Abstract:
This study aimed to address the challenges associated with identifying inborn errors of metabolism (IEM) in resource-constrained environments, focusing on amino acid and galactose metabolism disorders in the Bangladeshi infant population. Liquid Chromatography Mass Spectrometry (LC-MS) is a robust diagnostic tool but faces limitations due to cost, complexity, and accessibility. Therefore alternative approaches, including enzyme assays, genetic testing, and metabolite profiling, were explored for effective IEM detection. In pursuit of an affordable diagnostic solution, a low-cost kit was developed for early detection and intervention of amino acid and galactose-related disorders. A validated HPLC-based method was established for accurate quantification of amino acids. The method exhibited strong linearity (R2 = 0.9999) and high accuracy (99.10% average recovery), establishing its suitability for amino acid analysis. Precision was confirmed through repeatability and intermediate precision tests, with low RSD values (0.30% to 1.53% and 0.23% to 0.66%, respectively). Amino acid cutoff ranges were determined for the Bangladeshi population using the validated HPLC method. Noteworthy variations in amino acid counts were observed in positive samples, emphasizing the importance of accurate cutoff values. A lateral flow paper-based diagnostic kit was designed for phenylalanine detection, offering a cost-effective means for phenylketonuria (PKU) monitoring. The kit demonstrated a minimum detection limit of 2 mg/dL, catering to PKU therapy needs. The study also concentrated on Galactosemia as a model disorder, underlining the necessity of early intervention. Molecular analysis of the GALT gene unveiled specific mutations in the Bangladeshi population, contributing to Galactosemia's molecular diversity. Evaluation of silent mutations' impact on splicing shed light on potential gene expression and protein function alterations, influencing Galactosemia outcomes. An easy-to-use lateral flow diagnostic kit for Galactosemia was developed, showing specificity for galactose and delivering rapid results within 5 minutes. Stability assessment revealed promising results at 2-8°C storage over a year, with signal degradation at higher temperatures, emphasizing proper storage conditions. The study highlights the potential of the HPLC method for amino acid detection, paving the way for accessible and cost-effective amino acidopathy detection facilities. The Galactosemia kit, with further refinement, holds promise for broader use. Insights into alternate GALT gene splicing patterns provide avenues for therapeutic exploration. Future research should fine-tune kits and establish precise phenylalanine and galactose cutoff ranges for the Bangladeshi population, contributing to improved IEM management.
