J7ournal ofNeurology, Neurosurgery, and Psychiatry 1995;58:209-214

Rhabdomyolysis and acute encephalopathy in lateonset medium chain acyl-CoA dehydrogenasedeficiencyW Ruitenbeek, P J E Poels, D M Tumbull, B Garavaglia, R A Chalmers, RW Taylor,F J M Gabreels

AbstractA previously asymptomatic 30 year oldman presented with rhabdomyolysis,muscle weakness, and acute encephalo-pathy after strenuous exertion in the coldwithout adequate food intake. Serum andmuscle carnitine concentrations weredecreased. Urinary excretion of carnitineand glycine esters and biochemicalexamination of skeletal muscle andfibroblasts led to the diagnosis ofmediumchain acyl-CoA dehydrogenase (MCAD)deficiency. A point mutation atnucleotide position 985 of the codingregion of the MCAD gene was found. TheMCAD protein was synthesised in thepatient's fibroblasts at a normal rate, butwas unstable. In general, patients inwhom the 985 point mutation has beenestablished show much more severe clini-cal symptoms and other symptoms thanthose seen in this patient. The relation ofthe 985 point mutation and the residualMCAD activity to the symptoms is not asstraightforward as previously thought.

(7 Neurol Neurosurg Psychiatry 1995;58:209-214)

University HospitalNijmegen, TheNetherlandsInstitute of PediatricsW RuitenbeekInstitute ofNeurologyP J E PoelsF JM GabreelsSchool ofNeurosciences,University ofNewcasde, UKD M TumbullRW TaylorNational Institute ofNeurology "'C Besta",Milan, ItalyB Garavaglia

St George's HospitalMedical School,University of London,UKR A ChalmersCorrespondence to:Dr W Ruitenbeek,Department of Pediatrics,University HospitalNijmegen, PO Box 9101,NL-6500 HB Nijmegen,The Netherlands.Received 2 August 1994.Accepted 9 September 1994

Keywords: rhabdomyolysis; medium chain acyl-CoAdehydrogenase deficiency; encephalopathy

Rhabdomyolysis (acute muscle necrosis) canbe due to various acquired or hereditarycauses.' 2 The hereditary causes include spe-cific defects of metabolism-for example,enzyme defects in glycogen catabolism andglycolysis.3 Of the disorders of muscle lipidmetabolism, camitine palmitoyltransferasedeficiency is one of the most common meta-bolic causes of rhabdomyolysis.4 Recurrentattacks of rhabdomyolysis have been found inthree patients reported to have myopathic car-nitine deficiency.5-7 Recently a few otherdefects of fatty acid oxidation have been asso-ciated with rhabdomyolysis.8'0Medium chain acyl-CoA dehydrogenase

(MCAD) deficiency is thought to be the mostcommon disorder of fatty acid oxidation.'1-14As an inborn error of metabolism it has beenrecognised with increasing frequency in chil-dren with a Reye-like syndrome. As a conse-quence of the disturbance in fatty acidoxidation, MCAD deficiency is associatedwith acute attacks of hypoketotic hypogly-caemia and C6-C,O dicarboxylic aciduria. Theattacks are provoked by stress, fever, viralillness, and fasting. Some patients, earlier

classified as having carnitine deficiency, laterseemed to have MCAD deficiency.'3 In urinea characteristic profile of C6-C,0 acylcarnitinescan usually be shown, as well as acylglycines(for example, suberylglycine). The presenceof octanoylcarnitine in plasma may be aspecific indication for MCAD deficiency.'5The age of initial presentation of MCAD

deficiency varies widely. Most patients pre-sent acutely, usually between infancy and thesecond year of life. Duran et al described anasymptomatic adult man.'6

Recent DNA studies have shown a highprevalence of a point mutation at nucleotideposition 985 of the coding region of theMCAD gene in patients deficient in MCAD.'7

Here we describe the first patient with aninitial clinical presentation of rhabdomyolysisand acute encephalopathy in adult life, whowas later proved to have MCAD deficiency,established both at the enzyme and DNAlevel.

Case historyA 30 year old man developed progressivemuscle weakness in his arms and legs after aday of strenuous exercise in the cold withouttaking any food. He complained of headacheand nausea, began vomiting, and produceddark brown coloured urine. At the end of theday he became lethargic, irritable, and agi-tated. He was admitted with lowered con-sciousness. He had had another episode ofvomiting, headache, and motor restlessnessone year before. This occurred during a longmotorbike trip during which he did not eat. Itwas said that he sometimes had an acetone-like breath, especially when he had not eaten.The family history was non-contributory.On admission the patient was delirious.

Blood pressure, pulse rate, and temperaturewere normal. Due to generalised muscleweakness, he was unable to stand upright.Muscle stretch reflexes were normal and sym-metric. His liver was not enlarged. There wasno focal neurological deficit. On admissioncreatine kinase activity in serum was 4000IU/l (normal range 15-91 IU/1) and myoglo-bin was 134 ug/l (normal < 85 ,ug/l). The cal-cium concentration of 2-20 mmol/l was lownormal (normal range 2 20-2 60). Phosphateconcentrations were increased to 1 70 mmol/l(normal range 0-76-1-24). Blood glucose wasnormal. Activity of transaminases in serumwas raised, whereas y-glutamyltransferaseactivity was normal. Potassium concentrationwas increased to 5-8 mmol/l (normal range

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3 3-4 4 mmol/l). The patient had a low uri-nary output for two days (500 ml per 24hours). A transient increase of plasma urea to26 mmol/l (normal range 3-1-7-5 mmol/l) andcreatinine to 193 umol/l (normal range70-106,umol/l) indicated a moderate renalinsufficiency. The protein and ketone bodyreaction in urine was slightly positive.

Protein in CSF was slightly increased (530mg/l; normal value for age 150-450 mg/l), aswas lactate (1690,mol/l; normal range1200-1600 ,mol/l). The concentration of glu-cose was normal. There was no clinical or lab-oratory evidence for endocrinological,immunological, and chronic infectious dis-eases, vitamin deficiency, or disorders causedby toxic agents.

Electroencephalography showed a gener-alised slowing background pattern consistentwith encephalopathy. During the acute phasecerebral CT showed thin lateral ventriclesindicative of cerebral oedema. Electro-myography showed normal results. Electro-cardiography showed signs of left ventriclehypertrophy and repolarisation disturbances.Two years later the results of EEG, cerebralCT, and ECG were normal.

Diagnostic investigationsCLINICAL CHEMICAL ASPECTSAfter the patient had made a completerecovery, biochemical studies were performedto determine the aetiology of the rhabdomyol-ysis. The activity of carnitine palmitoyltrans-ferase I and II in leucocytes was normal.Serial measurements of carnitine in serum'8showed a concentration of non-esterified car-nitine in serum fluctuating between 6 and 38jmol/l (table) with a mean level of 21 umol/l(controls > 20 (mean 38 ,umol/l)). Organicacids were determined in urine by gas chro-matography-mass spectrometry. Fast atombombardment mass spectrometry was used toanalyse the excretion profile of acylcar-nitines.'9 There was a moderate dicarboxylicaciduria with increased excretion of suberyl-glycine, with an increase in excretion on pro-longed fasting. The acylcarnitine analysisshowed increased excretion of almost exclu-sively octanoylcarnitine. The identity of theseacylcarnitines was confirmed by accuratemass measurements and by linked scanning ofthe isobutyl esters.Normal concentrations of amino acids were

found in serum and urine.

Serum and muscle carnitine concentrations before andduring carnitine treatment in the patient and controls

Patient Controls

- Carnitine + Carnitine

Serum (jimol/l):Total camitine 13-43 25-78 >25Non-esterified carnitine 6-38 13-41 >20

Muscle (umol/g wet weight):Total carnitine 0-84 0 97 2-74-6Non-esterified camitine 0-73 0-61 2-2-4-2

MORPHOLOGICAL STUDIESA biopsy from the vastus medialis muscle wastaken one month after the rhabdomyolyticperiod. Only 20% of the fibres were type I(normal 35-50%). In semithin toluidinestained Epon sections there was an increasedamount of lipid droplets between the myofib-rils, mainly in type I fibres. Electronmicroscopy showed numerous lipid dropletsas well as local glycogen accumulation.Morphometric studies2" showed that the vol-ume density of lipid droplets (Vj): volumedensity of the myofibrils (Vmt) ratio was twicethe control mean (V,iNmt patient 0-87 (SEM0-15), controls 0-36 (SEM 0 07)).

BIOCHEMICAL ASPECTSConcentrations of total and non-esterifiedcarnitine in muscle tissue 8 were about 25% ofcontrol values (table). The activities of carni-tine palmitoyltransferase I and II, citrate syn-thase, creatine kinase, cytochrome c oxidase,palmitoyl-CoA synthetase, and succinatedehydrogenase were normal in skeletal mus-cle.21The oxidation rates of radiolabelled palmi-

tate, octanoate, butyrate, and pyruvate byintact cultured fibroblasts were determinedaccording to Veerkamp et al.22 The rate ofoctanoate was diminished to 5% of thecontrol mean (0- 11 v 2-4 (SD 0 9)) nmol.h '.mg-I protein. MCAD deficiency was estab-lished in fibroblast homogenate (1-6 v 15-6(6-2) mU.U ' citrate synthase), using an elec-tron transfer flavoprotein linked assay.23 Theactivities of short chain and long chain acyl-CoA dehydrogenases were normal. In a 600 gsupernatant of a frozen skeletal muscle speci-men, with phenazine methosulphate anddichlorophenolindophenol as electronacceptor,24 MCAD activity was not above thedetection limit.

MOLECULAR BIOLOGICAL ASPECTSGenomic DNA studies were performed onmuscle tissue and skin fibroblasts. The DNAwas extracted from cultured skin fibroblastsand skeletal muscle homogenate; 2,ug wasused to amplify an 87 bp region using thepolymerase chain reaction (PCR). Theoligonucleotide primers used create a restric-tion site for NcoI in the mutant fragment.25After an initial eight minute denaturation stepat 94°C, the PCR reaction was repeated for30 cycles (one minute denaturation at 94°C;two minutes annealing at 50°C; and threeminutes extension at 72°C; the final extensionwas for eight minutes). The 87 bp PCR prod-uct was precipitated and resuspended in 20,lof H20. A 10 ,l aliquot was digested with 5Uof NcoI at 37°C for two hours. The originalPCR fragment and the products of the restric-tion digest were treated by electrophoresis on a3% agarose gel containing ethidium bromide.

Digestion with NcoI of the amplified DNAfrom the patient's fibroblasts and muscleshowed only 61 and 26 bp fragments and no87 bp fragment (fig 1, lanes 8 and 10).Therefore the patient was apparently homozy-gous for the 985 point mutation.

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1 3 4 5 6 7 8 9 10

Figure1 Electrophoretic pattern of the 87 bpfragments and their NcoI digafter PCR amplification of muscle andfibroblast DNA from patient and co?bp NcoI digestion product is distinct, the 26 bp product is hardly visible. Laicontrol muscle; lanes 7-8 = patient's fibroblasts; lanes 9-10 = patient's mu.numbers = original PCRfragments; even numbers = digested PCR produci

The synthesis and stability ofprotein was estimated in culturecblasts. The normal medium for gwas Eagle's minimum essential r

MEM) supplemented with 10°/serum (FCS) and antibic[I5S]methionine incorporation 4medium was removed from conflilayers. Each dish (area 65 cm2)twice with phosphate buffered s.

and incubated at 37°C for two hml labelling medium (E-ME1methionine but with 10% Fmedium was replaced by 5 n

labelling medium containing L-[3'ine (100 ,uCi). After incubation f

at 37°C the medium was removeexperiments the degradation ofsised protein was studied by inccells for another six or 17 hournmedium with added unlabelledThe cell layers were rinsed twic4containing 2% unlabelled methionubilised by the addition of 1medium (150 mM NaCl, 10 nr

0 5% Triton X-100, 0 25% sodiisulphate, arnd 2% unlabelled met]

.......:.:.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.HiFigure 2 Gel electrophoretic pattern of the

protein synthesised in fibroblasts from patieniLanes 1 2 and 3 patient lanes 4, 5, andlanes 1 and 4 =MCAD protein synthesisedlanes 2 and MCAD protein synthesised

followed by six hours incubation in unlabelhlanes 3 and 6 = MCAD protein synthesisedfollowed by 17 hours incubation in unlabelle

7 4). The cell extract was centrifuged for onehour at 50 000 g. The supernatant was mixedwith antiserum raised against pig liverMCAD,'6 and incubated overnight at 4°C.The antigen-antibody complex was precipi-tated by adding 100 pl of Staphylococcusaureus cell suspension, followed by centrifuga-

c- 87 tlp tion (10 minutes at 1700 g). The pellet was

washed four times with 2 ml of buffer contain-p ing 150 ml NaCl, 10 mM Tris-HCl, 1%

Triton X-100, 1% deoxycholate, and 0-1%sodium dodecylsulphate, pH 7-2. The washedimmunoprecipitate was solubilised by boiling(five minutes) in 50,ul of a denaturatingbuffer containing 8% mercaptoethanol and0-003% bromophenol blue. After 10 minutes

'estion products centrifugation at 6000 g the supernatant wasnztrols. The 61nes 1-6 = subjected to SDS-PAGE electrophoresisscle; odd according to Laemmli,27 with 12% gels. Thets. gels were treated for fluorography with Auto-

fluor (Amersham, Buckinghamshire, UK),the MCAD dried, and fluorographed according to theI skin fibro- supplier's instructions.rowing cells Figure 2 shows the representative MCADnedium (E- synthesis pattern. The MCAD protein was/o fetal calf synthesised by the patient's fibroblasts at a)tics. For normal rate (lane 1). The rapid disappearanceexperiments of the protein incorporated radiolabelled[uent mono- methionine suggests a reduced stability of thewas washed protein (lanes 2 and 3). This is likely due toaline (PBS) the impaired ability of the mutant 44 kDaLours with 5 monomers to form a stable tetramer.28'30M without'CS). Thisnl of fresh Carnitine treatment5S]methion- The patient was treated with 8 g DL-carnitineor one hour daily for nine months. During the first monthed. In some of carnitine supplementation the patientthe synthe- noticed an improvement in his general condi--ubating the tion. Total carnitine concentrations in serums in normal increased to near normal values and becamemethionine. more constant. The concentration of carnitinee with PBS in muscle after nine months of carnitine treat-line and sol- ment had not increased (table), and lipidml NETS accumulation between the myofibrils had notaM EDTA, disappeared. Supplementation was stopped asim dodecyl- no objective improvement was seen.hionine, pH Supplementation of 6 g/day riboflavin (the

precursor of the flavin moiety of MCAD) hadno beneficial effect.

Physiological testsThese tests were performed after informedconsent of the patient.

FASTING STUDIESProlonged fasting studies were performedbefore the diagnosis of MCAD deficiency wasestablished. The blood concentrations of themost important metabolites were determinedbefore and after the patient had been treatedwith carnitine for 40 days. Figure 3 shows theresults. The concentration of non-esterified

MCAD fatty acids and ketone bodies increased during6 = control; fasting, whereas the ratio of ketonein one hour; bodies:non-esterified fatty acids declined toIin one hour, 0-33 at 48 hours without carnitine supple-ed medium;I in one hour, mentaton. Glucose concentrations remaineded medium. above 4-2 mmol/l for 48 hours, but declined

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Figure 3 Concentrationsof lactate, ketone bodies,non-esterified (free) fattyacids, and esterified andnon-esterified carnitine inpatient's blood duringprolongedfasting. Left:without carnitinesupplementation; Right:during carnitinesupplementation (8 g DL-caritine).

40

20 -

03200 Withoutr carnitine ,2400 k-

C-

c;0

Ea)LO

Carnitine:FreeEsterified

NEFAS

Lactate

Ketonebodies

1600 e

800 k

o 12 24 36Hours of fasting

48 60

Duringcarnitine treatment

0 12 24 36Hours of fasting

at 55 hours to 3 0 mmol/l. A more pro-nounced ketogenic response to fasting was

found during camitine treatment, whereas thenon-esterified fatty acid concentrationremained normal. The ketone bodies:non-esterified fatty acids ratio was 0-68 after a 48hour fast. Glucose concentrations were

maintained at 4-4 mmol/l. Apparently, the lowcamitine concentrations had some patho-physiological consequences besides theMCAD deficiency.

EXERCISE TESTS

The patient participated in two prolongedexercise tests3' (again before the diagnosis wasmade), one before and one during camitinetreatment. The patient became confused andexperienced myalgia and nausea at the end ofthe exercise test without camitine. The nor-

mal glucose concentration could no longer bemaintained and lactate and ammonia concen-

trations showed a pronounced rise. Duringcamitine treatment he performed the pro-longed exercise test with only slight myalgiaand felt well after finishing. The glucose, lac-tate, and ammonia concentrations did notbecome abnormal on this occasion.

DiscussionSince 1982 MCAD deficiency has beenrecognised in young children with episodic ill-

ness resembling Reye syndrome, provoked byviral illness, fever, and fasting.'12 3234 The firstepisode is often before the age of 2 years.'235The patients show a broad range of clinicalpresentation-namely, intolerance to fasting,episodic vomiting, lethargy, altered conscious-ness, and even coma. The outcome may besevere and fatal, although complete recoveryhas also been described.'6 Dependent on thedegree of involvement of the liver, heart, andskeletal muscle, the clinical features mayrange from recurrent episodes of hypogly-

caemia to progressive muscle weakness andmyalgia. Serum creatine kinase may be mod-erately increased. In many patients the mus-

cular symptoms are not prominent, but some

have symptoms of muscle weakness.Techniques such as gas chromatography-

mass spectrometry and fast atom bombard-ment mass spectrometry make it possible toestablish this metabolic disorder easily.'9 36

Recently the value of measuring octanoylcar-nitine in plasma from patients suspected tohave MCAD deficiency has been stressed.'"Deficiency of MCAD has been associatedwith a molecular lesion involving an A to Gnucleotide replacement at position 985 of theMCAD coding sequence,'7 resulting inreplacement of lysine329 by glutamic acid.3"Although the prevalence of this mutation ishigh in patients deficient in MCAD, thisapproach is not yet appropriate to diagnose allpatients with MCAD deficiency.30 37

Until now only one asymptomatic adultwith MCAD deficiency has been described,'6but his genotype was not established. Wereport the first patient with the MCAD pointmutation at nucleotide position 985 whoseclinical symptoms appeared at an adult age,whose medical history did not include muscleproblems during childhood, and who did nothave a positive family history. The clinicalpresentation of MCAD deficiency in thispatient consisted of acute rhabdomyolysis andtransient encephalopathy, provoked by pro-longed exertion and fasting. Hitherto, rhab-domyolysis has not been associated withMCAD deficiency, although it has recentlybeen described in several other defects of fattyacid oxidation.8 "° Fatty acids are importantsubstrates for skeletal muscle, especially dur-ing prolonged exercise and fasting. Thedecreased oxidative capacity of muscle tissuecan result in a moderate accumulation of lipiddroplets.The transient encephalopathy in the pre-

Carnitine:Esterified

Free

NEFAS

Ketonebodies

Lactate

48

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sent patient may be considered as a complica-tion of rhabdomyolysis or a diminished supplyof ketone bodies to the brain. The metabolismof the brain depends on glucose oxidationand, during fasting, on the oxidation of ketonebodies. Fatty acids are hardly metabolised bybrain tissue. When the excess of mediumchain fatty acids can no longer be metabolisedby fl-oxidation, as occurs in MCAD defi-ciency, ketogenesis becomes impaired and a-oxidation takes place. The impaired oxidationof octanoate may also have contributed to thedevelopment of encephalopathic symptoms.Octanoate disturbs the excretion of organicanionic compounds and mitochondrial func-tioning, especially in the choroid plexus.38 Italso induces epileptic and encephalopathicsymptoms. Carnitine supplementation canstimulate detoxification of octanoate by esteri-fication, after which octanoylcamitine israpidly excreted.The main dietary advice for patients with

disorders of fatty acid oxidation is to have anadequate carbohydrate intake at regulartimes.'4 The supplementation of this patient'sdiet with carnitine may have improved exer-cise tolerance and protected against the meta-bolic consequences of prolonged fasting.

It is remarkable that in our patient, with theaberrant clinical symptoms, the same pointmutation and the same biochemical featureshave been found as in the far more severecases ofMCAD deficiency. Our patient showsthe mutation in both alleles. The questionarises as to which factor(s) determine thisclinical heterogeneity. It has been shown thatthe 985 point mutation results in a diminishedconcentration of MCAD protein as a conse-quence of an enhanced degradation rate of theprotein.2939 Recently, other point mutations,some of them being localised on exon 11 likethe 985 mutation, have been found especiallyin compound heterozygotes.'03740-42 Possiblyenvironmental factors or the relative activityof the other enzymes of fl-oxidation play apart in determining the phenotype in patientsdeficient in MCAD. It is important to realisethat in adults with unexplained rhabdomyolysisand acute encephalopathy a diagnosis ofMCAD deficiency, or other disturbances infatty acid oxidation, should be considered.

This investigation is part of the research programme Disorders ofthe Neuromuscular System of the University of Nijmegen.We are indebted to Professor JH Veerkamp (Department of

Biochemistry, University of Nijmegen), Dr H J Ter Laak(Institute of Neurology, University of Nijmegen), Professor AM Stadhouders (Department of Cell Biology, University ofNijmegen), Professor R C A Sengers, Professor JM F Trijbels,Professor L A H Monnens, Dr J A JM Bakkeren (Institute ofPediatrics, University of Nijmegen), and Dr S DiDonato(National Institute of Neurology "C Besta", Milan) for investi-gations, discussion, and advice. R A C is grateful to Drs K NCheng and B M Tracey for their assistance with the gas chro-matography-mass spectroscopy and fast atom bombardment-mass spectrometry studies.

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