Acta physiol. scand. 1973. 88. 1-7 From the Department of Physiology, Veterinary College of Norway, Oslo, Norway

Metabolism of “C-Histamine in Goats and Pigs Treated with Aminoguanidine

By

KNUT ARNET ELIASSEN

Received 19 July 1972

Abstract

ELIASSEN, K. A. Metabolism of ‘%-histamine in goats and pigs treated with amino- guanidine. Acta physiol. scand. 1973. 88. 1-7.

The effect of aminoguanidine, an inhibitor of diamine oxidase (E.C. 1.4.3.6.) on the ca- tabolism of 14C-histamine has been studied in goats, a species in which oxidative deamination dominates, and pigs, a species in which methylation is the most important route of inactivation. In aminoguanidine treated goats the total 14C-imidazoleacetic acid ( 14C-ImAA) in urine w-as reduced from about 70 70 to about 4 % and in pigs from 18 % to 2 70. In goats the decrease in the formation of 14C-ImAA was partly compensated by an increased methylation though the fraction of 14C-histamine excreted unchanged was increased tenfold in the last-mentioned species. In pigs the decrease in 14C-ImAA seemed to be fully compensated by increased methyla- tion of histamine. The decrease in the excretion of non-radioactive 1.4-MeImAA in pigs and the change in the ratio: 14C-1.4-MeImAA/14C-1.4-MeHi in both pigs and goats during amino- guanidine treatment indicated that oxidation of 1.4-MeHi to 1.4-MeImAA is partly catalyzed by enzymes sensitive to aminoguanidine. The incomplete inhibition of the 14C-ImAA forma- tion during aminoguanidine treatment indicates that enzymes other than diamine oxidase might participate in the oxidation of histamine to ImAA.

Histamine is oxidatively deaminated by histaminase an enzyme believed by many to be identical with diamine oxidase (E.C. 1.4.3.6.) (Tabor 1954, Zeller 1938, and Zeller, Fouts and Voegtli 1953). Histaminase is efficiently inhibited by amino- guanidine (Schuler 1952, Schayer, Kennedy and Smiley, 1953).

Methylation is also a major pathway for histamine catabolism in many species. The ensuing oxidation of the methylhistamine is mainly or entirely catalyzed by another enzyme monoamine oxidase (E.C. 1.4.3.4) (Rothschild and Schayer 1958). For the methylation of histamine no specific inhibitors are known.

In most species, e.g. rats and man, the urinary excretion of histamine does not change significantly during periods of aminoguanidine treatment although oxidative deamination is of great quantitative importance for the detoxication of histamine in these species (Mitchell 1956, Westling 1958, Lindell et al. 1960 and Granerus, Wet- terqvist and White 1968). In sheep, on the other hand, the toxicity of histamine is completely altered by aminoguanidine treatment (Sjaastad 1967). This is in ac-

1-733003. Acfn physiot. scnnd. 88: I 1

2 KNUT ARNET ELIASSEN

cordance with the observations that ruminating species detoxicate i.v. injected histamine mainly by oxidative deamination (Eliassen 1969 and 1971 a ) . One of the main intentions of this paper was to examine if the observed effect of amino- gaunidine in sheep is typical for the ruminating species and further to examine how histamine is catabolized when diamine oxidase is inhibited by aminoguanidine.

Since there seems to be some controversy regarding the specificity of amino- guanidint on histamine metabolism, experiments on this aspect are also included.

It was believed that a possible effect of aminoguanidine on metabolic pathways other than oxidative deamination could be most easily demonstrated in species in which oxidative deamination is of minor quantitative importance. Pigs were chosen for this purpose since methylation prior to oxidative deamination has previously been found to be the major catabolic pathway for histamine detoxication in this species (Eliassen 1971 b) .

Abbreviations Hi: histamine, 4 (5) - (P-aminoethyl) imidazole. AcHi: N-acetylhistamine, 4 (P-acetylarriinoethyl) - imidazole. 1.4-MeHi: 1.4-methylhistamine, 1-methyl-4- (8-aminoethy1)imidazole. ImAA: imidazoleacetic acid, imidazole-4 (5) -acetic acid. 1.4-MeImAA: 1.4-methylimidazoleacetic acid, 1-methyl-imidazole-4-acetic acid. AG: aminoguanidine. DAO: diamine oxidase (E. C. 1.4.3.6).

Materials and Methods Methods and reagents in the present study were mainly the same as those previously described (EIiassen 1969, 1971 a and b ) .

The methods used include determination of 14C-metabolites by means of isotope dilution technique. Bioassay of urinary histamine activity was performed on guinea-pig ileum subse- quent to ion exchange chromatography. Conjugated histamine was determined as the increase in urinary free histamine which occurred on hydrolysis with 10 N HCI for 1 1 / 2 h. 1.4- MeImAA was estimated by the method of Granerus and Magnusson (1965) and Granerus (1968 a),.

Materzals: Histamine (2-ring-14C) spec. act. 54 mCi/mmole was obtained from the Radio- chemical Centre, Amersham, England.

1.4 MeHi and 1.4-MeImAA were synthesized in collaboration with Maj-Britt Johanson, Clin. Physiological Laboratory, Lasarettet, Lund, Sweden. Aminoguanidine sulfate was obtained from Eastman Organic Chemicals, Rochester, N.Y., U.S.A.

Administration of I4C-Hi: The purity of the injected 14C-Hi was checked by two-dimen- sional paper chromatography followed by autoradiography. Only one spot was detected. The 14C-Hi was therefore, without further purification dissolved in 20 ml 0.9 5% NaCl and in- jected into the jugular vein of the goat or into an ear vein of the pig. The goats were given about 45 /rCi Hi and the pigs about 60 pCi Hi. One pCi corresponds to 2.06 p g Hi-base.

Administration of aminoguanidine: 1 mg per kg of aminoguanidine sulfate (calculated as aminoguanidine base) was injected i.m. 3 times daily. The first dose was given 2 to 3 days before the injection of 14C-Hi and the treatment continued throughout the experimental period. About 1 week lapsed between the control experiments and the start of the aminoguanidine treatment.

Animals and feeding: 2 healthy female goats of Norwegian breed weighing 40 kg and 2 healthy castrated male pigs of the Norwegian “Land-win” breed were used in the experiments. Their body weight increased from 70 to 90 kg during the experimental period. The animals were housed in metabolism cages which permitted separate collection of urine and feces. Suf- ficient HCI to bring the pH of the urine below 2 was added to the collection bottles. The goats were fed pelleted concentrates and hay. The pigs’ diet consisted of concentrates containing the antibiotic zincbacitracin. Water was freely available throughout the experimental period.

HISTAMINE METABOLISM IN AG TREATED GOATS A N D PIGS 3

Results

Urinary excretion of H i and 1.4-MeImAA Histamine: Free and conjugated Hi was determined in urine collected the first

24 h after injection of I4C-Hi. The results are shown in Table 11, the values, given in terms of the base, are corrected for analytical losses and for the fraction of the injected histamine that can be calculated to be excreted unchanged in the urine (Table I ) . At least one recovery experiment was done for each urine specimen. In the pig the recovery of histamine diphosphate ( l@-lOO ,~ig/25 ml) was 84.3f 10.3 % (S.D., 8 expts.). For N-acetylhistamine (10-100 ,ug/25 ml), believed to be

TABLE I. Quantitative analyses for histamine metabolites in the first 24 h urine ofaminoguanidine treated goats and pigs, after i.v. injection of 14C-histamine.

Animals Treatment Excreted Histamine metabolites, yo of total 14C- in the first 24 h radioactivity urine per 24 h in yo of Hi 1.4- ImAA 1.4- Conj. Sum injected dose MeHi MeIm of

Free Ribo- Total AA metab- side Hi olites

Goat 4 No 86 1 0.27 - - 77 19 -0 97 Goat 4 AG 85 11 24.0 - - 4 61 -0 100 Goat 5 No 84 1 0.32 - - 60 22 -0 83 Goat 5 AG 15 10 27.6 - - 4 53 (0.5 95 Pig 4 No 94 2 10.3 4 15 19 66 < I 97 Pig 4 AG 90 1 37.5 0.3 3.2 3.5 55 <0.5 97 Pig 5 No 81 3 11.4 0.4 17 17 62 <0.5 93 P1g 5 AG 80 4 36.9 0.1 1.2 1.3 69 (1 111

TABLE 11. Urinary excretion of Hi and 1.4-MeImAA in goats and pigs with and without treatment with aminoguanidine.

Animal Treat- Histamine 1.4-MeImAA Moles 1.4- l4C-l .4- 14C-1.4- ment pg/24 h mg/24 h MeImAA MeImAA MeImAA

Total Free Conj. Moles Hi 14C-Hi "C-1.4-MeHi

Goat 4 No Goat 4 No Goat 4 No Goat 4 AG Goat 5 No Goat 5 AG Pig 4 No Pig 4 AG Pig 5 No Pig 5 AG

- - 22 -* 5.7 -*

7,000 380 17,300 1,200 12,500 450 2,800 300

-* -* -* -*

0.9 - 2.6 - 1.6 - 0.9

- - -

6,620 65 16,100 42 12,050 100 2,500 32

- - 30

140 30

180 90

- 20 6

20 5

30 60 20 20

-

70

69 2.5

1.9 6.4 1.5 5.4 1.9

All values are expressed as base or acid. The values are corrected for procedural losses. * Hi could not be determined by the method used since the urine showed antihistamine-likeactivity

4 KNUT ARNET ELIASSEN

identical to conjugated histamine, (Tabor and Mosettig 1949), the recoveries were about the same for both species and were as low as 47 f 5 % (S.D., 6 expts.) . Be- cause of the presence of substances with antihistamine activity in most of the urine specimens from goats, in most cases neither free nor conjugated Hi could be estimat- ed in these urines.

1.4-MeImAA: The values for urinary 1.4-MeImAA the first 24 h after injection of 14C-Hi are given in Table 11. The recovery of 1.4-MeImAA hydrochloride added to urine (300-400 pg/25-100 ml) was nearly the same for both species and for the whole material it averaged 86 I 2 0 % (S.D. 35 expts.). The values were corrected on this basis. Further the values were corrected for the fraction originating from the injected '%-Hi (Table I ) .

Excretion of radioactivity after injection of '"-Hi. The radioactivity recovered in urine during periods of AG treatment was not significantly different from that recovered in the control experiments (Table I) . However, the excretion of known I4C-Hi metabolites tended to be lower during the periods of AG treatment than in the control experiments.

Quantitation of '"-Hi metabolites ii2 the urine. Table I gives the values of the urinary 14C-metabolites of i.v. injected l4C-Hi before and during periods of AG treatment. The metabolic patterns in the control experiments do not deviate much from those found in previous experiments (Eliassen 1969, 1971 a and b ) . In goats AG reduced the l4C-ImAA (free and riboside) in urine from 70 % to 4 c/c of the total "C-activity. The decrease in the formation of TmAA by AG treatment was partly compensated by an increase in the formation of 1.4-hfeImAA and even more so by an increased formation of 1.4-MeHi. In spite of this compensatory increase in the methylation pathway, the fraction excreted as unchanged histamine also in- creased.

AG also reduced the formation of I4C-ImAA in pigs. In this species, however, the decrease in ImAs4 seemed to be fully compensated by increased methylation of histamine (Table I ) . However, the percentage of 1.4-MeImAA formed by oxidation of 1.4-h4eHi was not increased (Table I ) .

AG treatment reduced the ratio 14C-1.4-MeImAA11"C- 1.4-MeHi in both species. This was most pronounced in goats (Table 11). In the last mentioned species the ratio "C-1 .4-MeImAA/14C-Hi was also reduced during periods of AG treatment; this was not the case in pigs. Antihistamine activity made it impossible to determine urinary free Hi in goats, and accordingly the molar ratio 1.4-MeImAA/Hi could not be calculated. In pigs this ratio decreased during the period of AG treatment.

Discussion

The present work has demonstrated that aminoguanidine has a marked effect on histamine metabolism both in pigs and goats. This effect was most pronounced in

HISTAMINE METABOLISM I N AG TREATED GOATS A N D P I G S 5

goats, in which aminoguanidine (AG) reduced urinary l4C-1mAA from about 70 "/. to 4 of the total urinary radioactivity, and increased 14C-1.4-MeHi and 14C-Hi 100 fold and 10 fold, respectively (Table I). A minor increase in urinary I4C-1.4- MeImAA was also observed in this species. In the pig AG markedly reduced the amount of 14C-ImAA excreted with the urine. This decrease seemed to be fully compensated by a corresponding increase in the excretion of "C- 1.4-MeHi alone, without any change in the fraction excreted as unchanged 14C-Hi (Table I) . The about 10 fold increase in the urinary excretion of unmetabolized 14C-Hi in goats during periods of AG treatment agrees well with findings in sheep (Sjaastad 1967). Sjaastad found that urinary free Hi increased to about 12 times the control level during periods of AG treatment. In pigs AG-treatment did not seem to have any significant effect on the urinary excretion of either exogenous or endogenous free Hi (Table I and 11). The observation of unchanged excretion of unmetabolized Hi in pigs is in accordance with findings in man (Granerus, Wetterqvist and White 1968) which metabolizes in a similar way to pigs. The finding that AG in the present study affected the urinary excretion of Hi in goats, but not in pigs most likely reflects the minor importance of AG sensitive enzymes in the metabolism of Hi in the pig. This would also be expected from the metabolic patterns of I4C-Hi in the two species (Eliassen 1969 and 1971 b) . AG did not completely inhibit the oxidation of i.v. in- jected 14C-Hi to 14C-ImAA either in goats or pigs. This has also been found to be SO

in rats (Westling 1958, Westling and Wetterqvist 1962). These authors found that even doses of 20-40 mg AG-sulfate/kg did not completely inhibit the formation of ImAA from Hi. In man, however, doses of 0.1 mg/kg administered orally abolished the I4C-IrnAA formation (Granerus 1968 b). The formation of ImAA during treat- ment with AG is hardly due to incomplete inhibition of histaminase. It is more like- ly that enzymes which are little sensitive to or insensitive to AG participate in the in v i m formation of ImAA in some species. In this connection it is worth men- tioning that both benzylamine oxidase, an enzyme found in blood plasma of most mammals, and spermine oxidase (E.C. 1.5.3.3.) an enzyme found in plasma of ruminants, are able to catalyze the formation of IinAA from Hi (Blaschko and Hawes 1959, and Buffoni and Blaschko 1964). These enzymes are inhibited by AG (Blaschko 1962). Oxidation of 14C-Hi to ImAA by enzymes insensitive to AG has also been demonstrated in non-ruminating species (Kobayashi 1957). The increased excretion of methylated 14C-Hi metabolites in both species during periods of AG- treatment, indicates that methylation partly takes over when the oxidation of Hi to ImAA is blocked. However, non-radioactive 1.4-MeImAA was not materially affect- ed by AG in goats. In pigs the urinary excretion of non-radioactive 1.4-MeImAA seemed to be somewhat reduced during periods of AG treatment. In experiments in which large doses of Hi are given i.v. to goats and pigs, the ratios 14C-1.4-MeImAA/ "C-Hi and 14C-1 .4-MeImAA/14C-MeHi increase (Eliassen to be published). This indicates that the decreased ratio I4C-l .4-MeImAA/14C-1 .4-MeHi during periods of AG treatment is not due to an increase of the primary substrate, Hi. A more likely explanation for the reduced ratio I4C-1 .4-MeImAA/I4C- 1.4-MeHi during periods of

6 KNUT ARNET ELIASSEN

AG treatment, is that AG inhibits the oxidative deamination of 1.4-MeHi to 1.4- MeImAA. This would be in agreement with the observation of Rothschild and Schayer (1958) that AG treatment increased the 14C-1.4-MeHi, but not the 14C-1.4-MeImAA excreted in the urine of mice. Further, 12 h infusion experiments in man (Granerus et al. 1968) also gave evidence for an impaired oxidation of 1.4-MeHi during periods of AG treatment.

Oxidation of 1.4-MeHi to 1.4-MeImAA by monoamine oxidase is well established (Rothschild and Schayer 1958). The impaired oxidation of 1.4-MeHi during periods of AG treatment referred to above indicates that enzymes other than monoamine oxidase participate in the oxidative deamination of 1.4-MeHi. In c i t r o studies have shown that both histamine (Lindell and Westling 1957) and benzylamine oxidase and spermine oxidase (Blaschko 1959) oxidise 1.4-MeHi.

This investigation was supported financially by the Agricultural Research Concil of Norway. The technical assistance of Miss Astrid Stafseng and Mrs. Liv B. Kjuus is appreciated.

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