Catabolism of polyamines in the rat

Biochimica et Biophysica Acta, 802 (1984) 175-187

175

Elsevier BBA 21890

CATABOLISM OF POLYAMINES IN THE RAT POLYAMINES AND THEIR NON-a-AMINO ACID METABOLITES GITA A. VAN DEN BERG, HENK ELZINGA, GIJS T. NAGEL, ANNEKE W. KINGMA and FRITS A.J. MUSKIET

Central Laboratory for Clinical Chemistry, University Hospital, Oostersingel 59, P.O. Box 30.001, 9700 RB Groningen (The Netherlands) (Received March 15th, 1984)

Key words: Polyamine catabolism; Polyamine metabolite; (RaO

The metabolic fate of stable isotopically labeled polyamines was investigated after their first and second intraperitoneai injection in rats. Using gas chromatographic and mass fragmentographic analyses of acid-hydrolyzed 24-h urines, some aspects of the polyamine metabolism could be elucidated. After the injections with hexadeutero-l,3-diaminopropane, only labeled 1,3-diaminopropane was recovered from the urine samples. The rat injected with tetradeuteroputrescine excreted labeled putrescine, y-amino-n-butyric acid, 2-hydroxyputrescine and spermidine, while the urine samples of the rat after the injections with tetradeuterocadaverine contained labeled cadaverine and 8-aminovaleric acid. The injections of hexadeuterospermidine led to the appearance of labeled spermidine, isoputreanine, putreanine, N-(2-carboxyethyi)-4-amino-n-butyric acid, putrescine, ~,-amino-n-butyric acid, 1,3-diaminopropane, fl-alanine and spermine. After the injections with octadeuterospermine, labeled spermine, N-(3-aminopropyl)-N'-(2-carboxyethyi)-l,4-diaminobutane, N,N'bis(2-carboxyethyl)-l,4-diaminobutane, spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-aminon-butyric acid, putrescine, 1,3-diaminopropane, fl-alanine, 2-hydroxyputrescine and possibly v-amino-n-butyric acid were recovered. Clear differences between the metabolism after the first and second injection were noted for putrescine, spermidine and spermine, which is suggestive for enzyme induction a n d / o r the existence of salvage pathways. Introduction

Polyamines are functional components of living cells which, most probably by their association with nucleic acids, are indissolubly united with growth processes [1-3]. The determination of polyamines and their metabolites in biological fluids is important for the diagnosis and follow-up of cancerous growth and for the estimation of the extent of chemo- and radiotherapeutically induced tumor cell death in cancer patients (Refs. 4 and 5, and private communications). Due to their involvement in cell proliferation, the biosynthesis of polyamines has been studied in 0304-4165/84/$03.00 © 1984 Elsevier Science Publishers B.V.

detail [3,7]. Relatively little attention has been paid to the catabolism of these potent growth factors. The most frequently mentioned reversible deactivating route is by N-conjugation with acetic acid, propionic acid, glutamic acid, peptides and pyridoxal phosphate [8]. The quantitatively most important catabolic pathway of polyarnines is their oxidation. The enzymes, capable of catabolizing polyamines, are mono-, di- and polyamine oxidases [1,9]. The mammalian amine oxidases catalyze the oxidative deamination of one or both terminal primary amino groups of the polyamines to the corresponding mono- or dialdehydes. The products of oxidized polyamines, the amino aldehydes,

176

are unstable and toxic and are subsequently metabolized by an aldehyde dehydrogenase or an alcohol dehydrogenase to an amino acid or an amino alcohol, respectively [8-10]. Polyamine oxidase catalyzes the oxidative cleavage of spermidine and spermine, which are split at the secondary amino groups to yield 3-aminopropionaldehyde and putrescine from spermidine, and 3-aminopropionaldehyde and spermidine from spermine. Little is known about the characteristics and the regulation of the activity of the amine oxidases. Increased levels of diamine oxidase in serum have been associated with a number of diseased states, including several types of cancer [11]. Another mode of oxidative catabolism is the hydroxylation of putrescine to 2-hydroxyputrescine [12]. The quantitative importance of the different catabolic routes of the polyamines in vivo is indistinct. In order to elucidate some aspects of the polyamine metabolism, we present the results of experiments in which the metabolic fate of intraperitoneally injected, stable isotopically labeled polyamines was investigated in rats. Using gas chromatographic and mass fragmentographic analyses of 24-h urine samples, some already known and a few postulated metabolites could be identified. Materials and Methods

Standards and reagents 1,3-Diaminopropane, putrescine, cadaverine, spermidine, spermine, putreanine, 1,6-diaminohexane, 1,7-diaminoheptane, /~-alanine, ~,-amino-nbutyric acid and Norit A (activated charcoal) were from Sigma, St. Louis MO, U.S.A.; bis(3-aminopropyl)amine and Dowex 1-X4 (200-400 mesh) from Fluka, Buchs, Switzerland, and N-(3aminopropyl)-2-pyrrolidinone (the ~,-lactam form of isoputreanine), 8-aminovaleric acid, 1-amino-1cyclopentane carboxylic acid (cycloleucine), 1,4-diamino-2-butanone and malononitrile were from Aldrich Europe, Beerse, Belgium. Sep-Pak silica cartridges were from Waters, Milford, MA, U.S.A.; 3 ml aromatic sulfonic acid disposable extraction columns from Baker Chemical Co., Phillipsburg, N J, U.S.A., and heptafluorobutyric anhydride was from Pierce Chemical Co., Rockford IL, U.S.A.

All other reagents were from Merck, Darmstadt, F.R.G.

Syntheses of internal standards, polyamine metabolites and deuterium-labeled polyamines The internal standards N-(3-aminopropyl)-l,5diaminopentane (nonsymmetrical homospermidine), N,N'-bis(3-aminopropyl)-l,5-diaminopentane (symmetrical homospermine), N l-methylisoputreanine, and the polyamine metabolites N-(3aminopropyl)-4-aminobutyric acid (isoputreanine), N-(3-aminopropyl)-N'-(2-carboxyethyl)-1,4-diaminobutane (sphermidic acid 1), N, N'-bis(2-carboxyethyl)-l,4-diaminobutane (spermic acid 2) were prepared as previously described (13). N-(2-Carboxyethyl)-4-amino-n-butyric acid (spermidic acid 2;) was prepared by cyanoethylation [14] of ~,-aminobutyric acid with acrylonitrile in alkaline solution, followed by acid hydrolysis in 6 M HC1 at 120°C. 2-Hydroxyputrescine was prepared by reduction of 1,4-diamino-2-butanone with excess sodiumborohydride in aqueous solution. 1,3-Diaminopropane-d 6 (H2N-C2H2-C2H2-C2H2-NH2), putrescine-d 4 (H2N-C 2H2-CH 2-CH 2-C 2H 2-NH 2) and cadaverine-d4 (H2N-C2H2-CH2-CH2-CH2 C 2 H 2-NH2) were prepared by catalytic reduction of their di-cyano precursors malononitrile, succinonitrile and glutaronitrile, respectively, with deuterium gas [14]. Spermidine-d 6 (H2N-C2H2-CH 2-CH2-NH C2H2-CH2-CH2-C2H2-NH2) and spermine-d 8 (H 2N-CZH2-CH2-CHz-NH-C2 H2-CH2-CH2-C 2H2NH-CHR-CaH2-NH2) were prepared by cyanoethylation of putrescine-d4, followed by catalytic reduction of the recrystallized mono- and dicyano intermediates, respectively, with deuterium gas [14]. Animal experiments Five, 3-months-old female albino rats of the Wistar strain were individually kept in metabolic cages. Each individual rat was used for the study of the metabolism of only one labeled polyamine. 24-h urine was collected for 8 consecutive days in flasks, containing 1 ml of 2 M HC1 to prevent bacterial growth. After 2 days, the rats were intraperitoneally injected with 15 t~mol of 1,3-diaminopropane-d6, 15 /,tool of putrescine-d4, 2.5 t*mol of cadaverine-d4, 10 ~mol of spermidine-d6

177 and 7/~mol of spermine-d8, respectively, dissolved in 1 ml of 0.9% (w/v) NaC1 solution. The same treatment was repeated after the fifth day. 24-h urine volumes were measured, and the samples were stored at - 2 0 o C until analysis. The creatinine concentration was measured by a picric acid method.

Prepurification and derivatization To 0.5 ml of acidified urine was added an internal standard cocktail containing 12.5 nmol each of 1,6-diaminohexane (internal standard 1, IS1), 1,7-diaminoheptane (IS2), Nl-methylisoputreanine (IS3), bis(3-aminopropyl)amine (IS4), N-(3-aminopropyl)-l,5-diaminopentane (IS5) and N, N'-bis(3-aminopropyl)-l,5-diaminopentane (IS6) and 50 nmol of cycloleucine. After the addition of 0.5 ml of 12 M HC1 and subsequent acid hydrolysis at 120 °C for 18 h, the polyamines and three of their non-a-di- and triamino acid metabolites were isolated by silica gel adsorption, and converted into their (methyl)heptafluorobutyryl derivatives [13]. 1.5-t~l aliquots of the derivatized silica gel extracts were analyzed by gas chromatography with nitrogen-phosphorus detection and gas chromatography with mass spectrometric detection (see below). From the passage of the silica gel column, containing the unretained compounds, the non-amonoamino acid metabolites of the polyamines and spermic acid 2 were isolated and converted into their isobutyl-heptafluorobutyryl derivatives by a modification of the method described by Desgres et al. [15]. The (8 ml) passage of the silica gel column was acidified to a pH of about 2.5 with eight drops of 6 M HCI, and the urinary pigments were removed by thoroughly mixing with 100 mg of a 'modified charcoal/resin' mixture [16]. The charcoal/resin mixture was prepared by the addition of 5 g of sulfosalicylic acid to 20 g Dowex l-X4 (200-400 mesh) and 10 g Norit A charcoal. This mixture was stirred for 2 h in water and washed with 6 M HC1, ethanol and diethyl ether. The treated charcoal/resin mixture was dried at 120°C and stored at 40°C until its usage. After removal of the charcoal/resin by centrifugation at 1000 × g for 15 min, the 8 ml supernatant was passed over a Baker extraction column, that had previously been washed with 4 ml of 1 M HC1, 8

ml of water, 4 ml of 0.1 M HCI and 8 ml of water. The Baker column was washed with 4 ml of water and eluted into a 14 ml Sovirel tube with 3 ml of 4 M ammonia. Regeneration was performed by the same washing procedure as described above. The eluate was evaporated to dryness at 90 o C under a stream of air. Isobutyl esters were prepared by the addition of 1 ml of an isobutanol/HCl solution (freshly prepared by cautiously adding 10 ml of acetylchloride to 100 ml of mechanically stirred isobutanol) and heating the tightly capped tubes at ll0°C for 45 rain. After evaporation at 40°C under a stream of air, 200/~1 of acetonitrile and 200 /~1 of heptafluorobutyric anhydride were added, and the tubes were mechanically shaken at room temperature overnight (about 18 h). The solutions were evaporated to dryness at room temperature under a stream of air, and the residues were dissolved in 0.5 ml of ethyl acetate. 1.5-/~1 aliquots of the derivatized cation exchange extracts were analyzed by gas chromatography with nitrogen-phosphorus detection and gas chromatography with mass spectrometric detection (see below).

Gas chromatography with nitrogen-phosphorus detection and quantification Gas chromatography with nitrogen-phosphorus detection was performed with a Hewlett-Packard model 5880 gas chromatograph equipped with a model 7672A automated sampler and interfaced with a Tracor 812 Analytical Processing data system. The column was a 35 m cross-linked methylsilicone-coated (film thickness 0.11 /~m), siloxanedeactivated, fused silica capillary (i.d. 0.2 mm) from Hewlett Packard, 1181 KK Amstelveen, The Netherlands. Gas flow-rate (helium) was 0.5 ml/min, split ratio 1:12, detector temperature 300 ° C and injector temperature 260 ° C. The quartz insert of the injector was filled with a plug of glass wool and deactivated in a solution of 0.2% (w/v) Carbowax 20 M in chloroform and dried at 150°C. In the case of profiling the polyamines and their non-a-di- and triamino acid metabolites (silica gel extract), the oven temperature program was: 120°C, 7°/min to 260°C, 20 min at 260°C; and in case of profiling the non-a-monoamino acid metabolites and spermic acid 2 (cation exchange extract): 90°C, 5°/min to 240°C, 20 rain at 240°C.

178

For polyamines and their non-a-di- and triamino acid metabolites, quantification was performed by comparing the peak area ratio of each analyte and its internal standard to that of the standard containing 12.5 nmol of all compounds. 1,3-Diaminopropane, putrescine, cadaverine and putreanine were quantified using IS2, isoputreanine on the basis of IS 3, spermidine and spermic acid 1 using ISs, and spermine with IS6. In the case of the non-a-monoamino acid metabolites and spermic acid 2 quantification was performed by comparing the peak height ratio of each analyte and the internal standard (cycloleucine) to that of the standard containing 12.5 nmol of /3-alanine, ~,-aminobutyric acid, cycloleucine, &valeric acid, spermidic acid 2 and spermic acid 2. The concentrations were expressed in relation to those of urinary creatinine.

Gas chromatography~mass spectrometry and calculation of relative labelings Gas chromatography/mass spectrometry was performed with a Varian 3700 gas chromatograph directly coupled to a MAT 44-S mass spectrometer and operated under the following conditions: injector temperature 250°C; split ratio, 1:10; oven temperature program, 110°C, 1 0 ° C / m i n to 260 o C; ion source temperature, 200 o C; ionization energy 70 eV. The column was the same as that mentioned above. Both silica gel extracts and cation exchange extracts were monitored in the electron-impact mode on the ions at m / z 226, corresponding to the [heptafluorobutyryl-NH=CH 2]+ fragment of naturally occurring polyamines and metabolites, and m / z 228, corresponding to the [heptafluorob u t y r y l - N H = C 2 H 2 ] + fragment of their deuterated analogues. In case of spermidic acid 2 and spermic 2 (only cation exchange extracts) the ions at m / z 286/288 and 539/543, corresponding to the respective [M-heptafluorobutyryl] + fragments, were monitored. The peak area ratios (228/226, 288/286, 543/539) were calculated using the Finnigan MAT SS-200 data system. The mean of the peak area ratios calculated from the two urine samples before the injection with deuterated polyamines (that corresponds with the normal ratio caused by natural isotope abundance and actual fragmentation)

was fixed at 1.00. Other ratios (relative labeling) were calculated relative to these mean peak area ratios. In some cases the peak height ratios were used. Results

Purity of the synthesized deuterium-labeled polyamines and their mass spectra The purity of all synthesized deuterium-labeled polyamine analogues was checked by gas chromatography and gas chromatography/mass spectrometry. The spermidine-d 6 solution was found to contain 0.56 # m o l / m l of spermic acid 1 (corresponding to a 5.6 mol% contamination of the spermidine-d 6 preparation). In Fig. 1 the mass spectra of the (heptafluorobutyryl)3 derivatives of naturally occurring spermidine-d 0 (top) and of its deuterated analog spermidine-d 6 (bottom) are shown.

Measurements Fig. 2 shows an example of the gas chromatographic profiling of the silica gel extract for the urine sample of a rat after an injection of spermidine-d 6. The capillary column effects a nonbaseline separation of spermidine-d 0 and spermidined 6. The metabolites isoputreanine-d 4 and putreanine-d 4 appeared as shoulders in the peaks of their endogenous analogs. The high peak of spermic acid 1 was caused by the impurity of the spermidine-d 6 preparation. In the first 24 h after the injections of 10 ~mol spermidine-d 6 the increase of the excretion of spermic acid I was calculated to amount to 0.60/~mol and 0.63/~mol, respectively. In Fig. 3 an example of the gas chromatographic profiling of the cation exchange extract for the urine sample of a rat after an injection of spermidine-d 6 is depicted. In most urine samples the peak of spermic acid 2 was too small to be accurately measured. Only urine samples of rats injected with spermidine-d 6 and spermine-d8 were found to contain measurable amounts of spermic acid 2. A mass fragmentogram of the silica gel extract for the urine sample of a rat after an injection of spermidine-d 6 by monitoring the fragment ions m / z 226 and 228, is shown in Fig. 4. A relatively large peak is observed for spermic acid 1 at m / z 228, which is due to the previously mentioned

179 226

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180

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Fig. 6. Concentrations (o . . . . . . o) and relative labelings (O o) of polyamines and their metabolites in the urine of a rat before and after intraperitoneai injection with 2 x 15/zmol putrescine-d 4. The arrows indicate the days of injection. For abbreviations see legend of Figs. 2, 3 and 4.

182

with the mean relative labeling + 2 S.D., calculated from measurements of the compound performed in other urines in which no labeling could occur on metabolic grounds. For instance, as 1,3diaminopropane and cadaverine cannot be converted into putrescine, the mean relative labeling + 2 S.D. for putrescine measured in the urine of the rats injected with 1,3-diaminopropane-d 6 and cadaverine-d 4, served as a cut-off value for the establishment of putrescine labeling in the urines of the rat injected with labeled putrescine, spermidine and spermine. These cut-off values were calculated to be 1.20 (hydroxyputrescine), 1.13 (1,3-diaminopropane), 1.18 (/3-alanine), 1.21 (putrescine), 1.58 (y-aminobutyric acid), 1.33 (cadaverine), 1.30 (8-aminovaleric acid), 1.09 (spermidine), 1.19 (isoputreaninine), 1.41 (putreanine), 1.08 (spermidic acid 2), 1.31 (spermine) and 1.41 (spermic acid 1).

impurity of the spermidine-d 6 preparation. Fig. 5 shows mass fragment•grams of the cation exchange extract for the urine sample of a rat after injection of spermine-d 8 by monitoring the fragment ions m/z 226/228, 286/288 and 539/543. Only in the urine sample of the rat injected with spermine-d 8 accurate measurement of the relative labeling of spermic acid 2 was found to be possible. In Figs. 6-8, the concentrations and relative labelings of 14 polyamines and expected metabolites in the urine samples of rats_ injected with putrescine-d4, spermidine-d 6 and spermine-d 8 are depicted as a function of time.

Criteria for establishing the labeling of the polyamines and their metabolites For establishing the labeling of a compound each calculated relative labeling was compared /~-Ala

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TIME(DAYS) Fig. 7. Concentrations (O. . . . . . o) and relative labelings (o o) of polyamines and their metabolites in the urine of a rat before and after intraperitoneal injection with 2 × 10 #mol spermidine-d6. The arrows indicate the days of injection. For abbreviations see

legend of Figs. 2, 3 and 4.

183

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T IMEIDAYS)'--~

Fig. 8. Concentrations (O. . . . . . e ) and relative labelings (o O) of polyamines and their metabolites in the urine of a rat before and after intraperitoneal injection with 2 × 7 #mol spermine-d 8. The arrows indicate the days of injection. For abbreviations see legend of Figs. 2, 3 and 4.

Urinary excretion of labeled polyamines and polyamine metabolites After the two injections with 15/~mol of 1,3-diaminopropane-d 6 only labeled 1,3-diaminopropane was recovered from the urine samples, amounting to about 35% of the injected quantity after the first injection and about 50% after the second. The rat injected with putrescine-d4, excreted about 13% of the labeled putrescine after the first and a not measurable quantity after the second injection, and labeled T-aminobutyric acid, hydroxyputrescine and spermidine in the urine (Fig. 6). The urine samples of the rat after injection with 2.5 /~mol of cadaverine-d 4 contained about 15% of the injected amount of cadaverine-d 4 after both injections, and labeled 8-aminovaleric acid. The injections of spermidine-d 6 led to the ap-

pearance of labeled spermidine (about 35% after the first injection and about 25% after the second), isoputreanine, putreanine, spermidic acid 2, putrescine, T-aminobutyric acid, 1,3-diaminopropane, fl-alanine and spermine in the urine (Fig. 7). The increase in the excretion of labeled spermic acid 1 could be completely assigned to its presence in the spermidine-d 6 preparation, indicating that the metabolites found in the urine of this rat cannot be ascribed to catabolism of spermic acid 1. After the injections with spermine-ds, labeled spermine (a non-measurable amount after the first and about 1.5% of the injected quantity after the second injection), spermic acid 1, spermic acid 2, spermidine, isoputreanine, putreanine, spermidic acid 2, putrescine, 1,3-diaminopropane, fl-alanine, hydroxyputrescine and possibly T-aminobutyric acid were recovered from the urine samples.

184 COOH i

N-C-C-E-C-N

[I]

SAM - -

{,,

~ C02

i N ac Sd

l,,l [21

~N acSp

[

[L.I

[l~l [3}

B N-C-C-C-C-N-C-C-C-N 7

/~1p y r ~ t i ;"e~ 6 ]"z,3)OH Pu

succinic acid

dec.SAM

o N oc Sd

I N ac.Pu

N_C.C_C_C_N ~

[51

is0-

[/~pyrroline

I FATTY ACIDS

12N-C-C-C- N-C-C-C- C-N-C-C-C-N

( ~ufreanine

DAP Pu'.---~ 13-AIa---~

~x~rn°~°n' ISdacidl' c.~/aci~/,'d~"I I [

~'----- Sp acid1

Sp acid z J

Scheme I. Synthetic and hypothetic degradative pathway of polyamines. Only (mono) N-acetyl conjugation of polyamines is shown. Possible conjugation of metabolites and metabolization of conjugates are not included. Except for putrescine, only combined activities of amine oxidases and aldehyde dehydrogenases are shown. 1, L-ornithine decarboxylase (EC 4.1.1.17); 2, spermidine synthase; 3, spermine synthase; 4, N-acetyltransferase; 5, S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50); 6, hydroxylase; DAP, 1,3-diaminopropane; Pu, putrescine (1,4-diaminobutane); Sd, spermidine (N-(3-aminopropyl)-l,4-diaminobutane); Sp, spermine (N,N'bis(3-aminopropyl)-l,4-diaminobutane; GABA, -/-amino-n-butyric acid; fl-Ala, fl-alanine; Sd acid 2, N-(2-carboxyethyl)-4-amino-nbutyric acid; Sp acid 1, N-(3-aminopropyl)-N'-(2-carboxyethyl)-l,4-diaminobutane: Sp acid 2, N,N'-bis-(2-carboxyethyl)-l,4-diaminobutane; SAM, S-adenosyl-L-methionine; dec. SAM, decarboxylated S-adenosyl-L-methionine; OH, hydroxy; ac, acetyl; *, further metabolization possible into compounds depicted elsewhere in the scheme.

Discussion

In Scheme I, the biosynthetic and hypothetic degradative pathways of putrescine, spermidine and spermine, that have been used as starting points for our study, are depicted. The possible formation of amino alcohols and the metabolism of conjugates have not been taken into account.

Comments on the methodology The present approach for the in vivo elucidation of the polyamine metabolism demonstrates the usefulness of deuterated analogues in combination with gas chromatography and mass fragmentography. The advantages of this method over classical methods with radioactive tracers are: (i) the use of stable isotopes, (ii) the selectivity offered by the combination of capillary gas chromatographic retention times and mass spectrometric data, and (iii) the possibility to establish simultaneously the labeling of a large number of possible

metabolites without the necessity of laborious prepurification. All mass spectra of the heptafluorobutyryl derivatives of polyamines and their mona-, di- and triamino acid metabolites, except for spermidic acid 2 and spermic acid 2, comprise a base peak at m,/z 226, corresponding to the [heptafluorob u t y r y l - N H = C H 2 ] + fragment ion. For this reason, the deuterium label was incorporated at the carbon atom next to the primary nitrogen. Consequently, the mass spectra of the derivatized deuterated polyamines and their metabolites show a base peak at m / z 228 (Fig. 1). By monitoring the fragment ions m / z 226 and 228, the relative labelings could be established for all polyamines and most of their metabolites (Figs. 4 and 5). In the gas chromatograms of the cation exchange extracts, the relatively high amounts of the co-eluted a-amino acids sometimes made quantification of the monoamino acid metabolites difficult (Fig. 3). On the other hand, in the mass fragmen-

185 tographic profiling, the chances of interference were considerably less (Fig. 5), as amongst the naturally occurring a-amino acids only glycine, ornithine and lysine possess the required CH 2-N H 2 configuration. Because of the loss of this primary amino group configuration the relative labelings of spermidic acid 2 and spermic acid 2 were established by monitoring their [M-heptafluorobutyryl] ÷ fragment ions m/z 286/288 and 539/543, respectively (Fig. 5). With respect to the establishment of major and minor polyamine catabolic routes, no definite statements could be made on the basis of the height of the relative labelings, as we did not establish the relationship between the relative labeling of a compound and the absolute amount of its labeled analogue. However, in those cases in which the excreted amount of labeled compound led to clear increase of normal endogenous levels, an estimation of its quantity was possible.

Metabolism of intraperitoneally injected deuterated polyamines 1,3-Diaminopropane, putrescine and cadaverine. The possible metabolism of the diamines include: (i) oxidative deamination of one or both amino groups, and (ii) aminopropylation to yield tri- or tetraarnines. The oxidative deamination of one amino group of 1,3-diaminopropane, finally yielding fl-alanine, is known to occur in bacterial systems [9]. In the present study, we were unable to prove the existence of such a pathway in the rat. The finding of the labeled intermediates 7-aminobutyric acid and 8-aminovaleric acid, which are formed by the oxidative dearnination of putrescine and cadaverine, respectively, is consistent with the previous report of Noto et al. (Ref. 12, putrescine), and Henningsson and Henningsson (Ref. 17, cadaverine). Oxidative deamination of both amino groups, finally yielding malonic, succinic and tartaric acid, respectively, leads to the loss of the deuterium label. To examine the arisal of these metabolites a labeling with 13C, or otherwise positioned deuterium in case of putrescine and cadaverine, could be useful. It is, however, questionable whether these dicarboxylic acids can be identified as intermediates of the polyamine catabolism, as they may

rapidly be further metabolized into CO2, or used as biosynthetic building blocks. Moreover, as at least the normal body pool sizes of succinic and tartaric acid are high, the establishment of an increase in their relative labeling will be inaccurate. Further metabolization to CO 2 of 1,3-diaminopropane by bacteria [9], and of putrescine [18] and cadaverine [17] in the rat, has previously been described. In accordance to the results reported by Noto et al. [12], labeled hydroxyputrescine was found to be excreted after the injection with putrescine-d 4. 1,3-Diaminopropane and cadaverine may potentially be aminopropylated to homologues of spermidine and spermine. The occurrence of such a pathway has recently been demonstrated for 1,3-diaminopropane in bacteria [19] and for cadaverine in cultured Ehrlich ascites carcinoma cells, in the case that cadaverine is supplemented in the medium [20]. However, the formation of the higher polyamines of 1,3-diaminopropane and cadaverine, which were used as internal standards in our GC assays, did not occur, as GC and GCMS analyses of the concerning urines without the addition of internal standards showed no accountable peaks, and no labeling at the corresponding retention times. After the injection of putrescine-d4, we were able to detect labeled spermidine, which can be further metabolized to spermine (see below). Noto et al. [12] also demonstrated the conversion of intraperitoneally injected radioactive putrescine and spermidine, into spermidine and spermine, respectively. The urinary recovery of 1,3-diaminopropane in its unaltered form or an acid hydrolyzable conjugate, was considerably higher than that of putrescine and cadaverine (see Results). This may be explained by differences in the efficiency of their respective catabolic a n d / o r salvage pathways. In addition, for putrescine, clear differences between the recoveries after the first and second injection were observed (Fig. 6), which may be indicative for the induction of catabolizing enzymes after the first injection. The possibility that the secondly injected amount was salvaged more efficiently seems less likely. Spermidine. The injection of spermidine-d 6 gave rise to the appearance of a number of deuterated compounds in the urine (Fig. 7). Oxidation of one

186 or both primary amino groups by polyamine oxidases, followed by the action of aldehyde dehydrogenases, explains the formation of isoputreanine, putreanine and spermidic acid 2. The conversion of spermidine into isoputreanine and putreanine has been established in different mammals by several investigators [12,21-24]. To date, spermidic acid 2 has not been identified. In mammals and bacteria, spermidine can also be cleaved by polyamine oxidases between the secondary amino group and the aminopropyl moiety, yielding putrescine and fl-aminopropionaldehyde [9,18]. The latter is further oxidized to /3-alanine [11]. Both compounds were found to be labeled in the present study. To date, cleavage between the secondary amino group and the aminobutyl moiety, finally resulting in the formation of 1,3-diaminopropane and yaminobutyric acid, has only been established in bacteria [1,9]. The present finding of labeled 1,3diaminopropane as a metabolite of spermidine-d6 is indicative for the existence of such a degradative pathway in mammals. The hereby additionally formed ~,-aminobutyric acid may also originate from the oxidative deamination of putrescine-d4, as previously outlined. Clear differences in the relative labeling of putrescine and the excreted amount of isoputreanine occurred between the first and the second injection, which may again be explained by the induction of degradative enzymes by the first injection. Differences in the kinetics of the appearance of labeled ~,-aminobutyric acid and flalanine between the first and second injection were also noted. Although detected with a low degree of sensitivity, the conversion of spermidine into spermine could be established beyond doubt. In both instances its appearance in the urine was found to reach a maximum in the fraction collected between 24 and 48 h after the injections. Spermine. The injection of spermine-d8 resulted in the labeling of almost all measured compounds, indicating a complex catabolism (Fig. 8). In harmony with the fact that spermine is the second quantitatively most important polyamine in the majority of cells, whereas it accounts for only a small fraction of the polyamines and their metabolites excreted in urine, the catabolism

a n d / o r salvage of spermine-d8 was found to be very efficient. As previously reported by Noto et al. [12], the combined action of polyamine oxidases and aldehyde dehyrogenases is reflected by the appearance of labeled spermic acid 1 and spermic acid 2. Analogous to the catabolism of spermidine, the polyamine oxidase catalyzed cleavage between the secondary amino group and the aminopropyl moiety led to the simultaneous formation of labeled spermidine and fl-alanine [1,9]. Both metabolites are likely to undergo further metabolization as described above. The arisal of labeled 1,3-diaminopropane may be explained by the subsequent catabolism of the formed spermidine (see above), and/or the direct similar cleavage of spermine between a secondary amino group and the N-(3-aminopropyl)-laminobutyl moiety. Until now, the existence of the latter degradative route has only been suggested in pea seedlings, in which the formation of 1-(3aminopropyl)-2-pyrroline was postulated [9]. As it cannot be distinguished whether the N-(3aminopropyl)-l-aminobutyl moiety originates from the subsequent catabolism of spermidine or the direct catabolism of spermine, no definite proof for the existence of such a degradative pathway in rats can yet be given. On the basis of the appearance of high amounts of labeled isoputreanine after the injection of spermidine-d6 (see above), and its only marginal increase after the injection of spermine-d8, it can be concluded that isoputreanine predominantly originates from the catabolism of spermidine, and not spermine. The formation of putreanine can be explained by the subsequent catabolism of spermidine (see above) and by the action of polyamine oxidase on the formed metabolite spermic acid 1, as previously established by Seiler et al. [22]. The latter possibility seems less likely as the amount of spermic acid 1-d 6 that was coinjected as an impurity of the spermidine-d6 preparation, was completely recovered in the urine. A comparison between the first and second injection revealed striking differences between the kinetics of the appearance of metabolites. These metabolic shifts, that were previously encountered for spermidine (see above), may be considered to be indicative for time-dependent differences in

187

salvage pathways a n d / o r between the relative ratios of the concentrations of the enzymes involved in the polyamine catabolism, caused by enzyme induction.

Conclusions In conclusion, it can be stated that the metabolism of polyamines is complex, comprising the action of a multitude of catabolic- and biosynthetic enzymes, of which the relative activities may be subject to time-dependent variation caused by enzyme induction. The existence of salvage pathways should also be taken into account. In the present investigations, only the acid-hydrolyzable urinary polyamines and their non-a-amino acid metabolites were studied. With the exceptions of 1,3-diaminopropane, fl-alanine and spermidic acid 2, the finding of all other metabolites was in agreement with the findings of Noto et al. [12] and Henningsson and Henningsson [17]. As only the administration by intraperitoneal route was studied, and the amounts of the injected polyamines may not be (patho)physiological, conclusions on the relative importance of the different metabolic pathways may be limited for the elucidation of the metabolism of polyamines that are liberated from cells during (patho)physiological processes. Whether the metabolic pathways found in rats are comparable with those in humans remains to be established.

Acknowledgments The authors thank the Central Animal Laboratory (head: Mr. H. Dikken) for their technical assistance with the handling of rats. Drs. H. Verwey and B.G. Wolthers are gratefully acknowledged for their useful discussions and advice, and Mrs. J.E. Koornstra-Rijskamp for her dedicated assistance with the manuscript. This work was supported in part by grant no GUKC 83-16 (Dr. G.A. Van den Berg) from the Koningin Wilhelmina Fonds (the Netherlands Cancer Foundation).

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