Am J Hum Genet 32:781-792, 1980

Review Article

An Introduction to Gas Chromatography-Mass Spectrometry andthe Inherited Organic Acidemias

STEPHEN I. GOODMAN1

INTRODUCTION

The organic acidemias comprise a group of inborn errors of amino acid metabolismcharacterized not by aminoacidemia or aminoaciduria but by the accumulation andexcretion of acids that do not contain an amino group and that, therefore, do not reactwith ninhydrin. As such, they can be detected only by procedures specifically designedto examine these "organic acids" in serum or urine. The organic acids in these fluidsare so numerous, however, and their concentrations so altered by the intake of drugsand various foodstuffs or by changes in intestinal flora, that their analysis requiresextremely complex instrumentation. Gas chromatography-mass spectrometry (GC/MS), an instrument combination that simultaneously separates and identifies thecomponents of a complex mixture, is particularly well suited to the analysis of organicacids and is now widely used to screen for and to investigate these diseases. This paperwill briefly review the elements ofGC/MS and provide some current information aboutdiagnosis, pathogenesis, and treatment of the organic acidemias.

GC/MS

Gas chromatography (GC) is a procedure in which the volatile components of amixture are separated by partitioning between a moving, inert gas (carrier gas) and anonvolatile liquid (the stationary phase) [1]. The latter is contained in a column and iscoated onto an inert, size-graded solid (the stationary support). The carrier gas ispassed through the column as column temperature is raised and, as components of themixture leave the stationary phase, they are carried to a detector and become recordedas a series of peaks. Most of the organic acids in physiological fluids are not volatileenough to be analyzed by GC, but volatile methylated or trimethylsilyl derivatives can

Received February 20, 1980; revised April 28, 1980.This research was supported in part by Maternal and Child Health Special Project No. 252.1 Department of Pediatrics, University of Colorado Health Sciences Center School of Medicine, 4200 East

Ninth Avenue, Denver, CO 80262.

© 1980 by the American Society of Human Genetics. 0002-9297/80/3206-0001$02.00

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be made quite easily, and an "organic-acid profile," as the one shown in figure 1, canbe generated in any laboratory with a gas chromatograph. Any of the many publishedspecific techniques can be used providing that one becomes accustomed to the normalpatterns and to the artifacts of the system.

It is rare that an organic acidemia can be diagnosed on the basis of the profile alone,however, because large peaks in almost any area of the chromatogram will be duemuch more often to variations in the normal excretion pattern or to drugs or foodadditives than to the abnormal acids characteristic of disease. Adequate interpretationof the profile, thus, requires that peaks be identified, and, while there are many ways todo this, most of them are complex and time-consuming. The combination of the massspectrometer with the gas chromatograph impacts at this point, since each constituentof the mixture can be identified almost immediately by passing the GC effluent into arepetitively scanning mass spectrometer.The technique of mass spectrometry (MS) rests on the observation that the ion

radical that is created when a compound is vaporized and exposed to an electron beamin a near vacuum will disintegrate in a manner absolutely characteristic of its parentcompound [2]. The array of small fragments that is created constitutes the massspectrum, and the mass spectrometer is merely an instrument, albeit an extremelycomplex and costly one, to create these fragments and to record their weights andrelative abundance. The ions are created in the source of the instrument and, in thesimplest case (low resolution), are accelerated and passed into a free-flight tube wherebeams of different mass are separated by a magnetic field that deflects ions of lowermass more than those of higher mass. The beams then impact on a recorder to producea "mass spectrum." More commonly, however, the strength of the magnetic field inthe free-flight tube is changed in a precisely controlled manner so that ion beams of onemass after another pass through a narrow exit slit and impact on an ion photomultipliertube. In this instance, the output is a bar graph of photomultiplier tube response against

C

D

BA

E

FIG. 1. -Gas chromatogram of trimethylsilyl derivatives of urine organic acids in a normal child; Amalonic acid (internal standard), B = urea, C = citric acid, D = hippuric acid, and E = C-24 paraffin(external standard). Column packing: 5% OV-22 on 80/100 mesh Supelcoport (Supelco, Bellefonte, Pa.).Carrier gas: nitrogen at 30 ml/min. Column temperature held at 800C for 4 min, increased to 280NC at8TC/min, then held at 280'C for 4 min.

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GAS CHROMATOGRAPHY -MASS SPECTROMETRY

time, and time can be related to mass. The quadrupole instrument functions in muchthe same way, except that ion beams are separated by changing direct and alternatingvoltages on precisely contoured pole pieces (usually four) that surround the free-flightpath. In either case, however, the spectrum may be examined as it is generated, or itmay be stored for retrieval and examination at a more convenient time (fig. 2).

The modem mass spectrometer can generate a spectrum every 2 or 3 seconds, whichis easily fast enough to "catch" individual compounds as they are eluted from a gaschromatograph. For example, the rapid and certain identification of the peak shown infigure 3 as methylcitric acid allowed the diagnosis of propionic acidemia to be made inan infant with ketotic hyperglycinemia, and for appropriate treatment and geneticcounseling to be given as well. Indeed, GC/MS has been crucial in developing ourcurrent knowledge that ketotic hyperglycinemia, a syndrome characterized byhyperglycinemia, neutropenia, thrombocytopenia, mental retardation, ketoacidosis,and protein intolerance [3-5], can be caused by organic acidemias, and, in particular,by propionic acidemia, methylmalonic acidemia, and 2-methyl-3-hydroxybutyricacidemia. The cause of the syndrome, first described in 1961, was not clear until theorganic acidemias were discovered and these three particular ones were noted to sharemany of its phenotypic features [6, 7].

Screening for Organic Acidemia

Organic acids accumulate in serum before being excreted in the urine but, since mostare poorly reabsorbed from the glomerular filtrate by the renal tubule, their urineconcentrations become much higher than those in serum and the organic acidemias aremore easily screened for when urine is examined. Accepted indications to screen forthese diseases are (1) an unusual odor, (2) metabolic acidosis, either persistent or

1003-HYDROXYPROPIONIC ACID dITMS 147

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GOODMAN

FIG. 3. -Urine organic acids in a child with propionic acidemia; A = malonic acid (internal standard),and B = C-24 paraffin (external standard). Crosshatched peak is due to methylcitric acid. (Gaschromatographic conditions as detailed in fig. 1).

intermittent, and whether or not associated with an anion gap, (3) recurrent vomiting,especially if associated with acidosis, (4) acute disease in infancy, especially ifassociated with hyperammonemia or metabolic acidosis, and (5) progressive ex-trapyramidal movement disorders in childhood. More debated indications include: (6)Reye syndrome when recurrent, familial, or occurring in infancy, and (7) any inheriteddisorder of obscure cause.

Screening of urine by GC can be performed in any laboratory and, if urineconcentration is corrected for, will exclude disease in 80% -90% of samples. Largepeaks in the remainder, although most often due to drugs, food additives, or normalvariation, will require their referral to a laboratory with GC/MS instrumentation and,preferably, to one with enough experience examining organic acids that the spectra canbe interpreted rapidly. Several laboratories in North America and overseas, theauthor's among them, now serve such a function, and their number is increasing asfamiliarity with the technology increases and as the cost of the instrumentation isbrought down by advances in microprocessor and computer design.

THE ORGANIC ACIDEMIAS

Some information on nomenclature, metabolic block(s), and clinical and labdiagnosis of the inherited organic acidemias is provided in table 1. Details aboutgenetics, pathogenesis, and treatment are provided in later sections.

GENETICS

Pedigree data and/or enzyme measurements on parents of affected patients haveestablished that most organic acidemias are inherited as autosomal recessive traits, butin a few instances, for example, in methylmalonic acidemia due to the cbl D mutation[41, 42] and some forms of glutaric acidemia type II [34], the pedigree data are tooscanty and knowledge of the primary defect too uncertain to exclude X-linkedinheritance. Whatever enzyme defects are known are demonstrable in cultured

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GAS CHROMATOGRAPHY -MASS SPECTROMETRY

fibroblasts, and in utero diagnosis based on enzyme studies in cultured amniotic cellsshould be possible; to date, however, only fetuses with methylmalonic acidemia,propionic acidemia, and glutaric acidemia have been diagnosed antenatally [43 -47].Changes in amniotic fluid organic acids have been demonstrated in fetuses affectedwith these three conditions and with pyroglutamic acidemia [44-49], but it wouldappear prudent to base fetal diagnosis on enzyme assay until this approach has beenevaluated more fully.

Genetic heterogeneity has been studied extensively in methylmalonic acidemia andpropionic acidemia but not in other disorders, primarily because assay techniques arenot yet sensitive enough to detect complementation in somatic cell hybrids. Fibroblastsfrom patients with methylmalonic acidemia can be divided into five complementationgroups, and the disorder can thus be caused by mutations in at least that number ofdifferent loci. One complementation group, designated mut, contains mutations of themethylmalonyl-CoA carbonylmutase apoenzyme, while the remaining four, termedcbl A, cbl B, cbl C, and cbl D, are due to defects in the biosynthesis ofadenosylcobalamin, the mutase coenzyme [42, 50]. The defect in the cbl B group is inATP:cob(I)alamin adenosyltransferase [51]. While the defects in the other cbl groupsare not yet known, those in the cbl C and cbl D cells also compromise the synthesis ofmethylcobalamin, the coenzyme for N5-methyltetrahydrofolate-homocysteine methyl-transferase. The latter is one of two enzymes that remethylate homocysteine tomethionine, and methylcobalamin deficiency in these patients causes them to excretehomocysteine as well as methylmalonic acid [31, 41].The same complementation and assay techniques have been employed to divide

fibroblasts from patients with propionic acidemia into three groups: two (pcc A and pccC) in which the mutations may involve different subunits of propionyl-CoA apocar-boxylase [52, 53] and one (bio) in which it presumably affects holocarboxylasesynthetase and leads to simultaneous deficiency of propionyl-CoA carboxylase,3-methylcrotonyl-CoA carboxylase, and pyruvate carboxylase [54]. Propionyl-CoAcarboxylase activity is decreased appropriately in leukocytes and fibroblasts of obligatecarriers of the pcc A mutation but is normal in pcc C heterozygotes [46, 55].

PATHOGENESIS

There is much debate but little understanding about how the complex phenotype ofvarious inborn errors of metabolism derives from the primary gene defect, and this isno less true of the organic acidemias than it is of such a long-known and extensivelystudied disorder as phenylketonuria. The anion-gap metabolic acidosis and odors thatoccur in some of these disorders are easy to explain, but it is considerably more difficultto account for the episodes of vomiting, hepatomegaly, encephalopathy, and fattyinfiltration of the viscera that occur in many of them, and for the chronic degenerationof the caudate and putamen that is seen in glutaric acidemia.

Vomiting, hepatomegaly, hyperammonemia, and encephalopathy are some of themore consistent features of Reye syndrome, a disorder whose specific cause isunknown but in which generalized mitochondrial dysfunction appears to play a role inpathogenesis [56]. The obvious similarity of this disease to the episodes observed inmany organic acidemias has prompted examination of the effects of the accumulated

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metabolic intermediates on mitochondrial enzymes and/or function. Observations that:(1) methylcitrate (which is accumulated in propionic acidemia and methylmalonicacidemia) inhibits the citrate-malate shuttle as well as several enzymes of citrate andisocitrate metabolism [57], (2) methylmalonic acid inhibits mitochondrial transport ofmalate, 2-ketoglutarate, and isocitrate [58], (3) methylmalonyl-CoA inhibits pyruvatecarboxylase [59], and (4) propionate and isovalerate inhibit mitochondrial oxidation ofboth pyruvate and 2-ketoglutarate [60] all support the notion that some of thesecompounds may indeed be mitochondrial toxins. A unifying hypothesis to explain whymany of these disorders are so similar clinically has not, however, been put forward.The cause of striatal degeneration in glutaric acidemia [61 ] is also obscure and may

be particularly important in understanding Huntington disease, a disorder in which thedistribution of lesions is remarkably similar. One possibility is that glutaric acid inhibitsthe removal of glutamic acid from the synaptic cleft, causing overstimulation andeventually death of the glutamatergic neurons which are especially abundant in thestriatum [62, 63].

TREATMENT

General measures used in the treatment of the organic acidemias include those aimedat correcting dehydration, acidosis, hypoglycemia, and hyperammonemia during acuteepisodes of vomiting and encephalopathy, as well as the use of drugs like sodiumvalproate (Depakene) and the p-chlorophenyl analog of gamma-aminobutyric acid(Lioresal) to treat the movement disorder of glutaric acidemia [64]. As in treating otherinborn errors, measures specific to particular disorders may be directed at: (1) reducingthe concentration of accumulated enzyme substrate, if it or a derivative is consideredtoxic, (2) increasing the concentration of a necessary enzyme product, or (3) increasingthe activity of the mutant enzyme itself. All three approaches have been tried in theseconditions.

Efforts to reduce the concentration of enzyme substrate by reducing the intake ofprotein or specific amino acids have been made in all but pyroglutamic acidemia. Theresults have been good in isovaleric acidemia [65] but less encouraging in the otherdisorders, where some patients do well [7, 22, 64, 66, 67] and some do not [68, 69,and S. I. Goodman, unpublished observations]. Substrate reduction may also beachieved, however, by providing alternate pathways for its disposal, and examples ofthis approach include the treatment of patients with methylmalonic acidemia due tocbl C and cbl D mutations with betaine, and of those with isovaleric acidemia withglycine. In the first instance, betaine fuels the alternate route of homocysteineremethylation (via betaine-homocysteine methyltransferase), but whether or notlowering homocysteine accumulation in these patients affects the eventual outcome ofthe condition is not yet clear (S. I. Goodman, unpublished observations). In isovalericacidemia, however, where glycine apparently reduces isovaleryl-CoA accumulation byincreasing its conversion to isovalerylglycine, the acute episodes that usually occurwith intercurrent infection or protein loads become far less severe and frequent [70].Where deficiency of enzyme product is the critical problem, as it is in patients with

methylmalonic acidemia due to blocks in adenosylcobalamin synthesis (cbl mutants),two approaches have been tried. In the cbl mutants, for example, the block might be

788 GOODMAN

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bypassed by direct administration of adenosylcobalamin or its synthesis might beincreased by providing enough vitamin B12 so that the mutant enzyme, presumably aKm mutant in which enzyme-substrate interaction is impaired, will be exposed to moresubstrate and will thus form more product. Some of these patients respond toadenosylcobalamin [71], but cyano- and hydroxycobalamin are so much cheaper andeasier to obtain that treatment usually takes the latter approach [72, 73] and, whensuccessful, allows protein restriction to be eased considerably. Similarly, when largequantities of biotin (about 10 mg/day) are given to patients with combined carboxylasedeficiency, enough biotin is evidently processed (even in the face of the block) tonormalize tissue carboxylase activities [74].The above situations show how enzyme activity, as judged by the rate of product

formation, can be increased by providing a Km mutant enzyme with additionalsubstrate; in some cases, however, the mutation is such that activity can be boosted byproviding it with additional coenzyme. For example, some mut methylmalonicacidemia fibroblasts increase their apomutase activity when grown in mediumsupplemented with hydroxocobalamin [75], presumably because the mutation de-creases the interaction of enzyme and adenosylcobalamin rather than that of enzymeand substrate. No patient with a mut defect, has, however, been responsive in vivo, noteven those whose fibroblasts respond to cobalamin in vitro.

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