Donald H. Chace

Medium-Chain Acyl-CoA Dehydrogenase (MCAD) Mutations Identified by MS/MS-Based Prospective Screening of Newborns Differ from Those Observed in Patients with Clinical Symptoms: Identification and Characterization of a New, Prevalent Mutation That Results in Mild MCAD Deficiency*

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently diagnosed mitochondrial beta-oxidation defect, and it is potentially fatal. Eighty percent of patients are homozygous for a common mutation, 985A-->G, and a further 18% have this mutation in only one disease allele. In addition, a large number of rare disease-causing mutations have been identified and characterized. There is no clear genotype-phenotype correlation. High 985A-->G carrier frequencies in populations of European descent and the usual avoidance of recurrent disease episodes by patients diagnosed with MCAD deficiency who comply with a simple dietary treatment suggest that MCAD deficiency is a candidate in prospective screening of newborns. Therefore, several such screening programs employing analysis of acylcarnitines in blood spots by tandem mass spectrometry (MS/MS) are currently used worldwide. No validation of this method by mutation analysis has yet been reported. We investigated for MCAD mutations in newborns from US populations who had been identified by prospective MS/MS-based screening of 930,078 blood spots. An MCAD-deficiency frequency of 1/15,001 was observed. Our mutation analysis shows that the MS/MS-based method is excellent for detection of MCAD deficiency but that the frequency of the 985A-->G mutant allele in newborns with a positive acylcarnitine profile is much lower than that observed in clinically affected patients. Our identification of a new mutation, 199T-->C, which has never been observed in patients with clinically manifested disease but was present in a large proportion of the acylcarnitine-positive samples, may explain this skewed ratio. Overexpression experiments showed that this is a mild folding mutation that exhibits decreased levels of enzyme activity only under stringent conditions. A carrier frequency of 1/500 in the general population makes the 199T-->C mutation one of the three most prevalent mutations in the enzymes of fatty-acid oxidation.

AUTOMATED TANDEM MASS SPECTROMETRY FOR MASS NEWBORN SCREENING FOR DISORDERS IN FATTY ACID, ORGANIC ACID, AND AMINO ACID METABOLISM

Development of acylcarnitine and amino acid profiling using tandem mass spectrometry, and its application for use with dried blood specimens collected on filter-paper cards, has introduced an innovative new technology for detecting inborn errors of fatty acid, organic acid, and amino acid metabolism. From November 1, 1992 through June 30, 1999 we screened more than 700,000 newborns in Pennsylvania, Ohio, North Carolina, and Louisiana. We have prospectively detected 163 inborn errors of metabolism. Eighty-six patients have amino acid metabolism errors. Among them are phenylketonuria, hyperphenylalaninemia, maple syrup urine disease, and several urea cycle disorders. Thirty-two have organic acid metabolism errors, including glutaric aciduria type 1; 3-methylcrotonyl coenzyme A (CoA) carboxylase deficiency, propionic acidemia, methylmalonic acidemia, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency; and 45 have fatty acid oxidation errors, including 36 with medium-chain acyl-CoA dehydrogenase deficiency. Details of the methodology are presented and the potential of this screening technology is discussed. (J Child Neurol 1999;14(Suppl 1):S4-S8).

Comparison of amino acids and acylcarnitines assay methods used in newborn screening assays by tandem mass spectrometry

BACKGROUND The analysis of amino acids (AA) and acylcarnitines (AC) by tandem mass spectrometry (MS/MS) is performed in newborn screening laboratories worldwide. While butyl esterification assays are routine, it is possible to detect AAs and ACs as their native free acids (underivatized). The Centers for Disease Control and Preventions Newborn Screening Quality Assurance Program provides dried blood spot (DBS) quality control (QC) and proficiency testing (PT) programs for numerous MS/MS analytes. We describe empirical differences between derivatization and non-derivatization techniques for selected AAs and ACs. METHODS DBS materials were prepared at levels near, above and below mean domestic laboratory cut-offs, and distributed to program participants for MS/MS analysis. Laboratories reported quantitative and qualitative results. QC DBS materials were assayed in-house following established protocols. RESULT Minor differences (<15%) between quantitative values resulting from butyl esters and free acid techniques were observed for the majority of the analytes. Mass spectrometric response from underivatized dicarboxylic acid acylcarnitines was less intense than their butyl esters. CONCLUSIONS The use of underivatized techniques may also result in the inability to differentiate isobaric acylcarnitines. Laboratories should establish their own protocols by focusing on the decisions that identify test results requiring additional follow-up testing versus those that do not.

Metabolomic Profiling of Amino Acids and β-Cell Function Relative to Insulin Sensitivity in Youth

CONTEXT In longitudinal studies of adults, elevated amino acid (AA) concentrations predicted future type 2 diabetes mellitus (T2DM). OBJECTIVE The aim of the present investigation was to examine whether increased plasma AA concentrations are associated with impaired β-cell function relative to insulin sensitivity [i.e. disposition index (DI)], a predictor of T2DM development. DESIGN, SETTING, AND PARTICIPANTS Metabolomic analysis for fasting plasma AAs was performed by tandem mass spectrometry in 139 normal-weight and obese adolescents with and without dysglycemia. First-phase insulin secretion was evaluated by a hyperglycemic (∼225 mg/dl) clamp and insulin sensitivity by a hyperinsulinemic-euglycemic clamp. DI was calculated as the product of first-phase insulin and insulin sensitivity. RESULTS DI was positively associated with branched-chain AAs (leucine/isoleucine and valine; r = 0.27 and 0.29, P = 0.001), neutrally transported AAs (phenylalanine and methionine; r = 0.30 and 0.35, P < 0.001), basic AAs (histidine and arginine; r = 0.28 and 0.23, P ≤ 0.007), serine (r = 0.35, P < 0.001), glycine (r = 0.26, P = 0.002), and branched-chain AAs-derived intermediates C3, C4, and C5 acylcarnitine (range r = 0.18-0.19, P ≤ 0.04). CONCLUSION In youth, increased plasma AA concentrations are not associated with a heightened metabolic risk profile for T2DM; rather, they are positively associated with β-cell function relative to insulin sensitivity. These contrasting observations between adults and youth may be a reflection of developmental differences along the lifespan dependent on the combined impact of the aging process together with the impact of progressive obesity.

Improved MS/MS analysis of succinylacetone extracted from dried blood spots when combined with amino acids and acylcarnitine butyl esters

BACKGROUND The utilization of succinylacetone (SUAC) as the primary metabolic marker for tyrosinemia Type I is now well known, thus new methods have been developed to analyze SUAC as a first tier test in newborn screening. One approach is to prepare a SUAC hydrazine derivative from the dried blood spots (DBS) previously utilized in the extraction of acylcarnitine (AC) and amino acids (AA). The final derivatized products of SUAC, AA and AC are combined in a single tandem mass spectrometric (MS/MS) analysis. However, butyl esterification techniques may result in contamination of underivatized acylcarnitines by as much as 20%. We have developed a simple wash step to improve the combined analysis of SUAC, AA and AC in DBS by MS/MS. METHODS AA and AC were extracted with methanol containing labeled internal standard from 3.2mm punches taken from the DBS specimen. The previously extracted blood spot that remains after removal of the methanol extraction solvent was used in the preparation of SUAC with and without additional washing of the blood spot. The butyl ester eluates of AA and AC, and SUAC hydrazine derivatives were recombined and measured by MS/MS. RESULTS Three additional methanol wash steps of the remaining DBS punches prior to SUAC derivatization reduced the presence of underivatized acylcarnitines, resulting in a 4-fold reduction of underivatized palmitoylcarnitine. Palmitoylcarnitine butyl ester is detected at m/z 456 while the underivatized species is detected at m/z 400, which is also the mass of dodecanoylcarnitine butyl ester. The linearity of the SUAC assay was unchanged by the additional wash steps. For butyl esterification methods, the preferred analytic procedure, the presence of AC can compromise the results of a newborn screen for the actual concentrations of acylcarnitines. It is essential to remove any underivatized acylcarnitines prior to SUAC analysis. CONCLUSION The additional methanol wash steps did not alter SUAC assay results but did remove underivatized acylcarnitines which could result in the incorrect quantification of acylcarnitines.

Microsample analyses via DBS: challenges and opportunities

The use of DBS is an appealing approach to employing microsampling techniques for the bioanalysis of samples, as has been demonstrated for the past 50 years in the metabolic screening of metabolites and diseases. In addition to its minimally invasive sample collection procedures and its economical merits, DBS microsampling benefits from the very high sensitivity, selectivity and multianalyte capabilities of LC-MS, which has been especially well demonstrated in newborn screening applications. Only a few microliters of a biological fluid are required for analysis, which also translates to significantly reduced demands on clinical samples from patients or from animals. Recently, the pharmaceutical industry and other arenas have begun to explore the utility and practicality of DBS microsampling. This review discusses the basis for why DBS techniques are likely to be part of the future, as well as offering insights into where these benefits may be realized.

Neonatal Blood Carnitine Concentrations: Normative Data by Electrospray Tandem Mass Spectometry

Despite a number of published reports, there is limited information about carnitine metabolism in the newborn. To establish normative data, we analyzed whole-blood carnitine concentrations in 24,644 newborns at age 1.85 ± 0.95 d and umbilical cord whole blood and plasma carnitine concentrations in 50 full-term newborns. Total carnitine (TC), free carnitine (FC), and acylcarnitine (AC) were measured by electrospray tandem mass spectrometry. AC/FC ratios were derived from these measurements. The entire cohort was stratified according to TC values into a middle TC group representing 90% of the population and lower and upper TC groups representing 5% of the population, respectively. Normative data were derived from the middle TC group of full-term infants (N = 19,595). TC was 72.42 ± 20.75 μM, FC was 44.94 ± 14.99 μM, AC was 27.48 ± 8.05 μM, and AC/FC ratio was 0.64 ± 0.19 (±SD). These values differed significantly from umbilical cord whole blood TC values of 31.27 ± 10.54 μM determined in 50 samples. No meaningful correlation was found between TC and gestational age or birth weight in any group. In controlled analyses, prematurity was not associated with TC levels, whereas low birth weight (<2500 g) and male sex were significantly associated with higher TC levels. The association of low birth weight with higher TC values may be related to decreased tissue carnitine uptake. The sex effect may be related to hormonal influences on carnitine metabolism. Our study provides normative data of carnitine values measured by the highly precise method of electrospray tandem mass spectrometry in a large cohort of newborns and provides the basis for future studies of carnitine metabolism in health and disease states during the neonatal period.

Expansion of newborn screening programs using automated tandem mass spectrometry

Developments and improvements in the application of tandem mass spectrometry for newborn screening of inherited metabolic diseases using dried blood specimens have been ongoing since the initial work with fast atom bombardment ionization tandem mass spectrometry for phenylketonuria and other amino acid disorders was started in 1990. Previous work with acylcarnitine analysis of clinical samples was easily adapted and combined with the amino acid analysis such that a single analysis for both acylcarnitine and amino acid screening was accomplished. With the introduction of electrospray ionization techniques, great progress towards full automation was made resulting in a state-of-the-art, cost-effective method for diagnosis of multiple metabolic diseases in a single analytical run including phenylketonuria, maple syrup urine disease, homocystinuria, medium chain acyl-CoA dehydrogenase deficiency, as well as other disorders in amino, organic, and fatty acid metabolism. Currently, more than 1,000 samples per day are routinely analyzed in our program. Over 139 clinically significant and treatable disorders have been detected out of over 560,000 newborns screened. MRDD Research Reviews 1999;5:150–154.

Quantification of malonylcarnitine in dried blood spots by use of MS/MS varies by stable isotope internal standard composition.

BACKGROUND The utilization of MS/MS for the analysis of amino acids and acylcarnitines from dried blood spots (DBS) is routine in many newborn screening (NBS) laboratories. Recently, malonylcarnitine (C3DC) was shown to be elevated in the DBS of affected infants with malonic acidemia. Quantitative features were unknown, so that its measurement was an approximation. Synthesis of malonylcarnitine enabled both a study in the analytical characteristics of C3DC and a survey of its measurement in NBS laboratories. METHODS Malonylcarnitine was enriched in blood and spotted onto filter paper cards. The DBS were sent to several laboratories for analysis, and the results were returned to the Centers for Disease Control and Prevention (CDC) for evaluation. Reports included a description of the MS/MS method utilized. RESULTS A pilot proficiency survey shows a bimodal distribution of data from 98 laboratories. Analysis of proficiency data reveals the use of different stable isotope internal standards for quantification. Analysis of standard, labeled or unlabelled ((2)H(3)-octanoylcarnitine (C8), glutarylcarnitine (C5DC) and malonylcarnitine (C3DC) revealed significantly different ion detection values. Quantification in laboratories is based on the ratio of the metabolite in question to a reference stable isotope standard. CONCLUSIONS Quantification of metabolites depends upon the reference isotope standard utilized. Quantification requires describing the standards used for estimation of concentration (a pseudo-concentration) and a notation that includes a reference to the isotope standard used. This descriptive method will enable harmonization of data in screening laboratories.

Metabolic disorders detectable by tandem mass spectrometry and unexpected early childhood mortality: A population-based study†‡

Investigators have reported that certain metabolic disorders (fatty acid oxidation (FAO) disorders and organic acidemias) contribute to unexpected early childhood deaths. We estimated the contribution of these metabolic disorders to a population‐based sample of unexpected early childhood deaths. The study population included children less than 3 years of age who died during 1996–2001 and whose deaths were investigated by the Virginia Office of the Chief Medical Examiner (ME). Dried post‐mortem blood on filter paper was sent to a reference laboratory for metabolic screening by tandem mass spectrometry. When molecular DNA analysis was available to identify known gene mutations, positive screens were confirmed. If molecular DNA analysis for a suspected disorder was not available, tandem mass spectrometry was performed on newborn blood spots when available. If DNA analysis was not available and newborn blood spots could not be obtained, an independent expert biochemical geneticist confirmed the post‐mortem interpretation. We obtained screening results for 793 (88%) of 904 children examined. Eight children had a positive screen for FAO disorders or organic acidemias. One child would not have benefited from identification in the newborn period. However, seven childrens outcomes might have been improved had they been identified during the newborn period and effectively treated. Post‐mortem metabolic screening may identify a cause of death for about 1% of children who die unexpectedly before 3 years of age, allowing for identification and treatment of affected siblings. Identifying and treating affected children during the newborn period may offer an opportunity to reduce early childhood mortality. Published 2006 Wiley‐Liss, Inc.