metabolism of thromboxane b2 in the cynomolgus monkey

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METABOLISM OF THROMBOXANE B2 IN THE CYNOMOLGUS MONKEY

Hans Kindahl

Department of Chemistry

Karolinska Institutet

S-104 01 Stockholm, Sweden

ABSTRACT

[5,6,8,9,11,12,14,15-3HaThromboxane B2 was injected into the saphenous vein of female cynomolgus monkeys, and blood samples were withdrawn from the contralateral saphenous vein. The compound was eliminated from the circulation with a half- life of about 10 min after an initial rapid disappearance. Some more polar products appeared with time, and also small amounts of material less polar than thromboxane B2; however, the dominating compound in all blood samples was unconverted thromboxane B2.

About 45% of the given dose of tritium was excreted into urine in 48 hrs. Several metabolites of thromboxane B2 were found. The major urinary metabolite was identified as dinor- thromboxane B2 (about 32% of urinary radioactivity). Uncon- verted thromboxane B2 was also found in considerable amounts (13% of urinary radioactivity).

It is concluded that 1) dehydrogenation at C-12 is not a major pathway in the degradation of this compound, in contrast to metabolism at the corresponding C-15 alcohol group of prostaglandins; 2) after having gained access to the circulation, thromboxane B2 is the main circulating compound; however, assay of thromboxane B2 in plasma will be complicated or precluded by large artifactual production of the compound by platelets during sample collection.

ACKNOWLEDGEMENTS

This project was supported by grants from the Swedlsh Medical Research Council (project no. 03X-217).

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INTRODUCTION

Recently, it was demonstrated that the prostaglandin endoperoxides are converted mainly into thromboxanes rather than into the classical prostaglandins in certain cells and tissues, e.g. platelets and lung tissue (1,2). During platelet aggregation the very active and unstable thromboxane A2 (TXA2) is formed and then rapidly converted into the stable metabolite thromboxane B2 (TXB2), and possibly also into 12L-hydroxy-5,8,1&heptadecatrienoic acid (HHT) (3,4). The amounts of prostaglandins E2 and F2e formed simultaneously are only a few per cent of those of TXB2 and HHT in this system (4). For studies on the role of the prostaglandin endoperoxides and the thromboxanes, a radioimmunoassay for TXB2 was developed (5) and has been applied to in vitro studies on platelet aggregation (6,7). However, for measure- ment of thromboxane production in vivo, it was necessary first to investigate the metabolism of TXB2, since similar problems might pertain to this field as to the measurements of prostaglandins: it is well known that for estimating the endogenous prostaglandin production, certain metabolites have to be monitored in plasma or urine instead of the primary prostaglandins themselves (8).

The cynomolgus monkey, which earlier has been shown to be a suitable model for human studies (9,lO) was chosen for the study on metabolism of TXB2 in vivo.

EXPERIMENTAL

Preparation of r5,6,8,9,11,12,14,15-3H&hromboxane B2.

[5,6,8,9,11,12,14,15-3H&TXB2, specific activity 65 Ci/ mole, was prepared from incubation of [j,6,8,9,11,12,14,15- 3H d -arachidonic acid with a suspension of washed bovine pla-

te ets as described earlier (5). The tritium labeled arachidonic acid (specific activity 87 Ci/mmole) was obtained from the Radiochemical Centre, Amersham, England, and was diluted with unlabeled arachidonic acid to a specific activity of 80 Ci/mole. Addition of arachidonic acid to the platelet suspension always induced aggregation and thus dilution of the formed tritium labeled TXB2 with endogenous material. To estimate the specific activity of the resulting product, separate incubations were carried out with platelets from the same suspension but using unlabeled arachidonic acid under otherwise identical conditions, and the amount of the unlabeled TXB2 formed was determined using the radioimmunoassay for TXB2 (5). From obtained data on the yield of TXB2 from labeled arachidonic acid and the contribution from the platelets under these conditions, the specific activity of the isolated ~,6,8,9,11,12,14,15-3H8]-TXB2 was calculated as 65 Ci/mole.

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Animal Experiments.

Two female cynomolgus monkeys, body weight 2.1 and 3.6 kg, were used in these experiments. Tritium labeled TXB2, 32 uCi (specific activity 65 Ci/mole) was injected as a bolus injection into one saphenous vein of monkey A. Blood samples were collected from the contralateral saphenous vein at the follow- ing times: Sample I was collected from about 1-4 min after the injection; sample II: 5-8 min; sample III: 11-15 min; and sample IV was collected from about 18-23 min. The blood, 5.0 ml in each sample, was collected in tubes containing heparin, and the plasma was isolated. Urine was collected in portions for 48 hours after the injection and was stored frozen until analysis. The experiment was repeated with monkey B, which was given 29 UCi of 3H-TXB

2. The blood samples were withdrawn at

the same times as in t e previous experiment, and urine was collected for 48 hours.

Processing of Blood Plasma and Urine.

After determination of the total radioactivity in the samples of blood plasma, they were extracted at pH 3.5 using 2 g columns of Aaberlite XAD-2 (11). After eluting the columns with 20 ml water, the tritium labeled products were eluted with 20 ml methanol and were taken to dryness. The extract was subjected to reversed phase partition chromatography, solvent system C-45, 4.5 g columns (12).

The urine portions containing tritium were combined, acidified and extracted using a 500 g Amberlite XAD-2 column in a similar way. The labeled compounds were eluted from the column with 2 1 of methanol. The recovery at this stage was 92% of the urinary radioactivity. The extract was subjected to silicic acid chromatography (20 g column), using 750 ml of CHC13:MeOH, 9:l (v/v), which eluted essentially all of the labeled material. Over all recovery: 88% of the total urinary radioactivity. Further purification was achieved by reversed phase partition chromatography, system C-45, column size 45 g. Several radioactive peaks appeared; the material in each was rechromatographed in the same solvent system prior to further purification.

The material in chromatographic peaks was treated with diazomethane and the methyl (Me) esters of the compounds subjected to thin layer chromatography (TLC), solvent system: 2% methanol in ether (v/v). Radioactive zones were detected by scanning with a Berthold Diinnschichtscanner II. The labeled compounds were converted into trimethylsilyl ethers (TMSi) (12) and analyzed bygas-liquid chromatography (GLC) (Barber-Colman Gas Chromatograph, with simultaneous regi- stration of mass and radioactivity) on 1% SE-30, and by mass spectrometry (LKB 9000 S) as described earlier (13).

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RESULTS

Disappearance of TXB2 from the Circulation.

After intravenous injection of 32 UCi of 3H-TXB2 into monkey A, the radioactivity initially disappeared rapidly from the circulation. Already in the first sample, collected from 1 to 4 min after the injection, analysis of the tritium content indicated that only 12% of the given dose of tritium remained in the circulation (assuming a blood volume of 220 ml for this monkey). Thereafter, the decline of the tritium content was slow: the radioactivity of the last sample, collected from 18-23 min after the injection, showed that 5% of the given dose still remained in the circulation.

The tritium content of the samples was always determined prior to extraction; thus the low levels could not be explai- z;;ezt?;Ez,br tow recoveries. Furthermore, in a separate

l-l TXB2 was added to normal monkey plasma and extracted by the same procedure. The recovery was always high, about 90%. The red blood cells from the four blood samples mentioned above were also treated with distilled water and completely hemolyzed. The tritium content of these preparations was determined both before and after extraction using Aberlite XAD-2. The red cells contained only l-2% of the total sample radioactivities. Thus, the rapid initial disappearance of TXB2 was not caused by uptake into red blood cells either.

The four plasma samples from the i-v. injection experiment were purified as described above. Reversed phase partition chromatography of the extracted samples showed a similar pattern in all the samples, with one major peak of radioactivity eluted with 60-90 ml of effluent. This effluent volume is identical with that of TXB in the same solvent system. Fig. 1 shows the chromatograms fr m sample I and IV, respec- 3 tively. Some more polar material appeared with time, repre- senting about 17%, 19%, 21% and 23% of the total radioactivities of samples I-IV, respectively. Small amounts of less polar products were found in all cases. The amounts of this non-polar material was 6%‘ lo%, 12% and 17% of the total sample radioactivities in the four samples.

The major peak of radioactivity was collected and pooled from all the four samples and was esterified with diazomethane. The Me ester of the compound was subjected to TLC (Rf = 0.48; Rf of TXB2 = 0.48); the material in this zone was eluted with methanol, converted into the TMSi derivative, and analyzed by GLC. The C-value of this derivative was 24.6 (C-value of authentic TXB2-Me-TMSi = 24.6). A small mass peak was obser- ved coinciding with this radioactive peak in the gas chromatogram. A mass spectrum was recorded of this material, and was

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-z a ”

P loo- .>

2 rr 18-23 min

2 200- TXB2

25 50 75 100 125 Elution volume (ml 1

Fig. 1. Reversed phase partition chromatography of plasma sample I, obtained 1-4 min after i.v. injection of 3H-TXB2 (upper panel) and plasma sample IV (18-23 min, lower panel). Column size: 4.5 g. Solvent system: C-45.

Table I. Radioactivity in each blood sample expressed as nCi; a) total and b) radioactivity due to TXB2.

Blood sample: I II III IV

Collection time (min after injection): l-4 5-8 11-15 18-23

Monkey A a) 79 63 37 33 b) 60 45 25 13

Monkey B a) 63 53 32 21 b) 50 37 21 12

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found to be identical with the mass spectrum of authentic TXB2-Me-TMSi (see below). The amount of material in this peak far exceeded that which could be expected to be formed artifactually from platelets during blood sample collection, since parallel processing and analysis of 20 ml of normal monkey blood (obtained prior to the experiment) revealed no detectable amounts of TXB2 using this procedure (limit of detection: about 100 ng).

The half-life of TXB2 in the circulation could be estimated from these data. Initially, the half-life must be very short, since the radioactivity of already the first sample was so low. Thereafter, the compound disappeared more slowly, and for this period (l-23 min after the injection) an average tl/2 of about 10 min could be roughly estimated.

The experiment was repeated with monkey B. The same rapid disappearance was seen initially, and from the first sample (l-4 min after injection) could be calculated that 14% of the given dose of tritium remained in the circulation at that time. The estimated half-life for TXB2 during the period of sample collection was 9 min. As in the first experiment, some metabolic conversion of TXB2 was seen: in the last sample (18-23 min) 26% of the total sample radioactivity was represented by products more polar, and 16% by products less polar than TXB2. However, in all the samples the dominating compound was identified as unconverted TXB2 (see Table I).

Excretion of TXB2 Metabolites into Urine.

After intravenous injection of 3H-TXB2, 43% and 45% of the given amount of radioactivity (monkey A and B, respectively) was excreted into the urine in 48 hours. After 6 hours, both monkeys had excreted a total of 40% of the given dose, and an additional amount of about 3% and 1% were found in urine during the next 18 and 24 hour period, respectively.

The urinary portions were combined for each monkey and purified as described in the EXPERIMENTAL section. Fig. 2 shows a reversed phase partition chromatogram of the extract from the experiment with monkey A. About 90% of the sample radioactivity was eluted with the moving phase; 10% were found in the stationary phase as non-polar products. Four major peaks of radioactivity were found in the chromatogram: Peak A (eluted with 120-300 ml of effluent, 25% of the radioactivity applied to the column), peak B (370-470 ml, 32%), peak C (740-940 ml, 13%) and peak D (1050-1220 ml, 16%). Total radioactivity of minor peaks: 4%. A similar profile was seen from the experiment with monkey B. After this stage, corresponding peaks from both monkeys were pooled.

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E 0 ” lOOO-

I 1 ! I t 100 200 300 400 600 600 700 600 900 1000 1100 1200 1300

Elution volume (ml)

Fig. 2. Reversed phase partition chromatography of an extract from urine. Column size: 45 g. Solvent system: C-45.

Analysis of Material in Peak A.

The material in this peak was further purified using reversed phase partition chromatography, system D (moving phase, water:acetic acid, 300:2 (v/v); stationary phase, n-butanol, 100). Several incompletely separated peaks appeared. Due to the small amounts of tritium labeled material and large amounts of impurities in this sample, no further work was carried out with these metabolites.

Structure of Compound B, the Main Urinary Metabolite.

The material in peak B was rechromatographed in the same solvent system and esterified with diazomethane. The methyl ester of compound B was further purified on a TLC plate using 2% methanol in ether as the moving phase. One single radioactive compound was found, Rf = 0.40 (Rf of TXB2-Me: 0.48). The material in this zone was eluted with methanol and converted into the TMSi derivative. On GLC, the Me-TMSi derivative of the compound had a C-value of 22.8. This is 1.8 C shorter than that of TXB2-Me-TMSi, indicating that metabolite B might be a dinor derivative. The mass spectrum of metabolite B-Me-TMSi is shown in Fig. 3, together with that of TXB2-Me-TMSi. Several ions were found at the same m/e values in the two mass spectra, viz. 301, 225, and 217 (for the -

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interpretation of these ions, see Ref. 1). These ions are all interpreted to be formed by loss of the carboxyl side chain. Other ions, interpreted to have structures retaining the car- boxy1 end, were found at m/e values 28 units lower in the mass spectrum of the metabolite derivative compared to that of TXB2- Me-TMSI, e.g. 557(M-151, 482(M-901, 467(M-(90+15)), 411(M-(90+ 71)1, 366, 323, 295,+and 228, the base peak (BMSiO-CH=CH-CH2- CH=CH-(CH2) that metabo

jyCOOCH3] ) (cf. Ref. 1). This difference indicated ite B had been formed from TXB2 by degradation of

two carbon atoms from the carboxyl end.

Thus, the major urinary metabolite of TXB2 was dinor-TXB,.

Fig. 3. Mass spectra of the Me-TMSI derivatives of TXB2 (upper panel) and urinary metabolite B (lower panel).

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Identification of Compound C.

The material in peak C was rechromatographed in the same solvent system, subsequently esterified with diazomethane and subjected to TLC. Only one peak of radioactivity was seen (R zone waf, eluted

- 0.48; Rf of TXB2-Me = 0.48). The material in this converted into the trimethylsilyl ether

derivative and injected into the gas chromatograph. One radioactive compound was seen , with an equivalent chain length of 24.6 C (C-value of TXB2-Me-TMSi = 24.6). A mass spectrum was recorded from this peak and was found to be identical with that of TXB2-Me-TMSi (Fig. 3).

Thus, compound C was unconverted thromboxane B2, which represented 13% of the urinary radioactivity.

Analysis of Compounds in Peak D.

The material in peak D was esterified with diazomethane and subjected to thin layer chromatography. Three peaks of radioactivity appeared. The major peak remained on the line of application of the sample; the Rf values of the other two were 0.58 and 0.72, respectively. No further work was carried out with these metabolites, due to the small amount of material.

DISCUSSION

In certain biological systems, the prostaglandin compounds seem to be of minor interest as compared to the thromboxanes, and assay of thromboxanes might thus give a more reliable picture of physiological or pathological events, involving these tissues. It is well known that prostaglandins should not be assayed as such in the peripheral circulation, due to rapid metabolism and artifactual formation. It was thus of considerable interest to undertake a similar study on the metabolic fate of thromboxane B2, the comparatively stable degradation product of TXA2.

This study on the in vivo metabolism of TXB2 in the cynomolgus monkey demonstrates that the compound is eliminated with a very rapid initial half-life from the circulation, and after this initial phase the half-life was about 10 min. For at least 20-25 min after the injection, TXB2 was by far the dominating compound in the blood, and comparatively little degradation seemed to take place.

Had TXB2 been a substrate for the 15-hydroxy prostanoate dehydrogenase, a 12-keto metabolite would be formed, which could be expected to be eluted later than TXB2 in the reversed phase partition chromatography. Some non-polar material was seen in the blood samples, but comparatively little. Even

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about 20 min after the injection of the compound, only about 17% of the total sample radioactivity was found to be less polar than TXBZ itself. In the case o-f e.g. PGFzs, which is a substrate for the dehydrogenase, by far the ma3or part of the radioactivity is found as the 15-keto-13,14-dihydro compound only a few min after injection.

Somewhat larger amounts of more polar material also appeared with time in blood. Part of this material was eluted with a volume of effluent similar to that of dinor-TXB2 (Fig. 1). In analogy with the metabolism of certain prostaglandin analogues, where dehydrogenation at C-15 is prevented, it can be expected that 8-oxidation is a major pathway in the metabolism of TXBZ. The most polar compounds were not stud- ied, however, it is likely that tetranor compounds occur among these. 8-Oxidation is a slow process in comparison with the dehydrogenation of the secondary alcohol group in the side chain, and this is likely to be the explanation for the longer half-life of TXB2 in the circulation (cf. also Refs. 10 and 14).

About 45% of the given amount of tritium was found in the urine after 48 hours. The fecal content of tritium was not measured. Among the urinary products, unconverted TXB2 was found, and represented about 13% of the total urinary radioactivity; this is also in contrast to the prostaglandins (8). The major metabolite was identified as dinor-TXB3, formed by one step of B-oxidation. No attempts were made to establish the positions of the two double bonds; however, these are likely to remain in their original positions. Dinor-TXB has also been identified as the major urinary metabolit P of TXB2 in the guinea pig (15).

In conclusion, the metabolic fates of TXB2 seem to differ markedly from those of the prostaglandins. In quantitative studies of these compounds in the peripheral circulation, earlier assays were directed at the primary prostaglandins, and thus great problems were encountered, since large amounts of PGF3c and PGE2 are formed artifactually during the sample collection. Several sources of the prostaglandins have been demonstrated; probably the platelets are the most important of these. In the case of the prostaglandin assays, this problem has been entirely overcome by focussing on the 15-keto-13,14-dihydro metabolites, which are not formed artifactually during blood sampling and reliably reflect the endogenous levels.

In the case of assay of thromboxanes in vivo, the problem of artifactual formation of the compound is even greater. By far the major part of the platelet arachidonic acid is converted via the thromboxane pathway (4). It can be expected that "peripheral plasma levels" of TXB2 will be very high, and will certainly not reflect true endogenous circulating amounts of the compound. Unfortunately, TXB2 does not seem

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to be a substrate for the dehydrogenase, or is at least not significantly metabolized via this pathway. It is thus obvious that the endogenous production of this compound can not be estimated by monitoring a major metabolite in blood, as is the case with prostaglandins.

Recently, 7-[2,4-dihydroxy-6-(3-oxo-l-octanyl)-tetra- hydropyran-5-yl]-cis-5-heptenoic acid (12-keto-lO,ll-dihydro-TXB2) was identified as the major product from arachidonic acid after immunological challenge of the sensi- tized guinea pig lung (16). However, it was not established that TXB2 was the immediate precursor.

Further studies on the metabolism of TXB2 in other species, including the human, are in progress in our labora- tory.

REFERENCES

1. Hamberg, M. and B. Samuelsson. Proc. Nat, Acad. Sci. USA, 71: 3400 (1974). -

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Hamberg, M. and B. Samuelsson. Biochem. Biophys. Res. Conunun. 61: 942 (1974). -

Hamberg, M., J. Svensson and B. Samuelsson. Proc. Nat. Acad. Sci. USA, 2: 2994 (1975).

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Hamberg, M., J. Svensson and B. Samuelsson. Proc. Nat. Acad. Sci. USA, 71: 3824 (1974). -

GranstrBm, E., H. Xindahl and B. Samuelsson. Anal. Lett. 2: 611 (1976).

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Malmsten, C., E. Granstrijm and B. Samuelsson. Biochem. Biophys. Res. Commun. 68: 569 (1976). -

Malmsten, C., H. Kindahl, B. Samuelsson, S. Levy-Tole- dano, G. Tobelem and J.P. Caen. Br. J. Haematol., in press.

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Samuelsson, B., E. Granstrijm, K. GrBen, M. Hamberg and S. Hammarstrom. Ann. Rev. Biochem. 44: 669 (1975). -

Granstrom, E. Prostaglandins 2: 19 (1975).

Hansson, G. and E. Granstrdm. Biochem. Med. 15: 95 (1976). -

Green, K. Biochim. Biophys. Acta 231: 419 (1971). -

Hamberg, M. Eur. J. Biochem. 6: 135 (1968).

Granstrom, E. and B. Samuelsson, J. Biol. Chem. 246: 5254 (1971).

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15.

Granstrom, E. and G. Hansson. In: Adv. in Prostaglandin and Thromboxane Research, Vo1.i (Eds. B. Samuelsson and R. Paoletti) Raven Press, New York, p. 215, 1976.

Svensson, J. and M. Hamberg. Submitted for publication.

16. Dawson, W., J. R. Boot, A. F. Cockerill, D. N. B. Mallen and D. J. Osborne. Nature 262: 699 (1976).

Received 12/l/76 - Approved z/14/77

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