The clinical relevance of novel biomarkers as outcome parameter in adults with phenylketonuria.

Recent studies in PKU patients identified alternative biomarkers in blood using untargeted metabolomics. To test the added clinical value of these novel biomarkers, targeted metabolomics of 11 PKU biomarkers (phenylalanine, glutamyl-phenylalanine, glutamyl-glutamyl-phenylalanine, N-lactoyl-phenylalanine, N-acetyl-phenylalanine, the dipeptides phenylalanyl-phenylalanine and phenylalanyl-leucine, phenylalanine-hexose conjugate, phenyllactate, phenylpyruvate, and phenylacetate) was performed in stored serum samples of the well-defined PKU patient-COBESO cohort and a healthy control group. Serum samples of 35 PKU adults and 20 healthy age- and sex-matched controls were analyzed using ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry. Group differences were tested using the Mann-Whitney U test. Multiple linear regression analyses were performed with these biomarkers as predictors of (neuro-)cognitive functions working memory, sustained attention, inhibitory control, and mental health. Compared to healthy controls, phenylalanine, glutamyl-phenylalanine, N-lactoyl-phenylalanine, N-acetyl-phenylalanine, phenylalanine-hexose conjugate, phenyllactate, phenylpyruvate, and phenylacetate were significant elevated in PKU adults (p < 0.001). The remaining three were below limit of detection in PKU and controls. Both phenylalanine and N-lactoyl-phenylalanine were associated with DSM-VI Attention deficit/hyperactivity (R2 = 0.195, p = 0.039 and R2 = 0.335, p = 0.002, respectively) of the ASR questionnaire. In addition, N-lactoyl-phenylalanine showed significant associations with ASR DSM-VI avoidant personality (R2 = 0.265, p = 0.010), internalizing (R2 = 0.192, p = 0.046) and externalizing problems (R2 = 0.217, p = 0.029) of the ASR questionnaire and multiple aspects of the MS2D and FI tests, reflecting working memory with R2 between 0.178 (p = 0.048) and 0.204 (p = 0.033). Even though the strength of the models was not considered strong, N-lactoyl-phenylalanine outperformed phenylalanine in its association with working memory and mental health outcomes.

LC-MS/MS in clinical chemistry: Did it live up to its promise?: Consideration from the Dutch EQAS organisation.

BACKGROUND: Over the past decade the use of LC-MS/MS has increased significantly in the hospital laboratories. Clinical laboratories have switched from immunoassays to LC-MS/MS methods due to the promise of improvements in sensitivity and specificity, better standardization with often non-commutable international standards, and better between-laboratory comparison. However, it remains unclear whether routine performance of the LC-MS/MS methods have met these expectations. METHOD: This study examined the EQAS results, from the Dutch SKML, of serum cortisol, testosterone, 25OH-vitaminD and cortisol in urine and saliva over 9 surveys (2020 to first half of 2021). RESULTS: The study found a significant increase in the number of compounds and in the number of results measured in the different matrices, with LC-MS/MS over a period of eleven years. In 2021, approximately 4000 LC-MS/MS results were submitted (serum: urine: saliva = 58:31:11%) compared to only 34 in 2010. When compared to the individual immunoassays, the LC-MS/MS based methods for serum cortisol, testosterone and 25OH-vitaminD showed comparable but also higher between-laboratory CVs in different samples of the surveys. For cortisol, testosterone and 25OH-vitaminD the median CV was 6.8%, 6.1% and 4.7% respectively for the LC-MS/MS compared to 3.9-8.0%,4.5-6.7%, and 7.5-18.3% for immunoassays. However, the bias and imprecision of the LC-MS/MS was better than that of the immunoassays. CONCLUSION: Despite the expectation that LC-MS/MS methods would result in smaller between-laboratory differences, as they are relatively matrix independent and better to standardize, the results of the SKML round robins do not reflect this for some analytes and may be in part explained by the fact that in most cases laboratory developed tests were used.

Functional disruption of pyrimidine nucleoside transporter CNT1 results in a novel inborn error of metabolism with high excretion of uridine and cytidine.

Genetic defects in the pyrimidine nucleoside transporters of the CNT transporter family have not yet been reported. Metabolic investigations in a patient with infantile afebrile tonic-clonic seizures revealed increased urinary uridine and cytidine excretion. Segregation of this metabolic trait in the family showed the same biochemical phenotype in a healthy older brother of the index. Whole exome sequencing revealed biallelic mutations in SLC28A1 encoding the pyrimidine nucleoside transporter CNT1 in the index and his brother. Both parents and unaffected sibs showed the variant in heterozygous state. The transporter is expressed in the kidneys. Compelling evidence is available for the disrupting effect of the mutation on the transport function thus explaining the increased excretion of the pyrimidine nucleosides. The exome analysis also revealed a pathogenic mutation in PRRT2 in the index, explaining the epilepsy phenotype in infancy. At present, both the index (10 years) and his older brother are asymptomatic. Mutations in SLC28A1 cause a novel inborn error of metabolism that can be explained by the disrupted activity of the pyrimidine nucleoside transporter CNT1. This is the first report describing a defect in the family of CNT concentrative pyrimidine nucleoside transporter proteins encoded by the SLC28 gene family. In all likelihood, the epilepsy phenotype in the index is unrelated to the SLC28A1 defect, as this can be fully explained by the pathogenic PRRT2 variant. Clinical data on more patients are required to prove whether pathogenic mutations in SLC28A1 have any clinical consequences or are to be considered a benign metabolic phenotype.