Metabolism of the steroid anaesthetic alphaxalone by the isolated perfused rat lung

Journal ~r/Swro~d Bh hrmlarv Vol. 14. pp. 45 to 51 Pcrpmon Press Ltd 1981. Prtnlcd in Great Britain

METABOLISM OF THE STEROID ANAESTHETI~ ALPHAXALONE BY THE ISOLATED PERFUSED RAT LUNG TERMCE E. NICHOLAS,MKHML

E. JONES+,DAVID

W. JOHNSON

and GEORGEPHILLIPOU~ * Department of Human Physiology and Human Morphology, The Fhnders University

of

South Australia and t Department of Obstetrics and Cynaecology, The Queen Elizabeth Hospital, South Australia (Received 31 March 1980) SUMMARY

We have investigated the metabolism of the steroid anaesthetic, alphaxalone (3a-hydroxy-k-pregnane11,20-dione), in the isolated rat lung perfused at 10ml mitt-’ with the plasma substitute, Haemaccel. When [‘4C]-alphaxalone was introduced into the perfusate during a 90 min recirculating perfusion, four metaboiites appeared in the reservoir. We have identified two of these by gas chromatographic-mass spectrometric (GC-MS) methods as 3~1 I-dihydroxy-5x,-pregnane-20-one (metabolite 2). the major metabolite and 5x-pregnane-3x.l1,20_triol (metabolite 3); precise assignment of the stereochemistry at Cl 1 and C20 was not possible. Following perfusion of up to 30 min with ~n~ntrations of alphaxalone ranging from 0.08 to 8.9 pM, we have measured metabolite 2 in both the lung tissue and in the reservoir. From this data we have obtained a KM of 0.25 &i and a V_ of 7.8 nmol mitt- t g dry lung- ‘. Extrapolating from reported plasma concentrations in humans, it would seem that this ability of the lung to metabolize alphaxalone would have little influence during induction of anaesthesia. However, it could significantly influence the concentration of alphaxalone in the cerebral circulation during administration of the considerably lower doses of alphaxalone used for sedation during regional anaesthesia.

Recently [Sj it was reported that this preparation tensively metabolized progesterone.

Although the lung is the first tissue barrier to confront a drug administered intravenously, the possibility that passage through the pulmonary vascular bed may influence the arm-brain circulation time and the metabolism of intravenous anaesthetics appears to have been ignored. This is particularly surprising as the importance of the lung in the bio-transformation of both endogenous and exogenous compounds is now well recognized [I]. in light of the large number of steroid metabolizing enzymes that have been identified in lung homogenates [2], we have investigated whether the lung metabolizes alphaxalone (‘kx-hydroxySa-pregnane-11,2Odione), the major component of Alfathesina, a steroid anaesthetic. Although it is reported that the short duration of action of this anaesthetic is due to rapid hepatic metabolism [3], little information is available regarding either the rate of meta~lism or the identity of the metabolites. An early report from Child et a[.[33 tentatively identified the glucuronide of Za-hydroxy-alphaxalone as the major biliary metabolite of alphaxalone in the rat. We have used the isolated perfused rat lung to assess the importance of metabolism by the lung Previously we have shown that this preparation rapidly metabolized cortisone included in the perfusate [4].

ex-

MATERIALSAND METHODS Perfusion methods were similar to those previously described [4]. Briefly, male Porton rats (180-25Og) were anaesthetized with intraperitoneal sodium methohexitone [Eli Lilly (Australia) and Co.] 5Omg/kg, tracheostomized and artificially ventilated with 5% CO1 in oxygen (tidal volume 2.5 ml, 60 breaths/min, end expired pressure 2 cm HZO). The thorax was then opened and cannulae were placed in the puImonary artery via the right ventricle and in the left atrium. Without interrupting the circulation, the lungs were perfused at ~Oml/min with Haemaccel viu a Holter pump(mode1 RL175-110, Extracorporeal Medical Specialities, Inc. PA). Finally, the lungs were removed from the thorax and placed in a closed chamber saturated with water vapour at 37°C. Perfusion and ventilation pressures were continuously monitored with two Statham transducers (model P23Dc) connected to a Grass Model 7C Polygraph. Following the perfusion (3-30min).

the lungs were

rapidly

dissected

free, weighed and placed in liquid nitrogen. Lungs with any sign of oedema were discarded. The lungs were then freeze-dried, weighed and a known portion extracted for steroid analysis. In all cases the lungs were perfused initially for

Atfathesine (alphaxalone + afphadalone acetate) Glaxo Australia Pty. Ltd. (Victoria) 45

TERENCEE. NICHOLASet al.

46

IOmin with a medium containing no steroid; the medium containing steroid was then introduced from a separate reservoir without interrupting perfusion, and the perfusion was continued for the predetermined period. In the nonrecirculating perfusions (3 and 5 min duration), the total effluent was collected. In the recirculating perfusion (10 to 30min). the medium was returned to the reservoir which was stirred vigorously during the course of the experiment. S&ions

and chemicals

[“Cl-Alphaxalone (SA 6.OCi/mol) labeled in the 21 position was a gift from Glaxo Research Ltd. (U.K.) and was found to be 98.5% pure. It was stored as a 0.1 M solution in ethanol at -20°C and was protected from light .at ailtimes. Volumes of this solution were added to an Erhknmeyer flask and evag orated to dryness under nitrogen before addition of 50 ml of Haemaccel. Unlabeled alphaxalone was a gift from Glaxo Australia. Haemaccel (Behring Institute, Marburg, Germany) is a 3.5% colloidal plasma substitute to which we added 5 mM glucose and, after equilibration with 5% CO1 in oxygen, adjusted the pH to 7.4 at 37°C. Analytical procedures

Triplicate lOO/.d aliquots of the reservoir were taken before and after perfusion and were counted in 5 ml of PCS me Radiochemical Centre, Amersham) with a Searle Mark III Beta Scintillation Counter (Model 6880). Fifteen ml of the final reservoir was extracted three times with 25ml of ether at 2°C. the ether was then evaporated to dryness under nitrogen and reconstituted in 0.5 ml of water at 40°C. This in turn was extracted three times with 5ml of ether at 2°C. Finally, the ether was evaporated to dryness and reconstituted in 1oOjd of chloroform at 2°C. Duplicate 20 ~1 aliquots of this were spotted on a thin layer chromatography plate (Silica gel 60, 0.25 mm thickness, Merck, Darmstadt) which was then developed with chloroform-methanol (46:4, v/v). Areas of radioacti’vity were located by scanning the plate on a Berthold Dunnschidt-Scanner II (model LB2723) and the spots were scraped into vials together with 5 ml of a scintillation cocktail (7 g of 2,Sdiphenyloxazole and 0.1 g of pbis-[~methylstryryil-ale per litre of toluene) for counting. The results were confirmed using two other solvents systems to develop the plate: ethyl acetate-N-hexane-glacial acetic acid (75:20:5. v/v) and chloroform-acetone (61:9, v/v). More than 95% of the alphaxalone was recovered using this extraction method. Following freeze-drying, a portion of lung tissue was weighed, minced with a scalpel and then homogenized in 8 ml of water with an Ultra Turrax TP 18-10. This homogenate was extracted three times with 25 ml of ether at 2°C and centrifuged (2OOg for 10 min). The supernatant was removed, evaporated to dryness under nitrogen, reconstituted in 0.5 ml water

at 40°C and extracted three. times with 5 ml of ether at 2°C. The extract was again evaporated to dryness and reconstituted in 200 fi of chloroform at 2°C. Chromatography was performed as described above. Glucuronide determination

After extracting the medium and lung tissue, we freezedried the remaining aqueous fraction and extracted it with 40 ml of methanol. Following 15 min centrifugation at 2ODOg.the supematant was evaporated to dryness at 40°C in a Buchi Rotovapor-R. Ten ml of acetate buffer at pH 5.0 was added together with 50,000 Fishman Units of /I-glucuronidase (Sigma, St. Louis, MI) and the mixture incubated for IO hr at 37°C. Three 100~1 aliquots were then taken for counting and the mixture was extracted three times with 15 ml of ether at 2°C. The ether was then evaporated to dryness and the radioactivity of the total residue was determined. Gas chromatography-mass spectrometric methods

Samples of the dried ether extracts obtained as described above were derivatized as the methoxime and n-propyloxime trimethylsilicate compounds [6]. Gas chromatography was performed on a Pye 104A instrument fitted with a splitless injector (SGE, Melbourne, Australia), and a 20 x 0.5 mm, SE-30 glass SCOT column (SGE, Melbourne) of 14,000-15,ooO effective plates, injector temperature: 250°C. column temperature: 210-265°C programmed at l”C.min- ’ ; overall linear gas flow (nitrogen) 16-20cm.second-‘. Mass spectrometry was performed on a Hewlett Packard 5992B instrument fitted with a 3 x 2.Omm. 2% OVlOl on 120/140 mesh Gaschrom Q, glass packed column and a membrane separator. RESULTS

There was a rapid accumulation of ‘*C in lung tissue during the perfusion of the isolated lung with [‘*CJ-alphaxalone. As an index of accumulation, we have determined the ratio of d.p.m. per gram of wet tissue to the dp.m. in 1 ml of medium in the reservoir at the end of the perfusion. In the case of the nonrecirculating perfusions we have taken the activity of the pre-perfusion reservoir. When expressed in this manner, the tissue:medium ratio was 13.5 f 0.49 (mean f SEM of 14 perfusions) and did not vary with either the duration of perfusion, or the concentration of alphaxalone. During perfusions of the isolated lung with 4pM [‘*CJalphaxalone for periods of up to 9Omin, at least four metabolites appeared in the reservoir (Fig. 1). We have designated these metabolites 2-5. As can be seen from this figure, there is initially an increase in the amount of metabolite 2, followed by an increase in metabolite 3 and finally an-increase in metabolite 4. In addition, over a 90min perfusion there appeared a small percentage of radioactivity as metabolite 5 at the origin; as yet we have made no

47

Alphaxalone metabolism in lung

Fig. 3. A Hofstee plot constructed from the data in Fig. 2 relating to the overall production of metabolite No. 2. Other details as for Fig. 2.

Fig. 1. The radiochromoscan trace of a thin layer chromatogram of the ether extract of the perfusate following recirculating perfusion of isolated rat lungs for 30mins (top) and 90min (bottom) at 10ml min-‘. The perfusate reservoir initially contained 4 pM [‘*C]-alphaxalone. AX: alphaxalone, No. 2: 3a-ll-dihydroxy-5a-pregnane-20-one, No. 3: 5a-pregnane-3a,11,20_triol. Peaks No. 4 and 5 have not been identified.

attempt to characterize this polar compound. We have characterized the ability of the lung to form metabolite 2 by perfusing the lung with concentrations of [*4C]-alphaxalone ranging from 0.08 to 8.9pM. The duration of perfusions was such, that

0

I

2 ALPHIXAWNE

3

negligible amounts of other metabolites formed, and that the amount of metabolite 2 remained small compared with the amount of alphaxalone in the medium during the perfusion. Figure 2 shows the rate of appearance of metabolite 2 in the perfusate and also the total amount of metabolite in pet&ate plus lung tissue. Figure 3 is a Hofstee plot of the data from the latter curve. The radioactivity that could be attributed to glucuronides in both the perfusate and the lung tissue together varied from 0.2% of the total radioactivity in the initial reservoir following a perfusion with 0.5 PM [14CJ-alphaxalone for 5 min, to 0.47% after 30 min perfusion with 8 PM alphaxalone.

4 COMCENlllAllON

5

5

“9

~111

Fig. 2. The rate of production of metaholite No. 2 (M) and the rate of its appearance in the perfusate (V) following perfusions of duration 3 to 30 min with a medium containing between 0.08 and 8.9 PM [“CJ-alphaxalone. Each point represents the mean of two experiments.

48

TERENCEE. NICHOLASet al.

Fig. 4. Gas chromatograph traces of the methyloxime trimethylsilyl (top) and n-propyloxime trimethylsilyl (bottom) derivatives of the evaporated ether extracts of the perfusion medium after 90min. GC retention indices: (top) I = 2827; 2 = 2870; 3 = 2913 and (bottom) 1 = 2980; 2 = 3012; 3 = 2912. Identificationof metabolites

aration

The gas chromatograph trace of the methyloxime trimethylsilyl (MO-TMS) derivatives of the extract after !JOmin perfusion (Fig. 4) confirmed the presence of two major metaboiites (labeled 2 and 3). Prep-

(PO-TMS) derivatives caused a shift in the retention time of (2) but not (3) indicating the presence of an unhindered carbonyl group in the former. GC-MS of the MO-TMS (Fig. 5) and the PO-TMS (Fig. 6) of metabolite 2 afforded spectra

of

the

n-propyloxime

trimethylsilyl

Fig. 5. H-MS of the methyloxime trimethylsilyl derivative of metabolite 2 scanned over two separate mass ranges. Note: The masses of important ions are marked since the scaling between each ten mass units is not linear.

49

Alphaxaloae metabolism in lung 128

!,,

-TMSOH

I 250

360

1 1:_I, 340

i”

k , rdo

-1MSOH

jv i,

450

i-2

1 ,““I”‘,1 500

53sfM+‘) L 550

600

m/7.

Fig. 6. E&MS of the n-propyloxime trimethylsilyl derivatives of metabolite 2 scanned over two separate mass ranges. See note for Fig. 5.

with ions at m/z 87,100 (MO-TMS) and m/z 115,128 (PO-TMS) which are characteristic of oximes of ZO-oxosteroids [7] (see Scheme 1). They both afforded molecular ions for a mono-oxime, di(trimethylsilyloxy) pregnane and dominant ions which could be interpreted as the consecutive loss of an alkoxy group and trime~ylsil~ol (twice). Coupled with the knowledge that C-l 1 is unreactive to oxime formation [14], we assigned metabolite 2 as 3u,l l-dihydroxy-Sa-preg nan-20-one. The GC-MS of the t~me~ylsilylat~ derivative of metabolite 3 (Fig. 7) exhibited an ion at m/z 552 which is the mass of the molecular ion of a tri(trimethylsilyloxy)pregnane. Its spectrum could be interpreted as the consecutive loss of three molecules of

-

\ =LNOR j;

lb

Scheme 1. Mass spectral fragmentations common to oximes of 20-oxosteroids (upper and oentral sections) and to 20-silyloxy steroids (bottom section).

trimethylsilanol from the molecular ion. In addition, it contained an ion of m/z 117 which is common to the mass spectra of 2~t~methylsilyloxy pregnane compounds (see Scheme 1, bottom section). Accordingly we assigned the structure of metabolite 3 as 5a-pregnane-3a,l1,20-triol, arising from the reduction of alphaxalone at C-11 and C-20. DISCUSSION

Our results clearly show that the rat lung can metabolize alphaxalone and that the metabolites rapidly appear in the vascular compartment. Moreover, the tissue:medium ratio of ‘*C indicates that the steroid accumulates in the lung to levels well in excess of that in the pet&sate. At the highest concentration of alphaxalone used (8.9 PM), 21% of the 14C label remaining in the tissue was as metabolite 2. At this stage we do not know the site of a~umulation or the Iocation of the metabolizing enzymes. Nevertheless, the fact that the tissue:medium ratio was constant over a range of concentrations suggests that specific binding sites are not involved. This conclusion would be consistent with the stochastic model we have developed to explain the handling of alphaxalone as it passes through the pulmonary vascular bed [S]. Briefly, this model predicts that the time an alphaxalone molecule spends in the extravascular compartment during one transit of the pulmonary bed, will be a random variable which follows a compound Poisson distribution. Reduction at C-11 by 1 lo-hydroxyst~oid dehydrogenase, the enzyme which also reduces cortisone to cortisol[4], forms the major metabolite of alphaxalone (metabolite 2); this we have identified as 3cql I-dihydroxy-5a-pregnane-20-one. We have. identified the second metabolite formed (metabolite 3) as Sa-pregnane-3cql1,20_triol. Precise assignment of the stereochemistry at Cl 1 and C20 was not possible as we did not have the relevant reference standards, however, it is highly likely that the

50

TERENCE E. NICHOLAS et al.

I

0

50

i

I

I I

0

282<

Jdr l1lgI, 200

150

-TMSOH

372 .

,I

y

250

,, 300

,

340

'400 m,r

-1MSOH

4:

I"'.,"' 200 250

/" '300

,,Ih. 350

400

/I,t/l

552lM+'l

I! ,,,,,

,,)

450

506

550

600 m/7.

Fig. 7. ELMS of the trimethylsilyl derivative of metabolite 3 scanned over two separate mass ranges. See note for Fig. 5.

11/?-alcohol is formed since 11/&hydroxysteroid dehydrogenase is present in the lung[4]. The probable configuration at C-20 is 2Ckxsince 208 stereochemistry is less common in biological systems. Since ll/?-hydroxysteroid dehydrogenase is present in the microsomal fraction of the rat lung (unpublished results), it would seem that alphaxalone is reduced to metabolite 2 within the cell. The findings of this study do not support those of Pastorino[9] who reported no trace of alphaxalone in lung tissue following infusion of the rabbit with the steroid He ascribes this to a high elimination rate from the lung via excretion and metabolic transformation, but gives no evidence to substantiate this statement_ We can give no explanation for this difference in tindings apart from citing the species differences and cautioning that we perfused the lungs with Haemaccel, which is devoid of plasma proteins. Little information is available regarding the plasma protein binding of alphaxalone. Child et a/.[ 10-j have reported that 35.5% of alphaxalone is bound to protein in rat plasma, however, without knowing the affinity of binding it is difficult to determine whether this binding would greatly hinder access of the steroid to the metabolizing enzyme. We chose to perfuse the isolated lungs with Haemaccel rather than Krebs bicarbonate solution containing albumin, as is more commonly used, because Haemaccel binds alphaxalone to only a very small degree. Equilibrium dialysis at 37°C with 2 a [ ‘*CJ-alphaxalone showed a maximum binding of only about 5% in 24 h. Hence by using Haemaccel we could eliminate plasma binding as a variable, so that the total concentration of alphaxalone in the perfusate was essentially equal to that of unbound steroid We could see no advantage in using albumin or diluted plasma in the perfusate, for to gain a precise index of the role of the pulmonary circulation in metabolism of alphaxalone, one must use undiluted rat blood perfused through the pulmonary bed at the normal cardiac output of the rat. In addition, we were primarily interested in the ability of

the lung to metabolize alphaxalone during the induction of anaesthesia when, in humans, about 25 mg of steroid are injected over a period of seconds. Hence it is possible that equilibrium binding is not attained during passage from the site of injection to the pulmonary vascular bed and we have no indication of the bound : unbound ratio upon arrival in the lung. In essence, therefore, we have attempted to characterize the ability of the lung to metabolize unbound alphaxalone. Our results also diRer from those of Card et a/.[1 l] who concluded from their whole-body autoradiographic study that alphaxalone was not conantrated in rat lung However, it is likely that their methods were not sensitive enough to detect accumulation of radiolabel in such a diffuse tissue as lung. Moreover, as there is no specific binding in lung, the amount of alphaxalone present would fall rapidly as the plasma concentration decreased. We have identified two metabolites of alphaxalone. The first, 3x,1 l-dihydroxy-5a-pregnane-2@one has an anaesthetic activity that is about 25% of that of the parent molecule [12]. Reduction of the Z@carbonyl results in a compound with little anaesthetic activity. The very small amount of radioactivity that could be attributed to ghrcuronides confirms our impression that the lung has a very small role in glucuronation. We previously reported that there was little glucuronide formation following perfusion of the isolated rat lung with cortisone [4]. Similarly, Hartiala et a/.[51 reported that there was no glucuronide or sulphate conjugation following perfusion of the isolated lung with progesterone. In the event that the human lung possessed a similar ability to metabolize alphaxalone and contained a dehydrogenase enzyme with similar kinetic constants to that which we have characterized in the rat lung, the possibility exists that pulmonary metabolism may affect induction of anaesthesia. No information is available regarding the concentration of alphaxalone in the pulmonary artery during induction of anaesthesia in the human. However, if 2.5 ml of alpha&sin

51

Alphaxalonc metabolism in lung

(9 mg/ml alphaxalone) is injected over a 10 s period, and if we assume a cardiac output of 5 I/min and complete mixing in the heart, then the concentration in the pulmonary artery would be of the order of 27 &ml (81 PM). Hence it is very unlikely that metabolic conversion during passage though the pulmonary circulation would affect rate of initial induction of anaesthesia. Whereas the main site of metabolism of alphaxalone is the liver 1131, depending upon the K, of the principal enzyme involved in the liver, the lung enzyme may play a major role in metabolizing the steroid during the recovery phase when the plasma concentration has fallen. The ability of the lung to metabolize alphaxalone may assume great significance when the drug is being used to sedate. We have found that an infusion of alphathesin of 0.05 to 0.1 ml/min is sufficient when the drug is being used to sedate in association with regional anaesthesia. Preliminary results in one patient show that after two hours of infusion the plasma concentration was O.O85~g/ml; this is very close to the K, value in the isolated rat lung. If the V,, value is taken to reflect maximum clearance, then it appears that the isolated lung metabolizes 0.052 pg of alphaxalone per millilitre of perfusate. This figure is of the same order of magnitude as the plasma level that we have found in humans. In conclusion, we have shown that the rat lung metabolizes alphaxalone and have identified the two major metabolites formed. We have postulated that if the human lung possesses a comparable enzyme, then transformation in the pulmonary circulation may be an important determinant of the plasma concentration during sedation with the anaesthetic. We hasten to add that any extrapolation of our results to the human situation remaihs highly speculative at this stage.

Acknowledgements-We are grateful for the technical assistance of Felicia Bolt0 for whom support was provided by Allen & Hanburys (Australia).

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