Urinary C6–C12 dicarboxylic acylcarnitines in Reye's syndrome

Clinica Chimica Acta, 175 (1988) 79-88

79

Elsevier CCA 04185

Urinary C,-C,, dicarboxylic acylcarnitines in Reye’s syndrome B.M. Tracey

a, K.N. Cheng a, J. Rosankiewicz and R.A. Chalmers a

a, T.E. Stacey

b

a Perinatal and Child Health, MRC Clinical Research Centre, Harrow, and b Department of Child Health, St. George’s Hospital Medical School, Cranmer Terrace, London (UK) (Received

Key worak Cs-C,,

19 October 1987; revision received 10 February 1988; accepted after revision 18 February 1988)

dicarboxylic

acid, Acylcamitine;

Reye’s syndrome;

FAB-MS

C&i, dicarboxylic acylcamitines have been identified for the first time in urine from a 2-year-old girl presenting with Reye’s syndrome. The acylcamitines were extracted by ion-exchange chromatography and analysed, both underivatised and as methyl esters using high-resolution fast-atom-bombardment mass spectrometry and B/E-linked scanning. The acylcamitines were quantified by capillary gas chromatography of the acids extracted after hydrolysis of the acylcamitine esters. Dodecandioylcarnitine was present in the highest concentration (35.9 mmol/mol creatinine) which exceeded the urinary free dodecandioic acid concentration. The adipic, suberic and sebacic acylcarnitine concentrations were < 10% of the respective free acid concentrations. It is possible that B-oxidation of dicarboxylic acids is partially inhibited in Reye’s syndrome leading to accumulation of precursor dodecandioyl CoA which is metabolised to dodecandioylcarnitine. The accumulation of these metabolic intermediates may be significant in the pathogenesis of Reye’s syndrome.

Introduction Excretion of unusual acylcarnitines has recently been demonstrated in several inherited metabolic disorders in which there is intracellular accumulation of the

Correspondence to: Mrs. B.M. Tracey, Perinatal and Child Health, Watford Road, Harrow, Middlesex HA1 3UJ, UK.

0009-8981/88/$03.50

MRC Clinical

8 1988 Elsevier Science Publishers B.V. (Biomedical

Division)

Research

Centre,

corresponding acyl-CoA esters [l-4]. Some of these disorders also present with episodes clinical indistinguishable from Reye’s syndrome (encephalopathy with fatty degeneration of the viscera and with common biochemical features of hypoglycaemia, hyperammonaemia, C,-C,, dicarboxylic aciduria acid&a and raised urinary acylcamitine excretion). Analysis of urinary acylcarnitines in dicarboxylic acidurias aids differential diagnosis because of the disease-specific pattern which has been found in several inherited metabolic diseases [2,6,7]. Most of the acylcamitines so far character&d have been short or medium chain aliphatic monocarboxylic acylcamitines and acids containing a second functional group were not thought to be substrates for camitine acyltransferases until recently when 3-methylglutarylcarnitine was identified [6,7] Recently it has been shown by ourselves and others [2,4,5] that high resolution fast atom bombardment mass spectrometry (FAB-MS) and constant B/E ratio linked scanning (daughter ion spectra) can be used to identify acylcamitines. In the present work, C&i, dicarboxylic acylcamitine esters have been identified for the first time using FAB-MS and constant B/E ratio linked scanning in urine from a 2-yr-old child presenting with an episode of Reye’s syndrome which was not associated with any inherited metabolic disorder. Patient The patient was a 2-yr-old girl who was obtunded on admission to hospital after a short prodromal illness due to upper respiratory tract infection. She was hypoglycaemic (0.3 mmol/l) and hyperammonaemic ( > 400 pmol/l) and urine organic acid analysis carried out 4 h after admission showed a marked lactic aciduria and ketonuria, moderate dicarboxylic aciduria and salicylate metabolites. Here urine free and esterified camitine values were 4.6 and 69.3 mmol/mol creatinine (control ranges (95% confidence limits) 6.2-21.6 and 23.4-27, respectively) and plasma free and esterified camitine were 20.3 and 41.0 pmol/l (control ranges (95% confidence limits) 15.5-51.1 and 0.2-15.4) indicating an abnormal amount of esterified camitine present in both blood and urine. Studies carried out after the patient’s recovery showed a normal response to a fat load. Normal fatty acid oxidation was found in cultured skin fibroblasts. Methods

Acylcamitines were extracted from a portion of urine equivalent to 0.6 mg creatinine using a previously described method [8]. The extract was divided into two parts. One half of the dried extract was redissolved in 50 ~1 methanol before analysis by FAB-MS, the other half was used to prepare methyl esters of the acylcamitines. Methyl esters were prepared by dissolved the residue in 1 ml methanol, cooling the solution to -70°C in a solid COJacetone bath and adding 400 ~1 thionyl chloride. The solution was left for 5 min at - 70 o C and then warmed at 45 o C for 30 min. Solvent and reagent were then removed by a stream of nitrogen at 45 o C [9]. Prior to FAB-MS the methyl esters were redissolved in 50 ~1 methanol.

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D,L-Dodecandioylcamitine was prepared by a modification of the method of Bshmer and Bremer for monocarbocylic acylcamitines [lo]. A 1.1 molar excess of D,L-carnitine hydrochloride in trifluoroacetic acid was added to 1,12-dodecandioylmonochloride prepared in situ from equimolar quantities of thionyl chloride and 1,12_dodecandioic acid and the mixture was allowed to stand at room temperature for 3 h. Acetone was added to the mixture at 0°C to precipitate unreacted camitine. The D,r.-dodecandioylcarnitine was precipitated by addition of diethyl ether, filtered, washed with diethyl ether and dried in vacua. Fast atom bombardment (FAB) mass spectra of acylcamitines and methylated acylcarnitines were obtained using a JEOL DX 303 double focussing mass spectrometer fitted with a JEOL FAB ion gun (JEOL UK. Grove Park, London, UK). The sample containing approximately 4-8 nmol of acylcarnitines was applied on to the stainless steel FAB target using glycerol as the sample matrix. High energy xenon atoms were generated at 6 kV and an emission current of 5 mA. Collision induced dissociation (CID) spectra were measured by B/E linked scanning using a gas collision cell in the first field-free region. Helium was introduced to the gas cell at a pressure to give a ten-fold decrease in the parent ion intensity. The resolution of the mass spectrometer was set at 500 for all the measurements. Accurate mass measurements of the parent ions were also recorded at a mass resolution of 5 000 to further confirm their identities. To confirm the identities of the acyl moieties, a dried extract containing acylcarnitines was subject to mild alkaline hydrolysis by warming in 1 ml 0.2 mol/l KOH at 40°C for 1 h, followed by neutralisation and extraction on a DEAE-Sephadex column [ll]. The organic acids were analysed and quantified as TMS-derivatives by capillary GC and GC-MS [ll]. Urinary organic acids were also analysed by the same method [ll].

RMlltS The first urine sample collected from the patient after admission was analysed for acylcamitines both as free acid (underivatised), and as methyl esters by FAB-MS. In both cases spectra showed persistent molecular ions for camitine and acylcamitines [4,5] (Fig. 1 and Table I). A comparison of spectra showed that the methyl ester derivatives gave more prominent molecular ions than the underivatised acylcarnitines, probably reflecting the increase in ionization efficiency due to the loss of zwitterionic structure. Of particular interest is the presence of ions at m/z 290, 318, 346 and 374 in the underivatised acylcamitine sample which correspond to the molecular ions of adipyl (C,), suberyl (C,) sebacyl (C,,) and dodecandioyl (C,,) camitines respectively (Fig. la). These compounds when methylated showed a gain of 28 mass units confirming the presence of two carboxylic functional groups (Fig. lb). A constant B/E scan of the precursor ion m/z 374 was recorded and compared with that of the synthesised dodecandioylcamitine standard (Fig. 2a, b). Both the sample and the standard gave virtually identical daughter ion spectra and contained the characteristic daughter ions of acylcarnitines at m/z 85, 100, 102 and 144 [5]. In

82

100

15 a

a > ._

I

c

m

a, a

156

200

250

300

350

400

450 M/Z

Fig. 1. FAB-MS spectra of (a) underivatised acylcamitines and (b) acyl-camitine methyl esters extracted from urine of a patient with Reye’s syndrome (See Table I for identification of ions).

addition a series of 9 ions from mass 203 to 314 differing by 14 mass units was found, probably derived from C-C bond cleavage of the acyl side chain. The M-59 ion previously used [5] to characterise the acyl moiety in acylcarnitines because it retains the ester linkage was not prominent in these spectra (Fig. la). The daughter ion spectra of the methylated sample (precursor ion m/t 402) and synthetic

TABLE I M+ ions of acylcarnitines Acylcamitine

Carnitine Acetylcamitine Isobutyrylcamitine Isovalerylcamitine Adipylcamitine Suberylcamitine Sebacylcarnitine Dodecandioylcamitine

in FAB-MS

spectra of a urine extract from a patient with Reye’s syndrome

m/z M+

Monomethyl ester (M+ )

162 204 232 246 290 318 346 374

176 218 246 260

Dimethyl ester (M+ )

318 346 374 402

83

100. I

a

3”Z

85 50.

55

m ii

a 50.

Fig. 2. Constant B/E ratio-linked scan spectra for 374 ion of (a) dodecandioyhrnitine (b) urine acylcarnitines and 402 ion of methyl esters of (c) dodecandioylcamitine and (d) urine acylcamitincs.

standard showed a very similar dissociation pattern with daughter ions at m/z 99, 100,102 and 158, a series of ions differing by 14 pm between 217 and 328 and also an ion at 343 due to M-59 (loss of trimethylamino group). In addition high resolution accurate mass measurements of the dodecandioylcamitine ions were in agreement with calculated values (Table II).

TABLE 11 Accurate mass measurements the patient

of dodecandioylcamitine

Sample

molecular ion in urine acylcamitine

Formula

extract from

Calculated mass

measured mass

Error

Cl9 H36 06 N Cl9 H36 06 N

314.2543 374.2543

314.2521 314.2526

-4.2 - 4.5

C21 H40 06 N

402.2856

402.2854

-0.4

C21 H40 06 N

402.2856

402.2854

-0.4

(ppm)

Underivatised Dodecandioylcamitine Urine acylcamitine

std.

Methyl ester Dodecandioylcamitine dimethyl ester Urine acylcamitine methyl ester

TABLE III Urine concentrations Acyl group

of free Ce-C,,

dicarboxylic

acids and Ce-C,,

Urine cone mmol/mol

Total a By radioenzymatic

Acylcamitine

680 191 210 24

13.2 13.2 14.8 35.9

1105 assay total acylcamitines

acylcamitines

creatinine

Free acid Adipic Subetic Sebacic Dodecandioic

dicarboxylic

77.1 LL = 69.3 mmol/mol

creatinine

Subsequent B/E linked scan measurement of the ions at m/z 290, 318 and 346 of the underivatised acylcamitine sample and m/z 318, 346 and 374 of the methylated sample confirmed their identity as dicarboxylic acylcarnitines. Analysis by capillary GC and GC-MS of the organic acids recovered after hydrolysis of a Bio-Bad AG50 extract of acylcamitines from urine confirmed the presence of adipic, suberic, sebacic and dodecandioic acids. When the acids were quantified the total amount present was in good agreement with the total amount of acylcamitines determined by radioenzymatic assay [3,12] (see Table III). Comparison with the urine C&i, dicarboxylic acids also quantified by capillary GC showed that the amount of dodecandioylcarnitine excreted was in excess of the free dodecandioic acid, in contrast to the lower chain length dicarboxylic acylcamitines which were less than 10% of the respective dicarboxylic acids in urine (Table III). Discussion

Characteristic acylcamitines have been identified in urine using FAB-MS in a number of inherited metabolic disorders [l-7]. In the disorders of fatty acid

oxidation identification of characteristic acylcamitines can be an aid to differential diagnosis where only the common organic acid pattern of C&C,, dicarboxylic aciduria is present. Reye’s syndrome associated with an underlying metabolic disorder must also be distinguished from ‘classical’ Reye’s syndrome. The latter is usually associated with a viral prodrome and with salicylate administration, and our work has shown that analysis of acylcamitines during an episode can be useful in this respect. Enhanced excretion of acetylcamitine has previously been found in Reye’s syndrome [13]. In the present work C&i, dicarboxylic acylcamitines have been identified for the first time in urine from a child presenting with Reye’s syndrome associated with a viral prodrome and salicylate therapy. No evidence for an underlying inherited metabolic disorder was found. The acylcamitines were identified by high resolution FAB-MS and B/E linked scanning which gave daughter ion spectra showing a characteristic fragmentation pattern due to cleavages in the acyl side-chain as well as the ions due to camitine [5]. The FAB spectra of the methyl derivatives confirmed the dicarboxylic nature of the acyl group by the mass increment of 28 mass U in the molecular ions compared with the underivatised acylcamitines. Dicarboxylic acylcamitines have not previously been observed in disorders which give rise to medium chain dicarboxylic aciduria [14-171. In most of these disorders adipic acid is present in the highest concentration and chain lengths longer than Cl0 are usually found in only trace amounts except in Reye’s syndrome [18,19]. In our patient a significant increase in dodecandioic acid was found in the urine. The urinary concentrations of the C&i, dicarboxylic acylcarnitines were low (< 10%) compared with the corresponding urinary free acids with the exception of dodecanedioylcamitine which had a higher concentration than dodecandioic acid (see Table III). There is some evidence that the relative amounts of free C&i, dicarboxylic acids excreted correspond closely to the relative hepatic intracellular concentrations [20], if this is also true for C&J,, dicarboxylic acylcamitines it indicates that in this patient there is intracellular accumulation of dicarboxylic acylcamitines, and their metabolic precursor dicarboxylic acyl-CoA esters. Dodecandioyl CoA is believed to be the precursor of &oxidation of dicarboxylic acids [21,22] and in the present case the predominant accumulation of the Crz moiety shows that dicarboxylic /3-oxidation is being inhibited in Reye’s syndrome. Dicarboxylic acyl-CoA esters can result from activation of free dicarboxylic acids, probably in the cytosol, or from Poxidation of longer chain dicarboxylic acyl-CoA esters either in peroxisomes or mitochondria. All the hepatic carnitine acyl transferases show some activity towards medium-chain fatty acyl-CoA esters [23-251 but no experimental data exists on their activity towards dicarboxylic acyl-CoA esters. The large amounts of free adipic, suberic and sebacic acids excreted by this patient are derived from dicarboxylic acyl-CoA esters chain-shortened by /3-oxidation in mitochondria or peroxisomes [21,22]. These are hydrolysed to give the free acids rather than being converted into acylcamitines by carnitiiie acyltransferases, and therefore a particular circumstance must cause the synthesis and excretion of dodecandioylcamitine in excess of the other dicarboxylic acylcamitines and of free

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dodecandioic acid. This may be an attempt to increase the excretion of dodecandioic acid which has a limited solubility, or it is possible that dodecandioylcarnitine cannot enter the altered mitochondria found in Reye’s syndrome [26] and cannot be metabolised further in peroxisomes. The accumulation of these essentially abnormal metabolic intermediates may be significant in the pathogenesis of Reye’s syndrome. Acknowledgement We would like to thank Miss Margaret Jones and Mrs. Shirley Bartlett for their excellent technical assistance. References 1 Chalmers RA, Roe CR, Stacey TE, Hoppel CL. Urinary excretion of L-camitine and acylcamitines by patients with disorders of organic acid metabolism: evidence for secondary insufficiency of L-GUI& tine.Pediatr Res 1984;18:1325-1328.

2 Roe CR MiBington DS, MaItby DA, Bohan TP, KahIer SG, Chalmers RA. Diagnostic and therapeutic implications of medium-chain acyl-camitines in the medium chain acyl-CoA dehydrogenase deficiency. Pediatr Res 1985;19:459-466. 3 DeSousa C, Chahnem RA, Stacey TE, Tracey BM, Weaver CM, Bradley D. The response to L-camitine and gIycine therapy in isovaIeric acidaemia. Eur J Pediatr 1986;144:451-456. 4 Roe CR, Hoppel CL, Stacey TE, Chalmers RA, Tracey BM, MiIhngton DS. Metabolic response to camitine in methylmalonic aciduria. Arch Dis Child 1983;58:916-920. 5 MiIIington DS, Roe CR Mahby DA. Application of fast atom bomardment and constant B/E ratio linked scanning to the identification and analysis of acylcamitines in metabolic disease. Biomed Mass Spectrom 1984;11:236-241. 6 Chalmers RA, Tracey BM, Stacey TE, Cheng KN, Madigan MJ, Lawson AM. The use of fast atom bombardment mass spectrometry to identify and study urinary acylcamitines in disorders of organic acid metabolism. B&hem Sot Tram 1986;14:967-969. 7 GaskeII SJ, Guenat C, MiBington DS, MaItby DA, Roe CR. Differentiation of isomeric acylcamitines using tandem mass spectrometry. Anal Chem 1986;58:2801-2805. 8 Tracey BM, Chalmers R4, Rosankiewicx JR, DeSousa C, Stacey TE. Acylcamitines in urine in medium-chain acyl-CoA dehydrogenase deficiency measured by quantitative high pressure liquid chromatography. Biochem Sot Trans 1986;14:700-701. 9 CaprioIi RM, Seifert WE, Sutherland DE. Polypeptide sequencing: Use of dipeptidylaminopeptidase I and gas chromatography-mass spectrometry. Biochem Biophys Res Commun 1973;55:67-75. 10 Bohmer T, Bremer J. Propionylcamitine: physiological variations in vivo. Biochim Biophys Acta 1%8;152:559-567. 11 Chalmers RA, Lawson AM. Organic acids in man. London: Chapman and Hall, 1982. 12 Cederblad G, Linstead S. A method for the dete rmination of camitine in the picomole range. CIin Chim Acta 1972;37:235-243. 13 Roe CR, MiBington DS, MaItby DA, Bohan TP. Relative camitine insufficiency in Reye’s syndrome and relative metabolic disorders. J Nat Reye’s Synd Foundation 1985;5:201-215. 14 Pettersen JE, JeIhun E EIdjam L. The occurrence of adipic and suberic acid in urine from ketotic patients. CIin Chim Acta 1972;38:17-24. 15 Liebich HM. Gas chromatographic profiling of ketone bodies and organic acids in diabetes. J Chromatogr 1986;379:347-366. 16 Gregersen N, Kevraa S, Rasmussen K, Mortensen PB, Divry P, David M, Hobolth N. General (medium-chain) acyl CoA dehydrogenase deficiency (non-ketotic dicarboxylic aciduria): quantitative urinary excretion pattern of 23 biologicaIly significant organic acids in three cases. CIin Chim Acta 1983;132:181-191.

87 17 Naylor EW, Mosovich LL, Guthrie R, Evans JE, Tieckehuann H. Intermittent non-ketotic dicarboxylic aciduria in two siblings with hypoglycaemia: an apparent defect in fi-oxidation of fatty acids. J Inher Metab Dis 1980;3:19-24. 18 Ng KJ, Andresen BD, Hilty MD, Bianchine JR. Identification of long-chain dicarboxylic acids in the serum of two patients with Reye’s syndrome. J Chromatogr 1983;276:1-10. 19 Tonsgard JH. Urinary dicarboxylic acids in Reye’s syndrome. J Pediatr 1985;107:79-84. 20 Mortensen PB. C,-C,,-dicarboxylic acids in liver and kidney tissue in normal, diabetic ketotic and clofibratstreated rats. Biochim Biophys Acta 1986;878:14-19. 21 Gregersen N, Mortensen PB, Kolvraa S. On the biologic origin of C,-C,, dicarboxylic and C,-C,o-o-l-hydroxymonocarboxylic acids in human and rat with acyl CoA dehydrogenation deficiencies; in vitro studies in the w- and o-l oxidation of medium chain (G-C,,) fatty acids in human and rat liver. Pediatr Res 1983;17:828-834. 22 Vamecq J, De Hoffmann E, Van Hoof E. The microsomal dicarboxylyl CoA synthetase. Biochem J 1985;230:683-693. 23 Markwell MA, Tolbert NE, Bieber L. Comparison of the carnitine acyltransferase activities from rat liver peroxisomes and microsomes. Arch Biochem Biophys 1976;176:479-488. 24 Miyaxawa S. Oxasa H, Osumi T, Hashimoto T. Purification and properties of camitine octanoyltransferase and camitine pahnitoyltransferase from rat liver. J Biochem 1983;94:529-542. 25 O’Farrell S, Fiol CJ, Reddy JK, Bieber LL. Properties of purified camitine acyltransferases of mouse liver peroxisomes. J Biol Chem 1984;259:13089-13095. 26 Partin JC, Schubert WK, Partin JS. Mitochondrial ultrastructure in Reye’s syndrome (encephalopathy and fatty degeneration of the viscera). N EngI J Med 1971;285:1339-1343.