Pilot study of gas chromatographic–mass spectrometric screening of newborn urine for inborn errors of metabolism after treatment with urease

Gas chromatographic–mass spectrometric (GC–MS) techniques for urinary organic acid profiling have been applied to high-risk screening for a wide range of diseases, mainly for inborn errors of metabolism (IEM), rather than to low-risk screening or mass screening. Using a simplified procedure with urease-pretreatment and the GC–MS technique, which allows simultaneous determination of organic acids, amino acids, sugars and sugar acids, we performed a pilot study of the application of this procedure to neonatal urine screening for 22 IEM. Out of 16 246 newborns screened, 11 cases of metabolic disorders were chemically diagnosed: two each of methylmalonic aciduria and glyceroluria, four of cystinuria, and one each of Hartnup disease, citrullinemia and α-aminoadipic aciduria/α-ketoadipic aciduria. The incidence of IEM was thus one per 1477, which was higher than the one per 3000 obtained in the USA in a study targeting amino acids and acylcarnitines in newborn blood spots by tandem mass spectrometry. Also, 227 cases were found to have transient metabolic abnormalities: 108 cases with neonatal tyrosinuria, 99 cases with neonatal galactosuria, and 20 cases with other transient metabolic disorders. Two hundred and thirty-eight cases out of 16 246 neonates (approximately 1/68) were thus diagnosed using this procedure as having either persistent or transient metabolic abnormalities.     

Diagnosis of inborn errors of metabolism using filter paper urine,urease treatment,isotope dilution and gas chromatography–mass spectrometry

This review will be concerned primarily with a practical yet comprehensive diagnostic procedure for the diagnosis or even mass screening of a variety of metabolic disorders. This rapid, highly sensitive procedure offers possibilities for clinical chemistry laboratories to extend their diagnostic capacity to new areas of metabolic disorders. The diagnostic procedure consists of the use of urine or filter paper urine, preincubation of urine with urease, stable isotope dilution, and gas chromatography–mass spectrometry. Sample preparation from urine or filter paper urine, creatinine determination, stable isotope-labeled compounds used, and GC–MS measurement conditions are described. Not only organic acids or polar ones but also amino acids, sugars, polyols, purines, pyrimidines and other compounds are simultaneously analyzed and quantified. In this review, a pilot study for screening of 22 target diseases in newborns we are conducting in Japan is described. A neonate with presymptomatic propionic acidemia was detected among 10,000 neonates in the pilot study. The metabolic profiles of patients with ornithine carbamoyl transferase deficiency, fructose-1,6-bisphosphatase deficiency or succinic semialdehyde dehydrogenase deficiency obtained by this method are presented as examples. They were compared to those obtained by the conventional solvent extraction methods or by the tandem mass spectrometric method currently done with dried filter blood spots. The highly sensitive, specific and comprehensive features of our procedure are also demonstrated by its use in establishing the chemical diagnosis of pyrimidine degradation defects in order to prevent side effects of pyrimidine analogs such as 5-flurouracil, and the differential diagnosis of three types of homocystinuria, orotic aciduria, uraciluria and other urea cycle disorders. Evaluation of the effects of liver transplantation or nutritional conditions such as folate deficiency in patients with inborn errors of metabolism is also described.     

Noninvasive human metabolome analysis for differential diagnosis of inborn errors of metabolism

Early diagnosis and treatment are critical for patients with inborn errors of metabolism (IEMs). For most IEMs, the clinical presentations are variable and nonspecific, and routine laboratory tests do not indicate the etiology of the disease. A diagnostic procedure using highly sensitive gas chromatography-mass spectrometric urine metabolome analysis is useful for screening and chemical diagnosis of IEM. Metabolite analysis can comprehensively detect enzyme dysfunction caused by a variety of abnormalities. The mutations may be uncommon or unknown. The lack of coenzymes or activators and the presence of post-translational modification defects and subcellular localization abnormalities are also reflected in the metabolome. This noninvasive and feasible urine metabolome analysis, which uses urease-pretreatment, partial adoption of stable isotope dilution, and GC/MS, can be used to detect more than 130 metabolic disorders. It can also detect an acquired abnormal metabolic profile. The metabolic profiles for two cases of non-inherited phenylketonuria are shown. In this review, chemical diagnoses of hyperphenylalaninemia, phenylketonuria, hyperprolinemia, and lactic acidemia, and the differential diagnosis of beta-ureidopropionase deficiency and primary hyperammonemias including ornithine transcarbamylase deficiency and carbamoylphosphate synthetase deficiency are described.     

Identification and quantitation of urinary dicarboxylic acids as their dicyclohexyl esters in disease states by gas chromatography mass spectrometry

Clinical studies were conducted by gas chromatography mass spectrometry selected ion monitoring of urinary dicarboxylic acids as dicyclohexyl esters. The dicyclohexyl esters of the dicarboxylic acids give characteristic electron impact mass spectra suitable for selected ion monitoring. The mass spectra exhibit a prominent acid + 1H ion and an (acid + 1H)-H2O ion for use as quantitating and confirming ions. The cyclohexyl esters are stable for days at room temperature and have excellent chromatographic properties. Dicarboxylic acid quantitation is performed within one hour using only 50 microliter of unpurified urine. A rapid method specifically for methylmalonic acid quantitation is described which has assisted physicians in the diagnosis of pernicious anemia and methylmalonic aciduria. This procedure is applicable for screening urinary organic acids for detection of inborn errors of metabolism. The detection of a child with elevated medium length dicarboxylic acids in the terminal urine specimen is reported. This condition, previously described as an inborn error, is attributed to a terminal event. Finally, an increase in urinary succinic acid paralleling putrescine levels is described during a response to cancer chemotherapy.     

Rapid and sensitive method for prenatal diagnosis of propionic acidemia using stable isotope dilution gas chromatography-mass spectrometry and urease pretreatment

Propionic acidemia is one of the most frequent inborn errors of metabolism caused by a deficiency of propionyl-CoA carboxylase. Methylcitric acid, a key indicator of this disorder, is increased in amniotic fluid when a fetus is affected. Therefore, the direct chemical analysis of cell-free amniotic fluid for methylcitric acid, using stable isotope dilution gas chromatography-mass spectrometry, was carried out for the prenatal diagnosis of propionic acidemia. We developed a simple, highly sensitive, and accurate method for quantitation of this polar methylcitric acid in amniotic fluids by applying a simplified urease pretreatment which we devised earlier for urine. As the recovery of methylcitric acid from amniotic fluid was as high as 91% with a coefficient of variation lower than 3% in this procedure, only 0.02 ml of sample was required for the analysis of the affected fetus. This new procedure takes 1 h for sample pretreatment, including derivatization, and 15 min for GC-MS measurement and provides final results within 1.5 h.     

Separation of free amino acids in human plasma by capillary electrophoresis with laser induced fluorescence: potential for emergency diagnosis of inborn errors of metabolism

Free amino acids (AAs) in human plasma are derivatized with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) and analyzed by capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The labeling procedure is significantly improved over results reported previously. Derivatization can be completed in 40 min, with concentrations as low as 4×10⁻⁸ M successfully labeled in favourable cases. Twenty-nine AAs (including 2 internal standards) are identified and can be reproducibly separated in 70 min. Migration time RSD values for 23 of these AAs were calculated and found in the range from 0.5 to 4%. The rapid derivatization procedure and the resolution obtained in the separation are sufficient for a semi-quantitative, emergency diagnosis of several inborn errors of metabolism (IEM). Amino acid profiles for both normal donor plasma samples and plasma samples of patients suffering from phenylketonuria, tyrosinemia, maple syrup urinary disease, hyperornithinemia, and citrullinemia are studied.     

Simplified method for the chemical diagnosis of organic aciduria using GC/MS

GC/MS is widely used for the analysis of urinary organic acids for the chemical diagnosis of organic acidurias such as methylmalonic acidemia, propionic acidemia, isovaleric acidemia, glutaric aciduria type I, and multiple carboxylase deficiency. In this study, a rapid and simple preparation method for this analysis was developed in order to improve the laboratory productivity and the working environment. The solvent extraction and trimethylsilyl derivatization steps of the conventional method were improved by reducing the volume of urine sample and extraction solvent and by applying the flash-heater derivatization, respectively. The new method was successfully applied to the chemical diagnoses of five organic acidurias.     

Diagnosis and follow-up of inborn errors of amino acid metabolism: Use of proton magnetic resonance of biological fluids

Summary Proton magnetic resonance spectra of biological fluids such as urine, plasma and cerebro-spinal fluid can be used for multi-component analysis of highly concentrated species, thus providing information about the general metabolism of the patient. Hydrogen containing analytes in concentration higher than 10µM are indeed often detectable in biological fluid in 15 minutes by means of an unexpensive 200 MHz spectrometer essentially without sample manipulation. Amino acids, keton bodies, organic acids and other metabolites can be easily estimated by this approach; consequently this technique represents a powerful tool particularly in the diagnosis of inborn errors of amino acid metabolism, when improving the prognosis often depends on a very early diagnosis and on an effective method for monitoring the effects of therapy.In the present paper, several cases of inherited diseases related to amino acid impaired metabolism will be presented to illustrate the importance in the diagnosis. Phenylketonuria, tyrosinemia, cystinuria, ornithinemia, argininosuccinic aciduria, maple syrup urine disease (MSUD), alkaptonuria, lysinuria and other genetic pathologies were in fact unambiguously and rapidly diagnosed by means of the identification in the biological fluids of the relevant accumulating amino acids and/or of their metabolites. The proposed technique is suitable to become, in the future, a useful routine tool for a wide neonatal screening.     

Biomarker discovery, disease classification, and similarity query processing on high-throughput MS/MS data of inborn errors of metabolism

In newborn errors of metabolism, biomarkers are urgently needed for disease screening, diagnosis, and monitoring of therapeutic interventions. This article describes a 2-step approach to discover metabolic markers, which involves (1) the identification of marker candidates and (2) the prioritization of them based on expert knowledge of disease metabolism. For step 1, the authors developed a new algorithm, the biomarker identifier (BMI), to identify markers from quantified diseased versus normal tandem mass spectrometry data sets. BMI produces a ranked list of marker candidates and discards irrelevant metabolites based on a quality measure, taking into account the discriminatory performance, discriminatory space, and variance of metabolites' concentrations at the state of disease. To determine the ability of identified markers to classify subjects, the authors compared the discriminatory performance of several machine-learning paradigms and described a retrieval technique that searches and classifies abnormal metabolic profiles from a screening database. Seven inborn errors of metabolism-- phenylketonuria (PKU), glutaric acidemia type I (GA-I), 3-methylcrotonylglycinemia deficiency (3-MCCD), methylmalonic acidemia (MMA), propionic acidemia (PA), medium-chain acylCoAdehydrogenase deficiency (MCADD), and 3-OH long-chain acyl CoA dehydrogenase deficiency (LCHADD)-were investigated. All primarily prioritized marker candidates could be confirmed by literature. Some novel secondary candidates were identified (i.e., C16:1 and C4DC for PKU, C4DC for GA-I, and C18:1 forMCADD), which require further validation to confirm their biochemical role during health and disease.     

Sample preparation with an automated robotic workstation for organic acid analysis by gas chromatography-mass spectrometry

We attempted to automate sample preparation for analysis of organic acids by gas chromatography-mass spectrometry using a computer-controlled, automated robotic workstation that is integrated and connected to the gas chromatography-mass spectrometry (HP-5890/5971) system. Of the two methods developed, one employed solvent extraction, while the other utilized a silica, solid-phase extraction cartridge. Both automated methods were compared to a manual, solvent extraction procedure used routinely in our laboratory. Normal, spiked urine, and urine from patients with a variety of metabolic abnormalities were analyzed. The robotic workstation did not meet all our requirements for a rapid, reliable, laboratory device. Recoveries with the automated procedure were less than with the manual method, and some organic acids important in the diagnosis of inborn errors of metabolism were not detected. Additionally, the robotic device had mechanical and design problems that made it slower and less reliable than the manual procedure.     

Prenatal diagnosis of propionic acidemia by measuring methylcitric acid in dried amniotic fluid on filter paper using GC/MS

Propionic acidemia is a frequent inborn error of metabolism. Methylcitric acid, a key indicator of propionic acidemia, increases in the amniotic fluid of affected fetuses. For prenatal diagnosis, the methylcitric acid in amniotic fluid can be measured by stable-isotope dilution GC/MS. Here, we quantified this indicator in samples of amniotic fluid that had been dried on filter paper and transported at ambient temperatures, and compared the results with data obtained from the original amniotic fluid. We then used the filter-paper method to screen at-risk fetuses and obtained a clear-cut diagnosis in each case.     

Molecular basis of enzyme abnormalities in urea cycle disorders. With special reference to citrullinemia and argininosuccinic aciduria

This paper deals with enzymological, immunochemical and molecular genetic analyses of citrullinemia and argininosuccinic aciduria. Citrullinemia has been classified by Saheki et al. [J. inher. Metab. Dis. 8: 155-156, 1985] into three types from the properties of the deficient argininosuccinate synthetase (ASS) of the patients. Analysis of hepatic mRNA coding for ASS revealed certain characteristics in type II and III citrullinemic patients whose hepatic ASS protein was low. A newly developed enzyme-linked immunosorbent assay (ELISA) of argininosuccinate lyase (ASL) protein showed that 8 out of ten cases of argininosuccinic aciduria had no detectable ASL protein in the liver, erythrocytes, cultured skin fibroblasts or cultured amniocytes.     

Biochemical Genetics and Mental Retardation

The known cases of mental retardation which exhibit a genetically determined biochemical lesion were reviewed. Twenty-two inborn errors of metabolism with associated mental defect have been described to date, 12 of these within the past decade. Improved procedures for diagnosis and therapy make this area of investigation a promising one for clinicians, biochemists and geneticists. During a screening program for amino aciduria, a “new” metabolic defect, citrullinuria, was detected in a mentally retarded child. This condition is characterized by the presence of citrulline in the urine, blood and cerebrospinal fluid in concentrations 50- to 100-fold greater than normal. Although the amounts of citrulline excreted appear to be related to the protein intake, it was not possible to reduce the high citrulline concentration in the blood by dietary restrictions.     

Hydroxyl negative chemical ionization mass spectrometry linked with collisionally activated decomposition. A modern analytical tool in inborn errors of metabolism

Two kinds of inborn errors of metabolism, dicarboxylic aciduria and hyperoxaluria, have been studied by means of hydroxyl negative ion chemical ionization [NICI(OH-)], linked with collisionally activated decomposition experiments on the [M-H]- species of the pathognomonic organic acids. This method has led to non-controversial qualitative determinations of C4-C10 dicarboxylic acids and oxalic, glyceric and glyoxylic acids. NICI(OH-) linked with collisionally activated decomposition mass analysed ion kinetic energy spectrometry (CAD MIKES) is proposed herein for diagnostic purposes, as a valid mass spectrometric alternative to standard gas chromatographic/mass spectrometric analysis. The procedure is characterized by simplified sample treatment and by fast execution.     

Diagnosis and therapeutic monitoring of inborn errors of creatine metabolism and transport using liquid chromatography–tandem mass spectrometry in urine,plasma and CSF

Biochemical detection of inborn errors of creatine metabolism or transport relies on the analysis of three main metabolites in biological fluids: guanidinoacetate (GAA), creatine (CT) and creatinine (CTN). Unspecific clinical presentation of the diseases might be the cause that only few patients have been diagnosed so far. We describe a LC–MS/MS method allowing fast and reliable diagnosis by simultaneous quantification of GAA, CT and CTN in urine, plasma and cerebrospinal fluid (CSF) and established reference values for each material.     

Screening tests for argininosuccinic aciduria,orotic aciduria,and other inherited enzyme deficiencies using dried blood specimens

Screening tests have been devised to detect the catalytic activities of at least 19 enzymes, primarily of the Emden-Meyerhof pathway, in normal blood specimens collected and dried on filter paper. Since these specimens can be mailed to a laboratory for assay, such screening tests may be useful in detecting individuals with inborn errors of metabolism among large populations or in certain types of genetic studies. Two new screening tests, for argininosuccinic aciduria and orotic aciduria, have been devised. These tests detect the normal enzyme activity in erythrocytes by means of a visible growth response of bacterial auxotrophs when nonutilizable substrates are converted by the enzymes into growth-promoting products. The use of the dried blood specimen in gel electrophoretic and immunoelectrophoretic procedures for genetic studies is also described.This investigation was supported in part by National Institutes of Health Grant No. NB-05290 and by the Children's Bureau Grant No. 435.     

Identification of the human and bovine ATP:Cob(I)alamin adenosyltransferase cDNAs based on complementation of a bacterial mutant

In humans, deficiencies in coenzyme B12-dependent methylmalonyl-CoA mutase (MCM) lead to methylmalonyl aciduria, a rare disease that is often fatal in newborns. Such deficiencies can result from inborn errors in the MCM structural gene or from mutations that impair the assimilation of dietary cobalamins into coenzyme B12 (Ado-B12), the required cofactor for MCM. ATP:cob(I)alamin adenosyltransferase (ATR) catalyzes the terminal step in the conversion of cobalamins into Ado-B12. Substantial evidence indicates that inherited defects in this enzyme lead to methylmalonyl aciduria, but the corresponding ATR gene has not been identified. Here we report the identification of the bovine and human ATR cDNAs as well as the corresponding human gene. A bovine liver cDNA expression library was screened for clones that complemented an ATR-deficient bacterial strain for color formation on aldehyde indicator medium, and four positive clones were isolated. The DNA sequences of two clones were determined and found to be identical. Sequence similarity searching was then used to identify a homologous human cDNA (89% identity) and its corresponding gene that is located on chromosome XII. The bovine and human cDNAs were independently cloned and expressed in Escherichia coli. Enzyme assays showed that expression strains produced 87 and 98 nmol/min/mg ATR activity, respectively. These specific activities are in line with values reported previously for bacterial ATR enzymes. Subsequent studies showed that the human cDNA clone complemented an ATR-deficient bacterial mutant for Ado-B12-dependent growth on 1,2-propanediol. This demonstrated that the human ATR is active under physiological conditions albeit in a heterologous host. In addition, Western blots were used to show that ATR expression is altered in cell lines derived from cblB methylmalonyl aciduria patients compared with cell lines from normal individuals. We propose that inborn errors in the human ATR gene identified here result in methylmalonyl aciduria. The identification of genes involved in this disorder will allow improvements in the diagnosis and treatment of this serious disease.     

The zinc,copper, and manganese status of children with malabsorption syndromes and inborn errors of metabolism

The zinc, copper, and manganese status of seven children with malabsorption syndromes of varied etiology (MVE) and 12 with inborn errors of metabolism (IEM) receiving semi-synthetic diets was investigated using serum and hair trace element concentrations, dietary trace element intakes, and anthropometric measurements as the principal indices. The hair zinc levels of both test groups and hair manganese levels of the IEM group were significantly lower (p<0.05) than those of their respective healthy controls matched by age, sex, and geographic location, despite comparable dietary zinc and manganese intakes in test and control subjects. Four subjects from the malabsorption and five from the inborn errors group had hair zinc levels below 100 μg/g (range 30–88 μg/g). Of these nine subjects, serum zinc levels were determined for six, and five were less than normal (range 64–74 μg/dL). In contrast, the copper status of the MVE and IEM subjects, as indicated by hair and dietary copper levels, was not lower than the controls. Mean serum copper levels were 136±30 and 171±40 μg/dL for the IEM and MVE groups, respectively. Levels for the MVE subjects were higher than published normal values. The suboptimal zinc and manganese status observed in some of these test subjects probably arose from malabsorption and decreased availability of dietary zinc and manganese. However, the zinc depletion was not severe enough to result in linear growth retardation.     

An Economic Evaluation of Neonatal Screening for Inborn Errors of Metabolism Using Tandem Mass Spectrometry in Thailand

Background

Inborn errors of metabolism (IEM) are a rare group of genetic diseases which can lead to several serious long-term complications in newborns. In order to address these issues as early as possible, a process called tandem mass spectrometry (MS/MS) can be used as it allows for rapid and simultaneous detection of the diseases. This analysis was performed to determine whether newborn screening by MS/MS is cost-effective in Thailand.

Method

A cost-utility analysis comprising a decision-tree and Markov model was used to estimate the cost in Thai baht (THB) and health outcomes in life-years (LYs) and quality-adjusted life year (QALYs) presented as an incremental cost-effectiveness ratio (ICER). The results were also adjusted to international dollars (I$) using purchasing power parities (PPP) (1 I$ = 17.79 THB for the year 2013). The comparisons were between 1) an expanded neonatal screening programme using MS/MS screening for six prioritised diseases: phenylketonuria (PKU); isovaleric acidemia (IVA); methylmalonic acidemia (MMA); propionic acidemia (PA); maple syrup urine disease (MSUD); and multiple carboxylase deficiency (MCD); and 2) the current practice that is existing PKU screening. A comparison of the outcome and cost of treatment before and after clinical presentations were also analysed to illustrate the potential benefit of early treatment for affected children. A budget impact analysis was conducted to illustrate the cost of implementing the programme for 10 years.

Results

The ICER of neonatal screening using MS/MS amounted to 1,043,331 THB per QALY gained (58,647 I$ per QALY gained). The potential benefits of early detection compared with late detection yielded significant results for PKU, IVA, MSUD, and MCD patients. The budget impact analysis indicated that the implementation cost of the programme was expected at approximately 2,700 million THB (152 million I$) over 10 years.

Conclusion

At the current ceiling threshold, neonatal screening using MS/MS in the Thai context is not cost-effective. However, the treatment of patients who were detected early for PKU, IVA, MSUD, and MCD, are considered favourable. The budget impact analysis suggests that the implementation of the programme will incur considerable expenses under limited resources. A long-term epidemiological study on the incidence of IEM in Thailand is strongly recommended to ascertain the magnitude of problem.