in the form of N2 gas, on the basis of both the recovery method used for exhaled gas and the in vitro experiments on the stomach contents of mice and on the presence of nitrite, as discussed in the next section.

Conversion of Nitrite and Nitrate in the Digestive Tract
The conversion of nitrite and nitrate in the digestive tract, especially in the stomach, should be evaluated in consideration of normal flora and injesta (36). A proportion of the NOin the blood is transferred to the oral cavity through saliva. Nitrate in the oral cavity is partly reduced to NOby oral bacteria. Thus, the produced NOreacts readily with amines from ingested foods or drugs under acidic conditions to form nitrosoamines. We observed that a large amount of N2 gas and a small amount of NO are produced by anaerobic incubation with mouse stomach contents and NOin a Smith tube at 37°C, pH 3.5 (Fig. 4) (7). The production of N2 gas is presumed to occur through the reaction shown below, which is used in the Van Slyke method of amine-N determination.
R R H2N-CH-COOH + HNO2-* HO-CHCOOH + N2 + H2O NOin the stomach was converted to N2 gas by the proteins of the mouse diet as in the in vitro experiment. That is to say, NO-entering the stomach is absorbed partly through the stomach wall, and some NOreacts with amines, ureides, or ascorbic acid (37), but a considerable amount of NOis changed to N2 gas by reaction with proteins of the diet and disappears from the body. The unrecovered 15N in our previous experiments (5) may have been the result of this reaction. feces. Mice feces of 11.3 g were homogenized with pepton-NaCl solution of 200 mL. After 35 mg Na'5NO3 was added to the homogenate, which was incubated at 37°C for 6 hr, part of the homogenate was centrifuged at 3000 rpm for 10 min, and the supernatant was used to determine 15NH3, NO-, and NO-. Time (min) FIGURE 4. Gas appearance by in vitro incubation experiments with stomach and its contents or food plus nitrite in stomach pH. Mice stomach and its contents of 7.5 g or food of 5.0 g were homogenized with 25 mL 0.85% NaCl solution, and the homogenate was adjusted to pH 3.5 by addition of 2N HC1. After 25 mg Na"NO, was added to the homogenate, which was incubated at 37°C for 5 hr in Smith tube, the gas produced was analyzed by gas-liquid chromatography on a Molecular Sieve 5A column. Nitrate in the stomach is transferred to the intestine without reduction and absorption in the healthy animal (34). However, in the stomach of ruminants such as cattle or sheep, NO is reduced to NO - (38).
A considerable number of studies have been conducted regarding the fate of NO and NO in the intestine. Tannenbaum et al. (33) suggested that nitrate and nitrite are produced de novo in the intestine as a result of heterophic nitrification. According to the research of Witter et al. (34), who studied 1NO in humans and rats, the 13N compound administered was not rapidly absorbed through the stomach wall; the concentration was increased in the lower intestine, and a portion of the 13N was retained in the body. The results of Wang et al. (35) suggested that NO and NO in the intestine are converted to the nitrogen compounds other than NO2 and NO3 by intestinal bacteria before reaching the large intestine. The investigations by Hill et al. (39) and Ishiwata et al. (40) showed that neither nitrite nor nitrate could be detected in the contents of the intestine nor in the feces.
We observed in Na15NO2 injection experiments that 15N in the intestine was retained for a relatively long time in comparison with retention in the liver and kidney (7). From more detailed investigations on '5N in the intestine, we showed that the greater part of 15N in the intestine is composed of trichloroacetic acid (TCA)-soluble and TCA-insoluble 15N, and that the amounts of NOand NOare very small except for those existing 1 hr after the injection.
The ratio of TCA-soluble 15N to total 15N increased with time. This suggests that low molecular weight 15N compounds in the intestine are eliminated relatively rapidly, while high molecular weight 15N compounds are retained for a considerable length of time. In vitro incubation experiments (7) with mice intestinal contents and NO-/NOsuggested that NO& in the intestine is reduced to NO-and converted to unknown nitrogen compounds by intestinal bacteria.
To investigate the end products from NO-/NOin the intestine, a mixed solution of mouse feces and a peptone-NaCl solution of Na15NO3 was incubated at 37°C for 6 hr (Fig. 5). The concentration of NOin the mixture decreased with time and disappeared after 6 hr. As for NO-, a temporary increase was found, but it disappeared after 6 hr. On the other hand, the concentration of 15N-NH3 (15NH3) in the incubation solution increased rectilinearly with time. The in vitro results suggested that NOin the intestine was converted to NH3 through NO7 by intestinal bacteria.
To confirm the conversion of NOj to NH3 in vivo, mice were given an IP injection of 15N-nitrite (0.8 mg 15N per animal), after which the concentration of 15NH3 in the intestine contents was estimated at 1 hr and 3 hr following the injection. As shown in Table 1, the atom percent excess of 15NH3 in the intestinal contents after the injection showed high values compared with the control, indicating the conversion of 15NOto 15NH3.
From these results, it is considered that NO-in the intestine is reduced to NH3 through NO-by the intes-tinal bacteria, and NH3 thus produced is absorbed through the intestinal walls and metabolized to urea.

Discussion
On the basis of the results mentioned above, the possible metabolic pathway of inhaled NO is illustrated in Figure 6. A small amount of inhaled NO reacts with tissue components in the lung, but most of it enters the blood through the alveoli and reacts with hemoglobin in erythrocytes. NO-and NO-are produced through NOHb (nitrosyl-hemoglobin) and these are transferred to the serum. The greater part of the NOis excreted into the urine through the kidney.
Part of the NOin the blood is secreted into the oral cavity through saliva, and is converted to NOby oral bacteria. Part of the NOthat reaches the stomach is converted to N2 gas with the proteins of the diet by the Van Slyke reaction and disappears. The intestinal NOtransferred from the blood and stomach is converted to NH3 or unknown compounds through NOby the intestinal bacteria. Ammonia thus produced is absorbed through the intestinal wall into the body. This reabsorbed ammonia is metabolized to urea through the urea cycle and is excreted into the urine.
The metabolism of NO/NO2 in the living body greatly assists in its detoxification and disposal. Most of the NO/NO2 is rapidly converted to low toxicity NOin the blood by oxidation with 02, etc., and is eliminated from the body. However, a portion of NO/NO2 and produced NOreact with the living components and tissues, resulting in various injuries. NOis thought to be involved not only in pathological effects on the respiratory system (41), but also in injury of the cell membrane (24,42), disturbance of the information-mediating system (43,44), alteration of immunological functions (45,46), peroxidation of cell membrane lipids (47,48), carcinogenesis, and aging (49)(50)(51). A number of important questions regarding these problems cannot as yet be convincingly answered and still await further study.