Polydeuterated compounds in metabolic studies

Analyrrca Chmuca Acra, 247 (1991) 277-281 Elsevter Sctence Pubhshers B V., Amsterdam

Polydeuterated

277

compounds in metabolic studies Tomas Cronholm

Department

of Physrologrcal Chemistry, Karolrnska Instltutet,

Box 60400, S-104 01 Stockholm (Sweden)

(Recetved 21st August 1990)

Abstract

Polydeuterated compounds are valuable as metabohc tracers when radtoacttvtty must be avotded. The tsotoptc composttton, determmed by mass spectrometry, may contam much useful informatron Tn- and tetra-deuterated pregnane denvattves have been used to study progesterone metabohsm m pregnant women. The high sensttivtty obtained wtth polydeuterated sterotds was utthzed m a study of estradtol metabolism m rat uterus Deutenum labelhng at oxrdtzable hydroxyls makes tt possible to study oxrdatton-reductton reactions, e.g., in studies on sulphated progesterone metabohtes m man and on C,, steroids m rats. Rapid ethanol-acetaldehyde exchange wtth mtermolecular hydrogen transfer has been demonstrated m expenments wtth mrxtures of [l,l-*HZ]and [2,2,2-2H3]-ethano1. Polydeuterated ethanol and glycerol have been used to study compartmentalization of precursors in the synthesis of ketone bodtes and glycerohpids. Chnal [1-*HIethanols have been used to study stereoselectivity m the oxldatron of ethanol depending on cytochrome P-450 as well as tsotope effects and coenzyme compartmentahzatron. Keywords

Mass spectrometry,

Deutenum

labelhng;

Metabohc

A major advantage of using stable isotopes with gas chromatography-mass spectrometry (GC-MS) rather than radioactive isotopes with radioactivity measurements is that the amounts of different isotopic species are determined separately. This allows several differently-labelled precursors to be used in one experiment, and it also makes it possible to study loss of hydrogen by oxidation-reduction reactions or by exchange with water, as well as the extent of intermolecular hydrogen transfer. Other advantages are the lower toxicity, which makes it possible to conduct studies on pregnant women, and the high sensitivity when the total amounts are low. The present paper gives different examples of applications based on these advantages.

EXPERIMENTAL

Deuterated ethanols were obtained from A. Hempel (Dusseldorf, Germany) and Merck and

studies

[l-‘3C]ethanol was from Merck Sharp and Dohme (Montreal, Canada). Chiral [l- * HIethanols [ 11, *H-glycerols [2], [ *H,]estradiol [3], C,,-steroid sulphates [4-61 and C,,-steroid sulphates [7] were prepared in the laboratory. In vivo studies were conducted on women in weeks 30-39 of pregnancy, on male volunteers, on female Sprague-Dawley rats or on deer mice (Peromyscw maniculatur) of strains reported to have (ADH+) or lack (ADH-) alcohol dehydrogenase [8]. The deer mice were kindly supplied by Dr M.R. Felder, University of South Carolina. Hepatocytes were isolated from the rats by using a modification [2] of the procedure described by Berry and Friend [9]. Ethanol was determined as the 3,5-dmitrobenzoate [lo], acetaldehyde as the 2,4-dinitrophenylhydrazone [ll], organic acids as their methyl esters [12] or more recently as the tert. -butyldimethylsilyl derivatives of the O-methyl oximes [13], and steroids (released from sulphates by solvolysis), sn-glycerol-3-phosphate, and diacylglycerol (obtained by phospholipase C-cata-

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lysed hydrolysis of phospatidylcholine) as their trimethylsilyl ethers [2-71. The isotopic compositions were determined by GC-MS with a LKB 9000 or Finmgan 4000 instrument (electron energy 22.5 or 70 eV, respectively).

RESULTS

AND DISCUSSION

Studres with polydeuterated steroids In studies on pregnant women, the use of radioactive isotopes is ruled out for ethical reasons. The production rates of major progesterone metabolites in these SubJects were therefore studied with polydeuterated pregnane derivatives [7]. The SubJects were given rapid intravenous infusions of the 3-sulphates of 3cY-hydroxy-Sa-[3P,ll,ll-*H,]pregnan-20-one and 5cY-[3~,11,11,20~-*H,]pregnane-3a,20a-diol and of the disulphate of the latter, and the isotopic composition of steroid sulphates was determined in consecutive plasma samples collected for about 15 h. A disadvantage of using stable isotopes is that the amounts administered may be large enough to disturb the steady state. However, this can be compensated for if it is assumed that the rates of outgoing flows are proportional to pool sizes. This is done by modifying the percentages of labelled molecules by the factor Z/( Z - K ), where Z and K are the fractions of the injected and isolated steroid, respectively, which can give rise to the specifically deuterated steroid determined [14]. The modified percentages were used to get pool-kinetic parameters by iterative testing in equations considered to give the labelled fractions at different times. The equations were obtained by transformations of published equations [15,16], and the parameters were obtained in the equation giving the least sum of squared deviations from the modified observed values. The injected steroid sulphates underwent oxidation-reduction at C-20 and 16a-hydroxylation, and the diol became hydroxylated at C-21. The production rate of the pregnanolone/ pregnanediol couple was 0.08-0.5 mmo1/24 h, as determined from the slow component in a two-pool model. The rapid component and the decrease in

tetradeuterated pregnanediol molecules both gave the half-life in the oxidation-reduction at C-20 as 0.1-0.4 h, with reduction being fastest. The disulphate of 5cu-pregnane-3a,20cr-diol was a metabolic end-product and accounted for a major part of the elimination of the injected steroids. Taken together with previous studies, these results indicate that the formation of sulphated steroids with a 3a-hydroxy-5a configuration may account for 50% of the metabolism of progesterone in late pregnancy. Stable isotopes may also be superior to radioactive ones in pool-kinetic studies where the amounts are very small. This was demonstrated in a study on the turnover of estradiol in rat uterus in vivo, which was done by intravenous infusions of [11,12,13-2H,]estradiol followed by unlabelled estradiol or vice versa and GC-MS of the estradiol isolated from cytosolic and nuclear fractions of the uterus [3]. With the use of a one-pool model, it was found that the uterine uptake of estradiol was about 0.7 fmol h-’ mgg’, and the half-lives of the estradiol in the nuclear and cytosolic fractions were 4 h and 3 h, respectively. The results indicate an uptake of 40-90% of all estradiol passing through the uterus in proestrus (period in the hormonal cycle of female mammals immediately preceding estrus). In the experiments on pregnant women, no monodeuterated steroids could be detected in plasma. This indicates that the *H removed from C-20 during oxidation of the 20a-hydroxyl group was not utilised in reductions of ketosteroids. In contrast, an extensive intermolecular transfer of hydrogen was observed to accompany oxidationreductions at C-17 in rats. Thus, intravenous infusion of a mixture of the 3-sulphates of 5a-[2,2,4,4*H,l- and 5cy-[17c+*H]androstane-3P,17/3-diol resulted in excretion in bile of pentadeuterated 5crandrostane-3/3,17P-diol disulphate [4]. This indicates that the dissociation of reduced coenzyme from the oxidoreductase was slow, which in turn provides a requisite for redox interactions between the metabohsm of different steroids [17], and also has to be taken into account when rates of oxidation-reduction are calculated [4]. Similar experiments with 17-sulphates of 17/3-hydroxy-Sty[16,16,17a-*H,]androstane-3-one and Scu-[3/3(or

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3a)-‘Hlandrostane-3a(or 3/?),17/3-diolshowed that oxidation-reduction at C-3 was not accompanied by intermolecular hydrogen exchange, indicating rapid dissociation of the coenzyme [6]. Epimerization of the 3&hydroxy- to the corresponding 3ahydroxy-steroid occurred with retention of 5040% of the ‘H at C-3, indicating that it was not caused by two separate oxidoreductases. Omdation-reduction of ethanol Mixtures of [l,l-‘H,]and [2,2,2-*H,]-ethanol (1 : 1) have been given to rats [lo], humans [18] and deer mice [19], and they have been used with isolated hepatocytes [20,21]. In these studies, [*H,]- and [‘H,]-ethanol were formed, which shows that extensive ethanol-acetaldehyde exchange had occurred and that the NADH was firmly bound to alcohol dehydrogenase. In order to estimate the extents of oxidation-reduction and intermolecular hydrogen transfer, a mathematical model was set up, which described the changes in the concentrations of the four labelled species of ethanol by differential equations [lo]. The concentrations at different times were obtained by stepwise calculations from the coupled differential equations with assumed values for the extent of oxidation-reduction, intermolecular hydrogen transfer and isotope effect on ethanol oxidation. These parameters were then obtained from the equation that gave the least sum of squared deviations from the observed concentrations, obtained by GC-MS. The results from the experiments with rats indicated that both the rate of reduction of acetaldehyde during oxidation-reduction and the rate of association of reduced nicotinamide adenine dinucleotide (NADH) with alcohol dehydrogenase were nearly as high as, or higher than, the net rate of ethanol elimination [10,20], which suggests that ethanol elimination in the rat is limited by the rates of acetaldehyde oxidation, dissociation of NADH from alcohol dehydrogenase and re-oxidation of cytosolic NADH. Attempt to quantify the relative importance of these rate-limiting factors were done by additions of drugs both in vivo and in incubations with isolated hepatocytes [21]. Penicillamine added to isolated hepatocytes decreased the rate of reduction of acetaldehyde, as

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expected from its binding to penicillamine [22]. Because the rate of ethanol elimination is not increased by penicillamine [22], it can be concluded that oxidation of acetaldehyde is not normally a major rate-limiting factor. The mixture of labelled ethanols was also administered to deer mice, which are considered to lack alcohol dehydrogenase (ADH). Similar and high rates of oxidation-reduction were observed in these animals and the corresponding animals which do have ADH [19]. The extent of intermolecular hydrogen transfer was about 70% which shows that oxidation and reduction were catalysed by a common dehydrogenase. It could be calculated that at least 50% of the ethanol elimination in the animals lacking ADH was due to this dehydrogenase. Thus elimination of ethanol in the deer mice cannot be taken as evidence for an in vivo role of non-ADH pathways in ethanol metabolism. In similar experiments on humans, the dose of ethanol (2 mmol kg-‘) was too small to result in oxidation-reduction for more than the initial 30 min [18]. The apparent isotope effect was also very small. These results indicate that at low concentrations (below 1 mmol l-i), ethanol is completely eliminated from the blood passing through the liver. About half of the ethanol in blood had been formed from acetaldehyde during the initial oxidation-reduction, and the hydrogen incorporated was mainly derived from ethanol. The results indicate that, at higher concentrations, the rate of ethanol elimination is limited by acetaldehyde oxidation and by dissociation of NADH from alcohol dehydrogenase. Isotope effects and stereoselectrvtty In ethanol oxidation The isotope effect on elimination of [l,l‘H,]ethanol could be determined in vivo in the experiments described above [lo], and the value (about 3) is in good agreement with that reported for the isolated alcohol dehydrogenase [23]. The isotope effect on oxidation in isolated systems is preferably evaluated by determining the primary product, in this case acetaldehyde. A method of quantifying acetaldehyde by GC-MS was developed; in this method, the exchange of hydrogens

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at C-2 was eliminated as much as possible by preparing the derivative, the 2,4-dinitrophenylhydrazone, under neutral conditions in dimethylformamide [ll]: The residual exchange could be corrected for, because it produced dideuterated acetaldehyde molecules when [2,2,2-‘H,]ethanol was incubated. This correction was made essentially as described previously [24]. This made it possible to use 1 : 1 mixtures of [l,l-2H2]and [2,2,2-2H,]-ethanol or [‘Ha]- and [l-‘3C]-ethanol to measure the isotope effect by quantifying the acetaldehyde formed in different systems. The results were about the same with the two sets of ethanols and were in good agreement with results obtamed from non-competitive experiments where the rates of oxidation of unlabelled and [l,l2H2]ethanol were compared. The primary isotope effect on ethanol oxidation which depends on cytochrome P-450, was about 4 with liver microsomes or reconstituted systems enriched in ethanol-inducible forms of cytochrome P-450. Lower isotope effects were observed with other forms of cytochrome P-450, approaching the value 1.4 that was observed when the oxidation was due to free hydroxyl radicals formed in the xanthinexanthine oxidase system. Thus oxidation catalysed by the ethanol-inducible forms does not appear to be mediated by free hydroxyl radicals. The stereoselectivity in the oxidation of ethanol was determined by GC-MS of the acetaldehyde formed from (lR)- and (1S)-[l-2H]ethanol [ll]. No significant stereoselectivity was observed in the different cytochrome P-450 systems or the xanthine-xanthine oxidase system. The well-established stereoselectivity in the alcohol dehydrogenase-dependent oxidation of ethanol has been used to label separately the NADH pools at the alcohol and aldehyde dehydrogenases by administration of the chiral [l‘HIethanols to rats in vivo or to isolated hepatocytes, followed by analysis of the Krebs cycle and related acids [12,25]. In addition, [l,l-*H,]ethanol was used for comparison and to increase the 2H content in studies on pools. Experiments with aldehyde dehydrogenase inhibitors indicated that acetaldehyde was only oxidized in the mitochondrial compartment, because the inhibitors did not increase the relative transfer of ‘H from the l-pro-

T CRONHOLM

S position to acids formed by reduction in the cytosol. The ‘H could spread from the initial cytosolic compartment with alcohol dehydrogenase to the mitochondrial compartment and vice versa, apparently through malate. This exchange via shuttle systems appeared to be less efficient in the isolated hepatocytes than in vivo. Studies wrth polydeuterated precursors During continuous administration of [2,2,22H 31 ethanol to rats, about 50% of the acetyl-CoA used for cholesterol biosynthesis is formed from ethanol [24]. This was utilized to demonstrate that different pools of cholesterol were used for excretion in bile and for bile acid formation, and it was also possible to study the extent of exchange of the ‘H in the acetyl groups during the biosynthesis of cholesterol [26]. This exchange was 20-30% as calculated from the isotopic composition of a fragment ion obtained in the GC-MS of the derivative of the bile acid /?-murichohc acid, which contains one methyl and one methylene group derived from C-2 of acetate. Several organic acids of low molecular weight are excreted in bile. These have now been analysed in rats given [2,2,2-2H3]ethanol [13]. Acetate was formed from ethanol to an extent of about 82% and retained all *H, whereas 15% of the ‘H was lost in the citrate, 2-oxoglutarate and succinate intermediates of the Krebs cycle, and 24% in 3-hydroxybutyrate. More of the citrate (41%) than of the 3-hydroxybutyrate (11%) was formed from ethanol. The results indicate that different pools of acetyl-CoA are used for the synthesis of ketone bodies and citrate in the liver. Analysis for sn-glycerol-3-phosphate and phospholipids in rats given [1,1-2H2]ethanol has revealed that a specific pool of sn-glycerol-3-phosphate is used in the synthesis of phospholipids [27]. Because this might have been due to heterogeneities in the liver lobules, e.g., as a result of passage of bile acids mainly through the periportal cells, similar experiments were done with isolated hepatocytes. However, the resulting labelling was too low, and [1,1,3,3-‘H,]glycerol was instead used as a precursor [2]. It was then found that the glycerol moiety of phosphatidylcholine had lost 2H to a larger extent than the free sn-glycerol-3-

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phosphate, which indicates that a separate pool of sn-glycerol-3-phosphate was used as phospholipid precursor also in the isolated hepatocytes. Incubating a 1 : 1 mixture of [2-*HIand [1,1,3,3-*H,]-glycerol with isolated hepatocytes resulted in the formation of sn-[ *H,]-glycerol-3phosphate to an extent indicating that about 25% of the molecules had received a *H atom at C-2 [2]. This shows that rapid oxidation-reduction took place. The glycerol moiety of phosphatidylcholine was pentadeuterated to a higher degree, indicating that the phospholipid was formed by acylation of sn-glycerol-3-phosphate rather than dihydroxyacetone phosphate. Unpublished work presented here was supported by the Swedish Medical Research Council (Grant No. 2189) and the Swedish Alcohol Research Fund.

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