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Objectives: Aminoacidopathies are inborn errors of metabolism (IEMs) that cause intellectual disability in children. Luckily, aminoacidopathies are potentially treatable, if diagnosed earlier in life. The focus of this study was the screening of aminoacidopathies in a cohort of patients suspected for IEMs. Methods: Blood samples from healthy (IQ > 90; n = 391) and intellectually disabled (IQ < 70; n = 409) children (suspected for IEMs) were collected from different areas of Northern Punjab, Pakistan. An analytical HPLC assay was used for the screening of plasma amino acids. Results: All the samples (n = 800) were analyzed on HPLC and forty-three out of 409 patient samples showed abnormal amino acid profiles mainly in the levels of glutamic acid, ornithine and methionine. Plasma concentration (Mean ± SD ng/mL) were significantly high in 40 patients for glutamic acid (patients: 165 ± 38 vs. controls: 57 ± 8, p < 0.00001) and ornithine (patients: 3177 ± 937 vs. controls: 1361 ± 91, p < 0.0001). Moreover, 3 patients showed abnormally high (53.3 ± 8.6 ng/mL) plasma levels of methionine. Conclusion: In conclusion, biochemical analysis of samples from such patients at the metabolites level could reveal the underlying diseases which could be confirmed through advanced biochemical and genetic analyses. Thus, treatment to some of such patients could be offered. Thus burden of intellectual disability caused by such rare metabolic diseases could be reduced from the target populations.

Objectives: Inborn errors of metabolism (IEMs) are rare genetic disorders. Generally, IEMs are untreatable; however, some IEMs causing intellectual disability are potentially treatable if diagnosed earlier. In this study, levels of some clinically important biochemical parameters in intellectually disabled children suspected for IEMs were tested to see their association with intellectual disability, which could be helpful in preliminary screening. Methods: This comparative cross-sectional observational study was carried out from 2014 to 2017. Blood samples from 800 boys and girls (aged 4-24 years) were collected, of which 391 were healthy (IQ >90) and 409 were intellectually disabled (IQ <70) children with unknown cause. Clinically important (Liver and kidney enzymes etc.) biochemical parameters were analyzed in sera samples using commercial kits on semi-automated clinical chemistry analyzer. Results: Serum analysis showed the levels of ALP (p < 0.00001), ASAT (p = 0.001), ALAT (p = 0.016), albumin (p < 0.001), uric acid (p < 0.001), cholesterol (p < 0.001), triglycerides (p < 0.001), and hemoglobin (p = 0.005) were significantly different between healthy and intellectually disabled children. Conclusion: Changes in the liver function test and lipid profile parameters were significantly different in children with intellectual disability; however, it requires further detailed analysis for complete characterization of these diseases.

Classical homocystinuria is an inborn amino acid metabolism disorder resulting from mutations in the Cystathionine-β-Synthase (CBS) gene. These mutations lead to elevated homocysteine and methionine levels and reduced cysteine levels in the blood. Typically, diagnosis occurs after patients display symptoms, and various lab methods confirm it. DNA sequencing is the best option for early detection of genetic variants in asymptomatic suspected individuals. Unfortunately, its high cost can hinder its use, especially in low-income countries like Pakistan. Aim of this study was to devise a robust low-cost diagnostic/screening assay based on Tetra-ARMS-PCR for five prevalent genetic variants found in Pakistani classical homocystinuria patients. In the current study, T-ARMS-PCR assays were developed for five mutations (c.975G > C, c.770C > T, c.752T > C, c.1039 + 1G > T, c.451 + 1GG > TA), which were characterized previously in classical homocystinuria patients. These low-cost T-ARMS-PCR assays were then used to screen the affected individuals and their family members to identify their genotypes for pathogenic variations in the asymptomatic patients and carriers in their respective families. The outcomes were entirely consistent with those obtained from Sanger DNA sequencing, confirming the sensitivity, specificity, and reliability of the T-ARMS-PCR assay for detecting CBS mutations. T-ARMS-PCR has wide applications for low-income countries for the screening and early diagnosis of asymptomatic patients and carriers in the homocystinuria affected families as well as other inherited diseases. [ABSTRACT FROM AUTHOR]

Plasma amino acids are generally analyzed through ion exchange chromatography, a reproducible but time‐consuming method. Here, we report the optimization of a reverse‐phase‐high‐performance liquid chromatography with fluorescence detector (RP‐HPLC‐FLD) assay for the detection and quantification of plasma amino acids for potential applications in metabolic disorders (e.g., aminoacidopathies, a rare group of Inborn Errors of Metabolism). For assay development, initially standard amino acids were derivatized with ortho‐phthalaldehyde‐3‐mercaptopropionic acid (OPA‐3‐MPA) and filtered through a 0.20 μm syringe filter. The excitation and emission wavelengths of 240–450 nm (λex—λem) were used for the detection of amino acids. Chromatographic separation was achieved by gradient RP‐HPLC‐FLD through C18 symmetry column (150 × 4.6 mm, particle size 3.5 μm). HPLC assay was successfully optimized and was able to detect amino acids in the range of 10–400 ng/mL and good linearity (R2 > 0.98) was achieved in the mixture for each standard amino acid. Moreover, the current assay showed great efficiency with two additional advantages: the use of low‐cost mobile phases, and the detection and quantification of amino acids at low level (ng/mL) concentration in biofluids. This assay could be applied for the analysis of human plasma to identify aminoacidopathies in newborn screening programs, and other metabolic disorders. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN), also known as diabetic kidney disease (DKD), is a leading cause of morbidity and mortality in diabetic patients. A major cause of DKD is high blood pressure (hypertension) which remains uncontrolled chronically. Renin angiotensin aldosterone system (RAAS) is a major regulator of the blood pressure, electrolytes and fluid homeostasis. RAAS plays a key role in modulating the sodium metabolism, vasoconstriction, vascular tone and renal hemodynamics. Angiotensin converting enzyme (ACE) is a key member of RAAS pathway and target for anti-hypertensive drugs. ACE (I/D) polymorphism studies for its association with kidney function and risks of DN have been reported in different populations but with varying results. Hence, the objective of current study was to study the association of ACE (I/D) polymorphism with risk of DN in Pakistani subjects. For this study, 702 subjects were recruited who were divided into three groups; healthy control (n=222), diabetics without nephropathy (n=230) and diabetics with nephropathy (n=250). Clinically important biochemical parameters including glucose, uric acid, urea, creatinine, albumin, total protein cholesterol, and liver enzymes (ALP, ALT, and AST) were measured for all subjects. Genotyping for ACE (I/D) polymorphism was done by PCR assay. Statistical analysis showed that subjects with DN had higher (67%) frequency of ID genotypes and the ID genotype carriers also exhibited higher levels of creatinine (6.2±3.9 mg/dL) and urea (99±41 mg/dL). Moreover, logistic regression analysis also indicated that ACE ID genotype carriers had a 2.5-fold higher risk of DN as compared to the II genotype carriers. Hence the present study shows a strong association (OR 2.53, CI: 1.65-3.88, p<0.001) between ACE (I/D) polymorphism and DN in Pakistani subjects. Genetic polymorphism of ACE (I/D) is associated with diabetic nephropathy in Pakistani subjects. ACE ID genotype might also be considered as an important factor in risk stratification of the renal function for DKD and high blood pressure patients. [ABSTRACT FROM AUTHOR]

Owing to unique physical and optical properties, fluorescent carbon dots (C-dots) are used in various biomedical applications. In this study, C-dots have been integrated as sensing agent on paper surface for colorimetric determination of clinically important analytes. Polyacrylamidebased C-dots coated C-dips showed visible color change from white to brown for iron (Fe2+, Fe3+) sample. Moreover, iron and copper (Cu2+) samples showed significant quenching of C-dots under UV. Images captured with/without UV were analyzed to study the relationship between different iron and copper concentrations and colorimetric intensity values. Color intensity and fluorescence showed a linear relationship with concentrations of iron and copper (Range: 1×10-4 to 1×10-2 M, Linearity: R² = 0.94). LOD from the paper sensor showed a sensitivity of 3.89×10-5 μM, 4.65×10-5 μM and 6.7×10-5 μM for Fe3+, Fe2+ and Cu2+ respectively, reflecting its reliability as low-cost sensing solution to analyze clinically relevant analytes as reported in current study. [ABSTRACT FROM AUTHOR]

Apolipoprotein B (APOB) is the major part of low density lipoprotein (LDL), with two major isoforms: APOB100 and APOB48 found in the human body. Both isoforms are involved in the formation and transport of chylomicron and LDL-cholesterol. Point mutations in APOB may lead to change in protein stereochemistry, which may result in premature coronary artery disease, familial hypobetalipoproteinemia, hypocholesterolemia, mono-genic dyslipisimias and other atherogenic events in CVD. Here we evaluated the impact of all missense and non-coding single nucleotide polymorphisms (SNPs) of APOB retrieved from dbSNP using 17 different computational tools and further evaluated the structural impact of these convergent deleterious SNPs on APOB through HOPE. We found 9 missenses, 15 intronic or regulatory region SNPs and 2 were found in miRNA target sites of APOB. Out of these variant, the rs13306194 (Arg532Trp) was found in the conserved region of protein domain, which can potentially disrupt overall chemical structure and function of the APOB. Six missense SNPs in the coding, and 17 SNPs in noncoding regions are proposed as novel most deleterious variants of APOB. We also try to predict the structural model of APOB through protein docking. The results indicate the applicability of in silico approach to propose the most deleterious SNPs of APOB that should be prioritize for future genetic association studies in cohort of cardiovascular patients. While their structural impact on APOB may suggest these predicted nsSNPs possibly be a better drug target and contribute to the treatment and better understanding of human cardiovascular disease. [ABSTRACT FROM AUTHOR]