Search

Treatment of phenylketonuria (PKU) has evolved since the initial introduction of a phenylalanine (Phe) restricted diet. The most recent option for adults affected with PKU is treatment with an alternate enzyme, phenylalanine ammonia lyase (PAL), that metabolizes excess Phe. Proper management of all patients with PKU relies on accurate measurement of Phe levels in blood, to comply with guidance intended to minimize the neurological symptoms. Recently, our laboratory was notified of discrepant results for a patient with PKU who is treated with pegvaliase. Two specimens were collected at the same time but yielded unexpectedly different Phe concentrations. After exclusion of specimen mix-ups or analytical errors, we suspected that there was residual pegvaliase activity in the specimens continuing to degrade Phe after collection. To investigate this possibility, we performed spiking studies that showed the degradation of Phe over time at ambient temperatures. Sample preparation by protein crash appears to deactivate pegvaliase and prevents further Phe degradation. However, because pegvaliase deactivation would be required immediately following blood collection, appropriate mitigation measures must be implemented, including stringent pre-analytical requirements, alternate sample matrices such as dried blood spots, or point of care testing. Until then, health care professionals need to be cautious in their interpretation of Phe levels in their patients with PKU that are treated with pegvaliase.

OBJECTIVES/GOALS: Untargeted metabolomics platforms are powerful biomarker discovery tools. However, the absence of uniform study design, data analysis, and reporting standards limits translation of this research into the clinical lab. The goal was to critically appraise existing untargeted metabolomics platforms that analyzed inborn errors of metabolism. METHODS/STUDY POPULATION: A search strategy was conducted in MEDLINE via PubMed from January 16, 2013, to January 16, 2023. The search strategy was limited to primary literature articles written in English that evaluated human subjects with inborn errors of metabolism (IEMs). Articles that performed targeted metabolomic analysis or analyzed non-human samples were excluded. Information on patient cohorts analyzed, sample types, and method design were extracted using a template. Categorical data are summarized as frequencies and percentages. RESULTS/ANTICIPATED RESULTS: A total of 96 distinct IEMs were evaluated by the different untargeted metabolomics methods included in this review. However, most IEMs (55/96, 57%) were evaluated by a single platform, in a single study, with a limited cohort size. Only one study validated their results using a separate, validation cohort. There was considerable diversity in the separation techniques and mass spectrometry instrumentation used by the studies to create their untargeted metabolomics methods. Slightly over half (59%) of the studies identified at least some of the metabolites detected in their samples with the highest level of confidence. Importantly, most of the included studies reported adherence to quality metrics, including use of quality control material (65%) and internal standards in their analysis (61%). DISCUSSION/SIGNIFICANCE: Future studies analyzing IEM patient samples with untargeted metabolomics platforms should progress beyond single-subject studies and evaluate the reproducibility of the research using a prospective, or validation cohort as well as confirm metabolite annotations with reference metabolites standards to generate clinically useful data.

Abstract Background Previous work has identified age-related negative correlations for γ-hydroxybutyric acid (GHB) and γ-aminobutyric acid (GABA) in plasma of patients with succinic semialdehyde dehydrogenase deficiency (SSADHD). Using plasma and dried blood spots (DBS) collected in an ongoing natural history study, we tested the hypothesis that other biomarkers would follow a similar age-related negative correlation as seen for GHB/GABA. Samples (mixed sex) included: patients (n = 21 unique samples, 1–39.5 yrs) and parallel controls (n = 9 unique samples, 8.4–34.8 yrs). Archival control data (DBS only; n = 171, 0.5–39.9 yrs) was also included. Results Metabolites assessed included amino acids (plasma, DBS) and acylcarnitines, creatine, creatinine, and guanidinoacetate (DBS only). Age-related negative correlations for glycine (plasma, DBS) and sarcosine (N-methylglycine, plasma) were detected, accompanied by elevated proline and decreased levels of succinylacetone, argininosuccinate, formaminoglutamate, and creatinine. Significantly low acylcarnitines were detected in patients across all chain lengths (short-, medium- and long-chain). Significant age-dependent positive correlations for selected acylcarnitines (C6-, C12DC(dicarboxylic)-, C16-, C16:1-, C18:1-, C18:2OH-carnitines) were detected in patients and absent in controls. Receiver operating characteristic (ROC) curves for all binary comparisons revealed argininosuccinate and succinylacetone to be the most discriminating biomarkers (area > 0.92). Conclusions Age-dependent acylcarnitine correlations may represent metabolic compensation responsive to age-related changes in GHB and GABA. Our study highlights novel biomarkers in SSADHD and expands the metabolic pathophysiology of this rare disorder of GABA metabolism.

Abstract Analyses of 19 amino acids, 38 acylcarnitines, and 3 creatine analogues (https://clir.mayo.edu) were implemented to test the hypothesis that succinic semialdehyde dehydrogenase deficiency (SSADHD) could be identified in dried bloodspots (DBS) using currently available newborn screening methodology. The study population included 17 post‐newborn SSADHD DBS (age range 0.8‐38 years; median, 8.2 years; 10 M; controls, 129‐353 age‐matched individuals, mixed gender) and 10 newborn SSADHD DBS (including first and second screens from 3 of 7 patients). Low (informative) markers in post‐newborn DBS included C2‐ and C4‐OH carnitines, ornithine, histidine and creatine, with no gender differences. For newborn DBS, informative markers included C2‐, C3‐, C4‐ and C4‐OH carnitines, creatine and ornithine. Of these, only creatine demonstrated a significant change with age, revealing an approximate 4‐fold decrease. We conclude that quantitation of short‐chain acylcarnitines, creatine, and ornithine provides a newborn DBS profile with potential as a first tier screening tool for early detection of SSADHD. This first tier evaluation can be readily verified using a previously described second tier liquid chromatography‐tandem mass spectrometry method for γ‐hydroxybutyric acid in the same DBS. More extensive evaluation of this first/second tier screening approach is needed in a larger population.

OBJECTIVES/GOALS: To develop an untargeted metabolomics assay that can holistically characterize the small molecule signatures of different inborn errors of metabolism (IEM) for biomarker discovery and identification of novel IEMs, with the goal of implementing the assay into the clinical laboratory to improve testing efficiency. METHODS/STUDY POPULATION: A hydrophilic interaction liquid chromatography (HILIC) column and reverse phase (RP) column were assembled in tandem on a SCIEX X500B quadrupole time-of-flight (QTOF) system to create a dual liquid chromatography (LC), tandem mass spectrometry method. The X500B was operated in data-independent acquisition mode with both positive and negative ionization. A mixture of 165 reference standards from eleven compound classes common to IEMs were used to evaluate the capability of the assay to resolve small molecules. Chromatographic resolution for each standard was determined qualitatively by comparison to a reference spectral database. External validation of the assay will be performed by analyzing a commercial library of reference metabolites. RESULTS/ANTICIPATED RESULTS: A total of 88% (146/165) of the standards were detected by the assay. The RP column alone resolved 71% (117/165) of the standards, the HILIC column resolved 33% (55/165), while 17% (29/165) of the standards were resolved by both columns. The HILIC column resolved standards that were more polar, while the RP column resolved more non-polar compounds. To evaluate matrix effects, the reference standard mixture was spiked into pooled plasma. In the presence of plasma 6/146 (4%) of the standards were suppressed to levels below the limit of detection. We expect external validation with the commercial metabolite library will corroborate these results, and that the high-quality spectral data attained from this reference library can be used to improve identification of unknown metabolites in patient samples. DISCUSSION/SIGNIFICANCE: We have shown our untargeted metabolomics assay can detect known biomarkers for IEMs. Clinical implementation of this method could streamline diagnosis of IEMs while simultaneously improving patient outcomes by leveraging the metabolome for biomarker discovery, and improved understanding of IEM mechanisms to inform novel treatment strategies.

The expansion of the recommend uniform screening panel to include more than 50 primary and secondary target conditions has resulted in a substantial increase of false positive results. As an alternative to subjective manipulation of cutoff values and overutilization of molecular testing, here we describe the performance outcome of an algorithm for disorders of methionine, cobalamin, and propionate metabolism that includes: (1) first tier screening inclusive of the broadest available spectrum of markers measured by tandem mass spectrometry; (2) integration of all results into a score of likelihood of disease for each target condition calculated by post-analytical interpretive tools created byCollaborative Laboratory Integrated Reports (CLIR), a multivariate pattern recognition software; and (3) further evaluation of abnormal scores by a second tier test measuring homocysteine, methylmalonic acid, and methylcitric acid. This approach can consistently reduce false positive rates to a

Homocystinuria (HCU) due to cystathionine-β-synthase deficiency is generally regarded as a rare disease, but within the Qatari population has an incidence of 1 in 1800 live births. Most newborn screening methods for HCU using dried blood spots (DBS) rely on the detection of an elevated methionine level or a rapid screen for total homocysteine (tHCY). However, screening based on methionine levels alone lacks specificity and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for tHCY exhibit variable results with high false positive rates. This report describes a LC-MS/MS method for detection of tHCY on DBS, with improved specificity. tHCY was extracted from DBS with a solution containing dithiothreitol and subsequently butylated with hydrochloric acid in n-butanol. The butyl esters were separated by liquid chromatography on a reverse-phase column and the homocysteine (HCY), detected by tandem mass spectrometry. The butyl ester of HCY eluted at 1.8 min. Total analysis time was 6.1 min per sample, including column flush and equilibration. This method allows for the quantification of tHCY over a linear range from 0.3 to 200 µM. Intraassay and interassay imprecision and recoveries were acceptable. Good concordance was observed with another LC-MS/MS method. Application of this method improves specificity and reduces false positive rates in screening for HCU.