a new chemical diagnostic method for inborn errors …

A NEW CHEMICAL DIAGNOSTIC METHOD FOR INBORN ERRORS OF METABOLISM BY MASS SPECTROMETRY- RAPID, PRACTICAL, AND SIMULTANEOUS URINARY METABOLITES ANALYSIS

I. Matsumoto and T. Kuhara Division of Humarz Genetics, Medical Research lnstit~cte, Kanazowa Medical University, Uchinada, Kahoku-gun, Ishikatz,a 920-02, Jclpon

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I. Introduction .............................................................................................................. 44

11. Present neonatal mass screening program in Japan ....................................................................... 44 A. Target diseases of neonatal mass screening program.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 B. Examination methods used in the present neonatal mass screening program.. ......................................... 45

111. Planar analysis-New methods with GC/MS.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 A. The use of urine filter paper and its advantages.. ..................................................................... 46 B. Sample preparation and GCFIS analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

..... C. Differences between the conventional methods and the GC/MS chemical diagnostic method for mass screening.. 47 D. Chemical diagnostic method by mass chromatography (MC) used in the project study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 E. Three alternative chemical diagnostic methods using a mass spectrometer.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

V. Conclusions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments 56

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

In ttrost daleloped countries, neotlatal mass screerlirlg pr~ogr~nn~s for tlte early diagnosis of inborn errors of nletaholisnl ([EM) have beerr in~plenlented and have been forrrld to be eflecti1xe for tllc prevention or signifrcarrt reduction of clinical .s~n~/~tonr.s such as rnental retardation. These programs rely pr.in1ar.ily on simple hac,terial inhibition assays (the "Guthrie tests"). We de1,eloped a new method for screening [EM using GCIMS, ~ ~ h i c h erlahles accurate chenlical diagnoses through urinary analyses with a sitnple practical procedure. The urine san~ple preparatiorz for GCIMS takes one hour for one sanrple or three hoirt.s for- a hatch of 30 sanlples (will beBllly arlton~ated shortly), and the follotc~ing GCIMS nleasurement is conlpletetl rtithirl 15 nlin per- satnple. This method a1lon.t'~ the simultaneous analyses of nnlino acids, organic acids, sugars, sugar alcolrols, sugar acids, and nucleic acid bases. Therefore, a large trunlber of n~etabolic disorders can be sin~ultaneously tested by tllis ckenzical diagnostic procedure. This method is quite con~pr~el~ensive and different fionl corr\~entional GCIMS organic acidenlia screening procedures, which are not well-suited to detect metabolic disorders except organic acidurias. Sample preparation includes urease treat-

hated ~,itlr ut.ense, follocr~ed by deproteinization with alcohol, el~aporatiori to dryness of tlzc su1~ernatant, and trin~ctl~yl,silyla- tion; the sanlples were applied to GCIMS. A pilot study of the applicatior~ of this diagnostic procedure to the neorlatal mass screening o f22 disorders was started in Japan on February 1, 1995 in cooperation with ,four n~edical institutes. This program is supported by the Japarzese Society for Bionledical Mass Spec- tron1etr.y and the Japanese Mass Screening Society. The initial twenty-two target n~etabolic diseases are: metlzylmalonic acidenlia; pr.opioilic acidenlia; iso~~aleric acidenzia; nzaple syrup urine disease; P-ketotkiolase deficiency; galactosevzia; phenylketonuria; I~yperphe~~ylalaninemia; Izomocystinuria; alkaptonuria; nlultiple carbo.vylase deficiency; rtonketotic hyperglycinenzia; lysinuria; cystirluria; tyrosirlemia; glutaric aciduria type I; P - hydrov-P-n~ethylglutaric acidemia; P-methylcrotonylglycinuria; a-anzinoadipic-a-ketoadipic aciduria; ornithine transcarba- nzylase deficiency (four urea cycle disorders can be screened); glutaric aciduria type 11; and neuroblastoma. Neuroblastoma is not an IEM, and is examined at ca. 6 months of age. The twenty- two target diseases will be reconsidered during tlte pilot study.

ment, deproteinization, and derivatization. The nzethod I~as also been applied to neonate urine specimens that are absorbed into frlter paper. The air-dried sanrples were mailed to the arlalytical Received 17 June 1996; laboratory and eluted with M8ater. The eluate (0.1 nrL) was incu- accepted 16 October 1996.

Mass Spectrometry Reviews, 1996. 15, 43-57 8 1997 by John Wiley & Sons. Inc. CCC 0277-7037/97/0 10043- 15

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MATSUMOTO AND KUHARA

An accurate chemical diagnosis and hence early treatnimt of not only organic acidemias bur also amino acidemias, and sugar-, polyol-, and nucleic acid base-accumulating metabolic disorders can be made at a very early stage of life. This procedure is also applicable to metabolic profiling of other body jluids that are potentially informative for the study and charactcrizatioti of a wide range of inlzerited and acquired metabolic disorders. O 1997 John Wiley & Sons, Inc.

I. INTRODUCTION

Inborn errors of metabolism (IEM) can be divided into two major categories: intoxication type (accumulation of intermediate metabolites) and energy deficiency type. In many of these disorders, pathological consequences can be prevented by appropriately intervening with early treatment of neonates or infants with IEM. However, if the treatment is delayed, then therapeutic effects are drasti- cally lowered. For example, if the treatment for phenylketonuria is started later than 18 days of life, then irreversible mental retardation results (1). Therefore, early diagnosis is critically important for achieving timely treatment.

The present neonatal mass screening program that is implemented in Japan as well as in other developed countries relies primarily on simple bacterial inhibition assays (the "Guthrie tests") (2, 3). The program is practical, sufficiently specific, and cost-effective, but has several disadvantages. The greatest disadvantage is the one- method-for-one-disorder analysis or one-index-compound-for-one-disorder from blood spots on filter paper, a concept that we will refer to here as "point analysis."

Through the studies on various analytical methods (i.e., GC/MS for organic acid analysis, liquid chromatography for amino acid analysis) for high-risk screening, it was found that simultaneous analyses of a series of compounds is possible. Analyses of a single category of compounds (e.g., organic acids or amino-acids) is denoted as "line analyses." Recently, the simultaneous analyses of blood acylcarnitine and amino acids by tandem mass spectrometry have emerged as a rapid and multicomponent analysis (4-6). By combining several analytical procedures for "line analyses," a definitive chemical diagnosis of an underlying disease can be made. Ideally, the combi-

- nation of multiple line analyses in a single method could provide a rapid, cost-effective, and specific biochemical diagnosis of a large number of IEM, as well as a better biochemical understanding of various disorders.

Since 1975, we have carried out studies on improve-

by GC/MS for a neonatal mass screening program in Japan was initiated in 1995 by six institutions (Kanazawa Medi- cal University, Imperial Gift Foundation Bosi Aiiku Kai, Kurume University Medical School, Shimane Medical University, Sapporo City Institute of Hygiene, and Chiba Prefectural Children's Hospital) in cooperation with Shi- madzu Seisakusho Ltd., JEOL Ltd., and Yokogawa Ana- lytical Systems Inc.

In order to make the chemical diagnostic method feasible for neonatal mass screening, three essential require- ments must be fulfilled: a completely automated sample preparation, the shortest possible analytical time, and a computer algorithm for the interpretation of profiles and the flagging of abnormal results. GC/MS instruments have been greatly improved, resulting in higher separation effi- ciencies of mixtures, higher sensitivity of ionization and detection, faster scan rates, and high-speed data pro- cessing. We developed a new sample preparation method to fully utilize these features, which can be finished within 1 h following a GC/MS measurement of 15 min. With this sample pretreatment and the newest bench-top GC/ MS, an accurate analysis of amino acids, organic acids, sugars, sugar alcohols, and nucleic acid bases can be made at one time. The simultaneous analyses of multiple categories of compounds is denoted as "planar analysis." From these analyses, very comprehensive diagnoses and metabolic studies are possible. For example, we have diagnosed galactose-galactitol-galactonic acid in galactosemia, phenylalanine-phenylpyruvic acid-phenyllactic acid-o-hydroxyphenylacetic acid in phenylketonuria, branched-chain- amino acid-branched-chain-cu-hydroxy acid for maple syrup urine disease, methionine-homocystine-methylma- Ionic acid for homocystinuria, etc.

II. PRESENT NEONATAL MASS SCREENING PROGRAM IN JAPAN

The IEM now targeted in Japan include phenylketonuria (PKU), maple syrup urine disease (MSUD), homocystinuria, and galactosemia. Congenital hypothyroidism (cretinism), and congenital adrenal hyperplasia (CAH) are also screened. All neonates born in Japan are screened for these disorders, and as a part of this program, screening for neuroblastoma is also performed for 6-month-old infants. Both examination rates are nearly 100%. The numbers of children discovered to have these disorders by this program and the incidence of their disease is shown in Table 1.

ments of a chemical diagnostic method by G C N S for IEM, and have conducted high-risk screening at the re- A. Target Diseases of Neonatal Mass

quest of many medical institutions in Japan and other Screening Program

countries. As the result of these studies and services, a These six disorders can cause a marked mental retardation joint pilot study on the feasibility of chemical diagnosis or even death if treatment is delayed. Early diagnosis and

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MS DIAGNOSIS OF INBORN ERRORS OF METABOLISM m

TABLE 1. Numbers of children and the incidence of eight diseases detected by mass screening. - - - -

Name of disease Fiscal year No. of newborns No. tested No. of patients Fraction

Phenylketonuria Maple syrup urine disease Histidinemia Homocystinuria Galactosemia Congenital adrenal hyperplasia Congenital hypothyroidism Neuroblastoma

(Ministry of Welfare: Bulletin on Special Formula No. 31: 84-85, 1995).

treatment are critically important. In October 1977, the pyruvic acid (PPA), and phenyllactic acid (PLA), also Ministry of Welfare initiated screening for PKU, MSUD, important indicators for PKU, cannot be analyzed. histidinemia, homocystinuria, and galactosemia in all neo- Accordingly, it is not possible to make a differential nates in Japan. Cretinism was added in 1979, and neuro- diagnosis of PKU and other hyperphenylalaninemia by blastoma in 1984 for all infants. In 1988, CAH was added, only Guthrie's method; when a high Phe concentration but histidinemia was deleted. in blood is detected by the neonatal mass screening,

a more accurate examination is required for definitive

B. Examination Methods Used in the Present Neonatal Mass Screening Program

In the present neonatal mass screening, the same testing procedures are used in many developed countries, such as the bacterial inhibition assay (BIA: Guthrie's method) developed by Dr. Guthrie (3, the Pagen phage method or Beutler's method (7), radioimmunoassay (RIA) (8), and enzyme-immunoassay (EIA) (9). A single assay method is used for each disorder.

Using the BIA developed by Dr. Guthrie, semi- quantification of phenylalanine (Phe) is used in mass screening for PKU and has been proven to be useful. Subsequently, BIA has been adapted for measuring blood leucine for an examination of MSUD, methionine (Met) for homocystinuria, and histidine (His) for histidinemia. For galactosemia, a blood galactose (Gal) concentration is determined by the Pagen phase method. For cretinism, thyroid-stimulating hormone (TSH) and thyroxine (T4) concentrations are determined by RIA or EIA. For CAH, 17-a-hydroxyprogesterone ( 1 7-OHP) is measured by EIA or RIA. For each disorder except cretinism, only one compound is measured as a parameter by a biological semi-quantitative assay or an immuno- logical method. These conventional methods have the following advantages: (1 ) many specimens can be examined at one time, (2) the procedures are relatively simple, and (3) the examination cost per specimen is low. On the other hand, their accuracy and sensitivity are not so high, and considerable time and cost are required for the personal training and for quality control of the analytical systems. Although Phe is used as a single parameter for hyperphenylalaninemia, phenyl-

diagnosis. The "point analysis" represented by BIA has been

used for mass screening, and the results have been fruitful for the past 17 years in Japan and in other developed countries. However, as described above, one assay method is required for each disorder. Whenever a new target disorder is to be added, an appropriate method must be tailored and evaluated in terms of accuracy. These processes heav- ily increase the costs. If the number of targeted diseases exceeds 20, then the point analyses are not practicable. Furthermore, BIA is not an ideal analytical strategy, because it gives false-positives, false-negatives, and incon- sistent diagnoses in inter-laboratory comparisons (9).

To overcome the problems of BIA, various analytical instruments have been tried actively in high-risk screening since about 1970. Compared with point analysis, these line analyses use multiple parameters that are characteristic of each disorder in chemical diagnostic methods such as gas chromatography (GC) (lo), high-performance liquid chromatography (HPLC) (1 I), liquid chromatography (LC) (12) such as amino acid analyzer (AAJ (13), liquid chromatography-mass spectrometry (LC/MS), or gas chromatography-mass spectrometry (GCIMS). For analyzing organic acids specifically, GC/MS has proven to be the most effective method (14-17). These methods have been proven effective in reducing the number of misdiagnosed cases and providing, at least for a number of disorders, almost conclusive biochemical diagnoses. These line analyses have mainly been used for the secondary analyses of positive cases that are detected in neonatal mass screening. In those methods, a series of organic acids, sugar, or amino acids are measured at one time, so that the simultaneous examination of metabolic disorders of several compounds


in the same category is possible. Their accuracy is moder- ately or markedly higher than that of point analysis.

In patients with PKU, for example, the aromatic acids, PPA, PLA, o-hydroxyphenylacetic acid, and phenylacetic acid (PAA), increase as well as Phe. For chemical diagnosis by line analysis, two instruments, GC/MS and AA, have been used in most screening programs, because aromatic acids are efficiently analyzed by GC/MS, and Phe, an amino acid, is conveniently detected by AA.

Generally, line analysis is made first for organic acids, amino acids, or sugars in cases when specific metabolic disorders are suspected. If the suspected metabolic disor-

- der is absent on the first line analysis, then a subsequent alternative line analysis is performed. When data on AA are normal but clinical evidence suggests a metabolic disorder, examinations for sugars are conducted using other analytical systems such as GC, HPLC, or GC/MS. Mean- while, however, patients and their parents have to endure not only mental and physical hardship, but also added examination costs, transportation expenses, and the loss of time.

Ill. PLANAR ANALYSIS -NEW METHODS WITH GC/NIS

Only ten years ago, GC/MS analysis of complex mixtures was hampered by inadequate chromatographic separation using packed columns and considerably slower scan rates (about 1-3 slscan). To overcome these limits, extensive extraction procedures were necessary by limiting analyses by functional groups. For example, in serum- or urine- organic acid analysis, when organic solvent extraction with diethyl ether or ethylacetate was used under an acidic condition, or the organic acid fraction alone was separated by Dowex-I, DEAE-Sephadex, components other than organic acids were excluded. In other words, compounds with other properties that might contain information on disorders in patients in the original samples were completely discarded.

Recent progress in separation science and mass spectrometry has brought about great improvements in GC/ MS efficiency and sensitivity. A higher degree of GC separation and sensitivity as well as faster scan rates in MS have made possible analyses of more complex mixtures. In the state-of-the-art GC/MS, various components (mixed components) of biological fluids in specimens are efficiently separated by capillary GC, and their chemical structures are immediately analyzed. Each component is sensitively measured by MS proportional to those ions that are characteristic of each compound. GC/MS is now suitable for studies on the metabolic disorders caused by defects of specific enzymes. In 1966, Kay Tanaka at the Massachusetts General Hospital was the first to detect iso-

valeric acidemia, an inborn error of organic acid metabolism, by GC/MS (18), showing the usefulness of this method. Since then, GC/MS has been widely used in studies on metabolic disorders of organic acids, and many new disorders have been discovered. GC/MS has made a great contribution to the study of inborn errors of metabolism, and has been recognized as a complement to AA analysis for amino acids.

In 1977 we initiated the chemical diagnosis of 22 inherited metabolic disorders by GC/MS at Kurume Uni- versity Medical School, and have, so far, analyzed 7,000 specimens referred from hospitals throughout Japan. About 80% of the organic acidurias found in Japan were diagnosed by us. After our institution was moved to Kana- zawa Medical University, the number of analyzable disorders was increased to 100 by combining amino acid data obtained from AA. Further studies have advanced our knowledge, and the number of analyzable disorders is cur- rently 130. We are making an effort not only to utilize but also to educate others about chemical diagnoses of inborn errors of metabolism for clinical medicine.

A. Use of Urine Filter Paper and Its Advantages

In the present neonatal mass screening program, a blood specimen is obtained by puncturing the sole of a neonate 5-7 days after birth and absorbing the blood sample onto filter paper. The historical reason for using a blood sample is that, in the course of selection of a mass screening method, urine samples would not allow detection of Phe, the target compound for PKU in the Guthrie's test.

From the aspect of metabolic biochemistry, the body rapidly excretes unnecessary or toxic compounds that ex- ceed the desired levels for the body by homeostasis to maintain normal intracellular metabolism. Therefore, sub- stances toxic to the body, such as those compounds sought in neonatal mass screening, do not increase so dramatically in blood, but are excreted in large amounts into urine. Accurate and sensitive GC/MS analyses enable the com- plete detection of these compounds in urine samples. Moreover, collection of urine is easily accomplished and avoids invasive procedures such as sole p;ncture.

For the above reasons, we have used urine filter paper for the chemical diagnosis in our present neonatal mass screening as Tuchman et al. used for screening newborns for multiple organic acidurias in 1991 (19). We have made a comparison of the results of GC/MS analysis using urine filter paper with those using blood filter paper. Urine filter paper is generally more useful, as Chamberlin et al. (20) reported in 1987.

B. Sample Preparation and GUMS Analysis

The method we developed for preparation of urine samples for GC/MS analysis is shown in Scheme 1. Our sample

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The standardization and testing at PreventiNe is on and soon 130 disorders should be integrated. The challenge is integration of process to keep the cost and TAT same as that of 101 disorders. Else, a separate process will reduce the throughput in a big way.

MS DIAGNOSIS OF INBORN ERRORS OF METABOLISM

Urine (0.1 ml) C. Differences between the Conventional

1 Methods and the GUMS Chemical

Urease treatment (type C3) 37°C for 10 min. Diagnostic Method for Mass Screening

1s (Iabelled-creatinreand A*. As described previously, point analyses, such as BIA, the HDA and 3HM)

Pagen phage method, RIA, and EIA, analyze one compo- Deprotenization with ethanol nent for each disorder. For example, only Phe is semi- + quantitatively measured in the examination of PKU by

Mixing, centrifugation, and removal of ppt. BIA, and only the blood leucine concentration is deter-

1 mined in the examination of MSUD. On the other hand, the conventional line analyses by

Evaporation to dryness AA, GC, GCIMS, HPLC, or LCIMS characterize groups

1 of related compounds. PLA, PPA, and PAA are accurately Trimethylsilylation with 100 pl reagent measured si~nultaneously by GCIMS in the examination of

I PKU. For MSUD, 2-hydroxyisocaproic acid, 2-hydroxy-3- v

GCIMS analysis

Scheme 1. Flow chart of sample preparation

preparation includes urease treatment, alcohol deproteinization, evaporation to dryness, and trimethylsilylation.

Neonate urine specimens absorbed into filter paper, dried with air, and sent to the institutes, were eluted with water. The eluate (0.1 mL) was incubated with urease for 10 min to remove urea. After addition of d,-creatinine (100 nmol), deuterium-labeled amino acids, d,-glycine (20

methylvaleric acid, and 2-hydroxyisovaleric acid are measured by GCIMS.

Our new GCIMS chemical diagnostic method (22- 25) can simultaneously analyze amino acids, organic acids, sugars, sugar alcohols, and nucleic acid bases, and thus many new as well as different kinds of metabolic disorders can be chemically diagnosed. This "planar" analysis (26) system is a more comprehensive diagnostic method than point or line analyses, because the entire image can be evaluated like an X-ray film. The details of this new method and its features are described below.

nmol), d3-leucine (20 nmol), d,-methionine (20 nmol), 4- D. Diagnostic Method by Mars phenylalanine (20 nmol), d,-tyrosine (20 nmol), d,-homo- Chromatography (MC) Used in the Project cystine (40 nmol). heptadecanoic acid, and 3-hydroxymyr- Study istic acid, deproteinization with ethanol (final 90%), centrifugation and removal of the precipitate, and evaporation to dryness, the residue was trimethylsilylated with 0.1 mL of BSTFA and TMCS (10:l) for 30 min at 80°C.

One to two yL of the derivatized extract were injected into the GCIMS by an automatic injection system. The components were separated on a 0.25 mm inner diameter, 30 m long, 5% phenylmethylsilicone capillary column with a film thickness of 0.25 ym. The oven temperature program was started at 60°C and increased by 17"Ctmin up to 325"C, where it was held for about 9 min, during which time the data were processed and the results reported. Injection port and transfer line temperatures were 260°C and 280°C, respectively. The split injection mode was used with a split ratio of 1:40. The flow rate of the helium carrier gas was approximately 1.1 mLlmin, and its linear velocity was 38.5 crn/s.

Creatine converts to creatinine under this pretreatment condition, but the total creatinine measurement has been found to be useful for neonatal mass screening, as shown in Fig. 1. Amino acid determination, using stable-isotope- labeled amino acids as their internal standards, gave good quantitative data, as previously reported by Schulman and Abramson (21).

In our study, 2-3 ions that are unique and characteristic are selected for each metabolite that might be excreted into urine due to the metabolic disorders listed in Table 2.

Abnormal metabolites are examined and quantified by mass chromatography (MC) for chemical diagnosis. Figures 2 and 3 show a case of PKU. The upper panel in Fig. 2 shows the RIC chromatogram of the urinary metabolites that are obtained by the new "planar" chemical diagnostic method using urease treatment, and the lower panel shows that obtained by conventional organic solvent extraction. By both methods, ihe marked peaks of PPA, PLA, and PAA that cannot be detected by Guthrie's point method are revealed. In addition, Phe, which is not detected by the conventional method, is conveniently detected by the new chemical diagnostic method. In this way, an accurate chemical diagnosis of PKU can be made.

In methylmalonic aciduria (MMA), which is caused by the abnormally reduced activity of methylmalonyl-CoA mutase and is the most frequently observed disorder among the organic acidemias, methylmalonic acid and methylcitric acid are detected as marked peaks, as shown in Fig. 4. In propionyl-CoA carboxylase deficiency (PCC; propionic acidemia), 3-hydroxypropionic acid, methylcit-

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Mass spectrometric method (mglrnl)

FIGURE 1. Urinary creatinine determination by GCiMS and by enzyme assay. Urine specimens were obtained o n day 5 after birth.

ric acid, propionylglycine, and a series of metabolites that are characteristic of propionic acidemia are detected (Fig. 5). Unlike the Millington's method (4, 27) or Rashed's method (5, 6) discussed below, this method allows accurate chemical differentiation (differential diagnosis) between MMA and PPC by a single analysis. In addition, patients with PCC during their remission exhibit markedly increased glycine, and this increase or decrease can be easily detected by the same procedure (Fig. 5). Figure 6 shows a case of isovaleric acidemia.

Urea cycle disorders, except N-acetylglutamate synthase deficiency and carbamoylphosphate synthase deficiency, can be screened by this method, because uracil and orotate are recovered efficiently. Which compound is deficient in either ornithine carbamoyl transferase, argininosuccinate synthase, argininosuccinate lyase, or arginase is not always distinguished clearly by this method alone. If arginine, which cannot be quantified by the present method, is found to be increased, then arginase deficiency is suspected. If citrulline is found to be distinctly increased, then argininosuccinate synthase deficiency is suspected. If argininosuccinate is found to be increased by a direct analysis of urine with FAB/MS, then argininosuccinate lyase deficiency is suspected. If neither arginine, argininosucci-

nate, nor citrulline is increased, then the ornithine carbamoyl transferase deficiency is suspected. Figure 7 shows a RIC chromatogram and a mass chromatogram of urinary metabolites from a patient with ornithine carbamoyl transferase deficiency.

E. Three Alternative Chemical Diagnostic Methods Using a Mass Spectrometer

Conventional chemical diagnosis using GUMS has two major disadvantages for mass screening: speed and limited analytical range. When an analytical apparaius is used for mass screening, the analytical time must be as short as possible. However, in the conventional GCIMS methods, sample preparation requires 4- 10 h (solvent extraction, DEAE-sephadex extraction) and the GCMS analysis required about 50-60 min. Further, the results are limited to the line analyses of single categories of compounds such as organic acids or amino acids.

The first problem, "speed", was solved by Prof. Mil- lington at Duke University (Millington's method) (27) in the U.S. This method does not use GCIMS, and acylcarnitines or amino acids are automatically extracted from blood filter paper and ionized by fast atom bombardment

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TABLE 2. Target compounds for twenty-two diseases.

Disease Target compounds

Methylmalonic aciduria

Propionic acidemia

Isovaleric acidemia

Maple syrup urine disease

3-Ketothiolase deficiency

Galactosemia

Phenylketonuria

Hyperphenylalaninemia Homocystinuria

Alkaptonuria Multiple carboxylase deficiency

Hyperglycinemia Lysinuria Cystinuria

Tyrosinemia

Methylmalonic acid Methylcitric acid Methylcitric acid Propionylglycine Tiglylglycine 3-Hydroxy-n-valeric acid 3-Hydroxypropionic acid 2-Methyl-3-hydroxyvaleric acid Isovalerylglycine 3-Hydroxyisovaleric acid Leucine Isoleucine Valine 2-Hydroxyisocaproic acid 2-Hydroxy-3-methylvaleric acid 2-Hydroxyisovaleric acid 2-Methyl-3-hydroxybutyric acid 2-Methylacetoacetic acid Tiglyglycine Galactose Galactitol Galactonic acid Phenylalanine Phenyllactic acid 2-Hydroxyphenylacetic acid Phenylpyruvic acid Phenylalanine Homocysteine Methionine Homocystine Homogentisic acid 3-Methylcrotonylglycine Methylcitric acid 3-Hydroxyisovaleric acid Glycine Lysine Cystine Lysine Omithine Tyrosine 4-Hydroxyphenyllactic acid Succinylacetone 4-Hydroxyphenylpyruvic acid 4-Hydroxyp:~enylacetic acid

(FAB). Homologous series are analyzed by a precursor scan using a tandem mass spectrometer (MSIMS). Milling- ton's method is completely controlled by a computer, and the analytical time per specimen is 2.5 min. This approach is truly revolutionary relative to conventional chemical diagnosis by GC/MS (14, 15). Following the success of FAB, newer soft ionization methods were developed, allowing the direct analysis of aqueous solutions. Dr.

Rashed in Saudi Arabia developed a chemical diagnostic method in which homologous series are analyzed by a precursor scan, using ion spray (IS) ionization MS/MS (Rashed's method) (5,6). In Rashed's method, the analytical time per specimen was further shortened to 1 min. Recently, more rapid and accurate methods (28, 29) were developed, and the use of MS/MS for mass screening would be an efficient method with reasonable cost (30).


TABLE 2. Corltinued.

Glutaric aciduria type I

3-Hydroxy-3-n~ethylglutaryl-CoA lyase deficiency

3-Methylcrotonylglycinuria

2-Ketoadipic 2-aminoadipic aciduria

Urea cycle disorders

Glutaric aciduria type I1

Neuroblastoma

Glutaric acid 3-Hydroxyglutaric acid Glutaconic acid 3-Hydroxy-3-rnethylglutaric

acid 3-Methylglutaconic acid 3-Hydroxyisovaleric acid 3-Hydroxyisovaleric acid 3-Methylcrotonylglycine 2-Oxoadipic acid 2-Hydroxy adipic acid 2-Aminoadipic acid Orotic acid Uracil Glutaric acid Ethylmalonic acid Adipic acid Suberic acid 2-Hydroxyglutaric acid Homovanillic acid Vanillylmandelic acid

FIGURE 2. RIC chromatograms of TMS derivatives of urinary metabolites from a patient with phenylketonuria (PKU) (upper: new urease treatment; lower: conventional diethyl-ether extraction). ( I ) alanine, (2) glycine, (3) phenylacetic, (4) serine, (5) threonine, (6) erythritol, (7) pyroglutamic, (8) creatinine, (9) 2- hydroxyphenylacetic, (10) phenyllactic, (1 1) phenylalanine, (12) 4-hydroxyphenylacetic, (13) phenylpyruvic, (14) arabitol, (15) 4-hydroxyphenyllactic, (16) manitol, (17) glucose, (18) 3-hydroxymyristic (I.S.), (19) 4-hydroxyphenylpyruvic, (20) heptadecanoic (I.S.).

FIGURE 4. RIC chromatogram (upper) and mass chromatogram (lower) of the TMS derivatives of urinary metabolites from a patient with methylmalonic acidemia.


IS

A , &aA ,.. TIC

220.00 2.00 193.00 2.00 308.00 10.00

327.00 10.00 6 7 8 9 10 11 12 13

FIGURE 3. RIC chromatogram (upper) and mass chromatogram (lower) of the TMS derivatives of urinary metabolites from a patient with phenylketonuria.

However, these methods are limited to narrow classes of ate between MMA and PCC, two of the most common analytes compared with our chemical diagnostic method organic acidemias. by G C N S (Matsumoto's method) (23, 26, 31). Their In 1991, Shoemaker et al. at St. Louis University in methods permit high speed analyses, but do not differenti- the U.S. reported that organic acids and sugars can be


FIGURE 5. RIC chromatogram (upper) and mass chromatogram (lower) of the the TMS derivatives of urinary metabolites from a patient with propionic acidemia.

separated clearly by GCIMS after excessive urea in the Cho (33) at our laboratory simplified this method, and urine was degraded with urease and removed (32). Shoe- extended the variety of analyzable compounds. As a result, maker's procedure, however, takes several hours, needs a the simultaneous analysis of amino acids, nucleic acid skillful hand, and is not practical. M. Matsumoto and Z. bases, nucleoside, sugars, and sugar alcohols in addition

FIGURE 6. RIC chromatogram (upper) and mass chromatogram (lower) of the TMS derivatives of urinary metabolites from a patient with isovaleric acidemia.


FIGURE 7. RIC chromatogram and mass chromatogram of the TMS derivatives of urinary metabolites from a patient with ornithine carba~noyl transferase deficiency.

to organic acids became possible. We further improved the procedure with a potential for neonatal mass screening. This new procedure takes one h for the pretreatment of one sample, or three h for a batch of 30 samples, as described in IV. B "Sample Preparation" in this article. In mass screening, many specimens should be treated; a fully automatic pretreatment system is almost completed by a research group of Yokogawa Analytical Systems, Inc.

Under the Japanese Society for Biomedical Mass Spectrometry, the Committee for the GCIMS Analytical Conditions for Clinical Mass Spectrometry was organized (including the GCIMS specialists from three companies, Shimadzu Seisakusho Ltd., JEOL Ltd., and Yokogawa Analytical Systems Inc.) for evaluating and improving the GCIMS analytical conditions (22). This Committee selected, as the preferred GC column, a DB-5, 30 m X 0.25 mm, 0.25-pm film thickness column to achieve satisfac- tory separation within 15 min. A 15-min analytical time per sample is feasible for mass screening. We are trying to further shorten the GCIMS measurement interval to 15- 20 min by using a column with a thinner film thickness and less diameter. When the entire analytical process is

I fully automated with an appropriate diagnostic program in the near future, 80 samples would be analyzed in a day per one GCIMS instrument, and about 20,000 samples annually, based on 250 work-days a year.

Our method is slower than either Rashed's or Milling- ton's method, but is comprehensive in terms of diagnosis rather than screening. The present method allows, in most cases, the definite chemical diagnoses of an IEM. There- fore, the total time needed before a patient is diagnosed using our method may not be longer than that using the other methods. For example, in the case of MSUD, this method enables not only the determination of a branched chain amino acid (leucine, valine, or isoleucine), but also those of the corresponding branched chain a-hydroxy acids. In addition, whether the metabolites formed after the oxidative decarboxylation of the branched chain u- 0x0 acids are increased or not can be examined at the

same time. For PKU, phenylalanine, phenylacetate, phe- nylpyruvate, and o-hydroxyphenylacetate are simultaneously measured. For galactosemia, galactose, galactitol, and galactonate are the target compounds. Figure 8 shows the RIC chromatogram of the TMS derivatives of urinary metabolites obtained from a patient with galactose-l-phosphate uridyl transferase (GALT) deficiency (upper). The increase in galactose was not observed because the urine was collected on the third day after treatment. However, galactitol and galactonate remained abnormally increased. It is known that this condition is accompanied by generalized amino aciduria and secondary tyrosinemia. The distinctly increased excretions of a number of amino acids, and p-hydroxyphenyllactate, phenyllactate, and p-hydro- xyphenylpyruvate were noticed from this chromatogram. In this new method, the number of diseases that can be diagnosed will be increased, because a large number of compounds from different categories are analyzed.

Combined cytochrome (aa,, b) deficiency, so-called severe infantile cytochrome deficiency, is not targeted in mass screening, and may not be appropriate because effective treatment is not available. This disease can also be screened with our method (34). It presents severe, chronic lactic aciduria, mild but persistent ketonuria, increased excretion of malate and fumate, glucosuria, phosphaturia, and generalized amino aciduria, becausre, not only in mus- cle but also in kidney, the electron transport system is affected (Fig. 9). For fructose- 1, 6-diphosphatase deficiency, the chemical diagnosis can be made from the increased amount of lactate, glycerol, and glycerol-3-phosphate, although it is limited to the urine sample that is collected during a hypoglycemic attack. For adenine phosphoribosyltransferase deficiency, the marked increase of adenine can be observed (35). ZHydroxyadenine is detect- able and 2, 8-dihydroxyadenine is found in trace amount (Fig. 10).

The present method can also be applied for the study of sugar metabolism. In diabetes mellitus, glucose, sorbi- tol, gluconic acid, and other important markers for the

admin

Underline

admin

Underline

IP

glycine

m -

phosphate

m - - 2-deoxytetronate

erythritol

erythronate creatinine

alanine - - glycine 2-hydroxybutyrate

m - 3-hydroxybutyrate

r - valine

leucine+phosphate

proline

(T) - threonine

pyroglutamate

- 0 -

t a t 1 glucose

glucose


adenine 65954401

FIGURE 10. Mass chromatogram of the TMS derivatives of urinary metabolites from a patient with adenine phosphoribosyl-transferase deficiency.

diagnosis and for the evaluation of treatments can be targeted. We think that this method has diverse application potentials for mass screening not only of infants but also of adults.

V. CONCLUSIONS

We believe that this method, technically practical and still comprehensive from the metabolic point of view, will be- come an important method in mass screening as well as in monitoring for various disorders of all age groups rang- ing from the neonate to the elderly. This method will be useful for screening not only endogenous but also exoge- nous compounds from humans and animals.

ACKNOWLEDGMENTS

This study was supported in part by the JAMW Ogyaa Donation Foundation and General C, Grant-in-Aid Scien- tific Research of Ministry of Education 07672503, 08672652. We express our deep gratitude to the staff in the project research group, the staff at our laboratory, those persons in charge at the apparatus manufacturers, and Miki Osabe for the preparation of this manuscript.

REFERENCES

1. Scriver, C. R.; Rosenberg, L. E. Amino Acid Metabolism and its Disorders, Vol. X irt the Series of Major Problems in Clir~icczl Pediatrics, W. B . Saunders: Philadelphia, 1987, 290-337.

The 23rd meeting of the Japanese Society for Mass Screening was held in Osaka on September I and 2, 1995, and the 20th annual meeting of the Japanese Society for Biomedical Mass Spectrometry in Nagoya on September 28-30, 1995. In the apparatus exhibition room of these meet- ings, a demonstration of the "chemical diagnosis of metabolic disorders by simultaneous GCIMS analysis of urinary metabolites" was performed jointly by three institutions (Kanazawa Medical University, Kurume Uni- versity Medical School, and Shimane Medical University), in cooperation with Shimadzu Seisakusho Ltd., JEOL Ltd., and Yokogawa Analytical Systems Inc. Urine samples from neonates with metabolic disorders were examined in the demonstration on September 1 and 2, and those from adults with diseases such as diabetes mellitus and renal diseases in addi- . tion to metabolic disorders were examined in the demonstration on Sep- tember 28-30. A notice of this demonstration had been given in medical journals several months earlier, and urine samples from patients with suspected metabolic disorders had been sent to Kanazawa Medical Uni- versity. On the day of the demonstration, samples that had been pretreated for GC/MS analysis were injected into one of the three GClMS systems that were developed by the above companies. After GClMS analysis. chemical diagnosis was made using the diagnostic program, and reports were automatically printed out. This procedure was an unprecedented demonstration of the chemical diagnostic system for mass screening.

2. Guthrie, R. Newborn Screening: past, present and future. In Generic Disease: Screening and Management; Carter, T . P . ; Wiley, A. M., Eds.; Alan R. Liss: New York, 1986, 319- 339.

3. Guthrie, R.; Susi A. "A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants," Pediatr. 1963, 32, 338-343.

4. Chace D. H.; Millington, D. S.; Terada, h*.; Kahler, S. G.; Roe C. R.; Hofman, L. F. "Rapid diagnosis of phenylketonuria by quantitative analysis for phenylalanine and tyrosine in neonatal blood spots by tandem mass spectrometry," Clin. Cheni. 1993, 39, 66-7 1 .

5. Rashed, M. S.; Ozand, P. T.; Harrison, M. E.; Watkins, P. J. F.; Evans, S. "Electrospray tandem mass spectrometry in the diagnosis of organic acidemias," Rapid Commun. Mass Spectrorn. 1994, 8, 129- 133.

6. Rashed, M. S.; Ozand, P. T.; Bucknall, M. P.; Little, D. "Di- agnosis of inborn errors of metabolism from blood spots by acylcarnitines and amino acids profiling using automated electrospray tandem mass spectrometry," Pediatr. Res. 1995738, 324-331.

admin

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MS DIAGNOSIS OF INBORN ERRORS OF METABOLISM w

7. Beutler, E.; Baluda, M. C. "A simple spot screening procedure for galactosemia," Lab. Clin. Med. 1966, 68, 137- 141.

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I sterdam, 1987. 1 10. Ettre, L. S.; Zlatkis, A., Eds. The Practice of Gas Chroma-

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highly efficient carboxylic acid analyser and its application," J. Chromatogr. 1976, 123, 129.

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13. Spackman, D. H.; Stein, W. H.; Moore, S. "Automatic re- cording apparatus for use in the chromatography of amino acids," Anal. Chem. 1958, 30, 1190.

14. Goodman, S. I.; Markey, S. P. Diagnosis of Organic Acide- mias by Gas Chronlatograplzy-Mass Spectrometry; Alan R. Liss: New York, 1981.

15. Charlmers, R. A.; Lawson, A. M. Organic Acids in Man; Chapman and Hall: London, 1982.

16. Matsumoto, I.; Kuhara, T. "Gas chromatography-mass spectrometry for chemical diagnosis of the inherited metabolic diseases-differential chemical diagnosis of lactic aci- dosis," Mass Spectronz. Rev. 1987, 6, 77-134.

17. Matsumoto, I.; Kuhara, T. Inborn Errors of Amino Acid and Organic Acid Metabolism. In Mass Spectrometry; Clinical and Biomedical Applications, Vol. I; Desiderio, D. M., Ed.; Plenum Press: New York, 1992, 259-298.

18. Tanaka, K.; Budd, M. A.; Efron, M. L.; Isselbacher, K. J. "Isovaleric acidemia: A new genetic defect of leucine metabolism," Proc. Natl. Acad. Sci. U.S.A. 1966, 56, 236- 242.

19. Tuchman, M.; McCann, M. T.; Johnson, P. E.; Lemieux, B. "Screening newborns for multiple organic acidurias in dried filter paper urine samples: Method development," Pediatr-. Res. 1991, 30, 3- 16.

20. Chamberlin, B. A.; Sweeley, C. C. "Metabolic profiles of urinary organic acids recovered from absorbent filter paper," Clin. Chem. 1987, 3314, 572-576.

21. Schulman, M. F.; Abramson, F. P. "Plasma amino acid analysis by isotope ratio gas chromatography mass spectrometry computer techniques," Bionzed. Mass Spectrom. 1975, 2, 9- 14.

22. Kuhara, T. In GCIMS Practical Chenlical Diagnosis; Matsu- moto, I.; Sakamoto, S.; Kuhara, T.; Sudo, M.; Yoshino, M., Eds.; Soft Science: Tokyo, 1995, 33 (in Japanese).

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graphic mass spectrometric analysis of urinary metabolites-application to the neonatal mass screening," Proc. Jay. Soc. Bion~ed. Mass Spectrom. 1995, 20, 45-51.

24. Kuhara, T.; Matsumoto, I. "Simple procedure for simultaneous metabolites analysis by mass spectrometry-Applica- tion to neonatal mass screening," Advanced Medicine 1995, 2, 37-40 (in Japanese).

25. Kuhara, T.; Ning, C.; Furumoto, T.; Zhang, C.; Matsumoto, M.; Inoue, Y.; Shinka, T.; Matsumoto, I. "Application of chemical diagnostic procedure using gas chromatography- mass spectrometry to neonatal mass screening for inborn errors of metabolism," J. Kanazawa Med. Univ. 1995, 20, 521 -526 (in Japanese).

26. Matsumoto, I. "Point, linear and planar analysis for chemical diagnosis," Jap. Soc. Biomed. Mass Spectrom. Circular 1994, 32, 7-8 (in Japanese).

27. Millington, D. S.; Kodo, N.; Norwood, D. L.; Roe, C. R. "Tandem mass spectrometry: A new method for acylcarnitine profiling with potential for neonatal screening for inborn errors of metabolism," J. Inherit Metah. Dis. 1990,13, 321 - 324.

28. Chace, D. H.; Hillman, S. L.; Millington, D. S.; Kahler, S. G.; Roe, C. R.; Naylor, E. W. "Rapid diagnosis of maple syrup urine disease in blood spots from newborns by tandem mass spectrometry," Clin. Chenz. 1995, 41, 62-68.

29. Chace, D. H.; Hillman, S. L.; Millington, D. S.; Kahler, S. G.; Adam, B. W.; Levy, H. L. "Rapid diagnosis of homocystinuria and other hypermethioninemias from newborns' blood spots by tandem mass spectrometry," Clin. Chem. 1996, 42, 349-355.

30. Sweetman, L. "Newborn screening by tandem mass spectrometry (MS-MS)," Clin. Chem. 1996, 42, 345-346.

31. Kuhara, T.; Shinka, T.; Inoue, Y.; Matsumoto, M.; Ning, C.; Zhang, C.; Furumoto, T.; Matsumoto, I. "Application of a new analytical procedure using GC/MS to the mass screening of metabolic disorders-Simultaneous urinary metabolites analysis," Proc. Jap. Soc. Biomed. Mass Spec- trom. 1996, 21, 73-82.

32. Shoemaker, J. D.; Elliott, W. H. "Automated screening of urine samples for carbohydrates, organic and amino acids after treatment with urease," J . Chromatogr. 1991, 562, 125-138.

33. Matsumoto, M.; Zhang, C.; Shinka, T.; Inoue, Y.; Furumoto, T.; Kuhara, T.; Matsumoto, I. "The chemical diagnosis of the metabolic disorders 1. Chemical diagnosis of propionic acidemia,"J. Kanazawa Med. Univ. 1994, 19, 213-219.

34. Ning, C.; Kuhara, T.; Matsumoto, I. "Simultaneous metabolic profile studies of three patients with fatal infantile mitochondria1 myopathy-de toni-fanconi-debre syndrome by GC/MS," Clin. Chim. Acta 1996,247, 197-200.

35. Suzuki, K.; Kobayashi, S.; Kawamura, K.; Kuhara, T.; Tsu- gawa, R. "A family study of 2,8-dihydroxyadenine stone- Report of two cases of a compound heterozygote for adenine phosphoribosyltransferase deficiency (APRT*J/APRT*QO)," Int. J . Urol., to appear.

Kanazawa Medical University

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a new chemical diagnostic method for inborn errors …

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