Development and validation of an ultra?performance liquid chromatography quadrupole time of flight mass spectrometry method for rapid quantification of free amino acids in human urine

An ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-qTOFMS)method using hydrophilic interaction liquid chromatography was developed and validated for simultaneous quantification of 18 free amino acids in urine with a total acquisition time including the column re-equilibration of less than 18 min per sample. This method involves simple sample preparation steps which consisted of 15 times dilution with acetonitrile to give a final composition of 25 % aqueous and 75 % acetonitrile without the need of any derivatization. The dynamic range for our calibration curve is approximately two orders of magnitude (120-fold from the lowest calibration curve point) with good linearity (r2 ? 0.995 for all amino acids). Good separation of all amino acids as well as good intra- and inter-day accuracy (<15 %) and precision (<15 %) were observed using three quality control samples at a concentration of low, medium and high range of the calibration curve. The limits of detection (LOD) and lower limit of quantification of our method were ranging from approximately 1–300 nM and 0.01–0.5 µM, respectively. The stability of amino acids in the prepared urine samples was found to be stable for 72 h at 4 °C, after one freeze thaw cycle and for up to 4 weeks at ?80 °C. We have applied this method to quantify the content of 18 free amino acids in 646 urine samples from a dietary intervention study. We were able to quantify all 18 free amino acids in these urine samples, if they were present at a level above the LOD. We found our method to be reproducible (accuracy and precision were typically <10 % for QCL, QCM and QCH) and the relatively high sample throughput nature of this method potentially makes it a suitable alternative for the analysis of urine samples in clinical setting

Propionic acidemia as a cause of adult-onset dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is extremely heterogeneous with a large proportion due to dominantly inherited disease-causing variants in sarcomeric genes. Recessive metabolic diseases may cause DCM, usually with onset in childhood, and in the context of systemic disease. Whether metabolic defects can also cause adult-onset DCM is currently unknown. Therefore, we performed an extensive metabolic screening in 36 consecutive adult-onset DCM patients. Diagnoses were confirmed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Measurement of propionyl-CoA carboxylase (PCC) activity was done in fibroblasts. Whole exome sequencing (WES) data of 157 additional DCM patients were analyzed for genetic defects. We found a metabolic profile characteristic for propionic acidemia in a patient with severe DCM from 55 years of age. Genetic analysis demonstrated compound heterozygous variants in PCCA. Enzymatic activity of PCC in fibroblasts was markedly reduced. A targeted analysis of the PCCA and PCCB genes using available WES data from 157 further DCM patients subsequently identified another patient with propionic acidemia. This patient had compound heterozygous variants in PCCB, and developed severe DCM from 42 years of age. Adult-onset DCM can be caused by propionic acidemia, an autosomal recessive inheritable metabolic disorder usually presenting as neonatal or childhood disease. Current guidelines advise a low-protein diet to ameliorate or prevent detrimental aspects of the disease. Long-term follow-up of a larger group of patients may show whether this diet would also ameliorate DCM. Our results suggest that diagnostic metabolic screening to identify propionic acidemia and related disorders in DCM patients is justified