Hepato-Protective Effects of Delta-Tocotrienol and Alpha-Tocopherol in Patients with Non-Alcoholic Fatty Liver Disease: Regulation of Circulating MicroRNA Expression.

MicroRNAs (miRNAs) play a key role in the regulation of genes for normal metabolism in the liver. Dysregulation of miRNAs is involved in the development and progression of non-alcoholic fatty liver disease (NAFLD). We aimed to explore changes in circulating miRNA expression in response to delta-tocotrienol (δT3) and alpha-tocopherol (αTF) supplementation and correlate them with relevant biochemical markers in patients with NAFLD. In total, 100 patients with NAFLD were randomized to either receive δT3 (n = 50) 300 mg or αTF (n = 50) 268 mg twice/day for 48 weeks. Plasma expression of miRNA-122, -21, -103a-2, -421, -375 and -34a were determined at baseline, 24 and 48 weeks of intervention using RT-qPCR. Both δT3 and αTF significantly downregulated expression of miRNA-122, -21, -103a-2, -421, -375 and -34a. Moreover, δT3 was more effective than αTF in reducing expression of miRNA-375 and -34a. A significant correlation was observed between miRNA expression and biochemical markers of hepatic steatosis, insulin resistance (IR), oxidative stress (OS), inflammation and apoptosis. δT3 and αTF exert hepato-protective effects by downregulating miRNAs involved in hepatic steatosis, IR, OS, inflammation and apoptosis in patients with NAFLD. Furthermore, δT3 has more pronounced effects than αTF in reducing miR-375 and miR-34a, which are linked to regulation of inflammation and apoptosis.

Diagnostic evaluation of heel prick newborn screening of thyroid stimulating hormone on dissociation-enhanced lanthanide fluorescence immunoassay with the establishment of reference value in Pakistani neonates.

OBJECTIVE: To develop and validate a method on Dissociation Enhanced Lanthanide Fluorescence Immunoassay for neonatal heel prick blood human thyroid stimulating hormone and the establishment of its reference value in the local population. METHODS: The multi-centre cross-sectional validation study was conducted from September 2016 to September 2018 at Zahra Beau Naqvi Foundation Welfare Trust laboratory, Islamabad, Pakistan, and comprised samples related to newborns aged 1 month or less taken from neonatal units of 39 hospitals based in Punjab, Khyber Pakhtunkhuwa, Gilgit-Baltistan and Azad Jammu and Kashmir. Samples were collected after 24 hours of birth using the heel prick test. The samples were dried and sent to the laboratory for assessment where Dissociation Enhanced Lanthanide Fluorescent Immunoassay was used to estimate thyroid stimulating hormone levels. Data recorded included age, gender, and birth detail, like gestational age, mode of delivery etc. Data was analysed using SPSS 21. Method validation and reference value were manually calculated. RESULTS: Of the 14,147 samples received, 8,207(58%) related to boys and 5,940(42%) to girls. Most samples 4903(34.6%) came from Peshawar. The overall mean age of the newborns was 5.6±4.8 days. Thyroid stimulating hormone data was divided into three groups; positive with median value 27.8±36.6 uIU/ml, negative with median 1.42±1.60 uIU/ml, and borderline with median 11.4±4.12uIU/ml. Prevalence of congenital hypothyroidism in high-risk population in the positive group was 39(0.3%), negative 14,012(99.0%) and borderline 96(0.7%). Reference cut-off was calculated as 7.06uIU/ml for screening of healthy and positive cases of congenital hypothyroidism. Method Validation results showed limit of detection -0.5uU/ml, limit of quantitation LOQ 0.8uU/ml, accuracy 100±5%, precision coefficient of variation at each level of calibrators -4, 8.8, 1.2, 11.3, 7.2 and 4.3% respectively, and linearity from to 0.8uU/ml to 254.1uU/ml. CONCLUSIONS: Neonatal human thyroid stimulating hormone by heel prick blood was found to be an affordable and highly sensitive method of screening for congenital hypothyroidism.

δ-Tocotrienol in Combination with Resveratrol Improves the Cardiometabolic Risk Factors and Biomarkers in Patients with Metabolic Syndrome: A Randomized Controlled Trial.

Background: Metabolic syndrome (MetS) is a cluster of central obesity, hypertension, hyperglycemia, and dyslipidemia. It is a global health issue with an increased risk of cardiovascular disease. Recently, a few natural products have been reported with promising anti-inflammatory and antioxidative effects. We aimed to evaluate the impact of δ-tocotrienol and resveratrol mixture (TRM) supplementation on cardiometabolic risk factors and biomarkers in patients with MetS. Methods: A randomized controlled trial was conducted at the hospitals of National University of Medical Sciences Rawalpindi, Pakistan. A total of 82 patients with MetS aged 18-60 years were enrolled based on International Diabetes Federation-2005 diagnostic criteria and randomly grouped into TRM (n = 41) and placebo (n = 41). Patients in the TRM group were given a 400 mg capsule (δ-tocotrienol 250 mg; resveratrol 150 mg), and a placebo (cellulose 400 mg) twice daily for 24 weeks. The biochemical tests were analyzed on ADVIA 1800 Chemistry® analyzer and inflammatory biomarkers by ELISA methods. Results: In the TRM group, a significant reduction in waist circumference, blood pressure, mean (95% confidence interval) of fasting plasma glucose -0.15 mmol/L (-0.22 to -0.08), serum triglyceride -0.32 mmol/L (-0.47 to -0.17), and increment in high-density lipoprotein cholesterol were observed as compared with placebo. TRM supplementation also improved biomarkers: high-sensitive C-reactive protein -0.61 mg/L (-0.89 to -0.33), interleukin-6-1.99 pg/mL (-2.50 to -1.48), tumor necrosis factor-α -2.19 pg/mL (-2.55 to -1.83), malondialdehyde -0.48 µmol/L (-0.65 to -0.30), and total antioxidant capacity 1.71 U/mL (1.29 to 2.13). Conclusion: TRM supplementations improved cardiometabolic risk factors and biomarkers of inflammation and oxidative stress without any significant side effects in the patients with MetS. Clinical Trials Registry: The clinical trial was registered in Sri Lanka Clinical Trials Registry (https://slctr.lk/trials/slctr-2019-021).

Role of δ-tocotrienol and resveratrol supplementation in the regulation of micro RNAs in patients with metabolic syndrome: A randomized controlled trial.

OBJECTIVE: To determine the effect of δ-tocotrienol and resveratrol mixture (TRM) supplementation in comparison to placebo for 24 weeks, on the relative expression of miRNAs (miRNA-130b-5p, miRNA-221-5p, miR-15b-5p, miRNA-122-5p, and miRNA-376b-5p) in patients with Metabolic syndrome (MetS). DESIGN: This randomized placebo-controlled trial was conducted at the tertiary care institute of the NUMS, Rawalpindi, Pakistan. A total of 82 adult MetS patients were enrolled and randomly grouped into the TRM group (n = 41) and the Placebo group (n = 41). Patients in the TRM group were given 400 mg capsules (δ-tocotrienol 250 mg; Resveratrol 150 mg) and placebo received (cellulose 400 mg capsule) twice daily for 24 weeks. RESULTS: The TRM supplementation revealed a significant (p < 0.001) upregulation of 3.05-fold in miRNA-130b-5p and 2.45-fold in miRNA-221-5p while miRNA-122-5p was downregulated by 2.22-fold as compared to placebo. No significant difference was observed in miRNA-15b-5p and miRNA-376b-5p. Moreover, TRM group participants with reverted MetS had significantly (p < 0.05) upregulated miRNA-130b-5p, miRNA-221-5p, and downregulated miRNA-122-5p relative to non-reverted patients with MetS. CONCLUSION: Daily TRM supplementation may improve metabolic syndrome by upregulated miR-130b-5p, which is involved in central obesity and inflammation, as well as miR-221-5p, which is involved in insulin resistance. Additionally, TRM downregulate of miRNA 122, which improved dyslipidemia.

Multiple Carboxylase Deficiency Organic Acidemia as a Cause of Infantile Seizures.

Multiple carboxylase deficiency organic Acidemia is a rare inherited metabolic disorder. It is autosomal recessive disorder of two types: Holocarboxylase deficiency and Biotinidase deficiency. It is the metabolic disorder resulting from deficiency of biotin as a co-enzyme or reduced activity of biotin-dependent carboxylases (propionyl CoA carboxylase, and 3-methylcrotonyl CoA carboxylase and pyruvate carboxylase). A case of two months' female child is reported, who presented with recurrent infantile seizures and skin rash since birth; and biochemically with metabolic acidosis, hyper-ammonemia (on and off) since birth with multiple hospitalization. She had past history of jaundice. One sibling's death at 2nd day of life due to similar complaints. Initial presentation of raised ammonia and lactate levels were the first indication to this organic academia, which was later proven by increased peak levels of various organic acids on urine organic acid analysis by gas chromatography-mass spectrometry. Key Words: Multiple carboxylase deficiency, Holocarboxylase synthetase deficiency, Biotinidase deficiency.

Late Diagnosis of Paracetamol Poisoning is Always Lethal in Young Adult.

Acetaminophen has a remarkable safety profile when prescribed in proper therapeutic doses, but hepatotoxicity can occur when misused or after an overdose. The principal toxic metabolite of acetaminophen is N-acetyl-p-benzoquinone imine (NAPQI). Toxicity should be considered in all suspicious cases because of the ubiquitous and initially asymptomatic nature of acetaminophen intoxication. A case of 29-year male soldier is discussed, who presented with pain in abdomen, vomiting and jaundice of sudden onset. The diagnosis of ischemic liver damage was made at initial presentation. Raised liver function tests and elevated prothrombin time were the first indication to this condition, which were proven by detection of acetaminophen in blood and urine by liquid chromatography-mass spectrometry. Further supportive evidence of hepatic necrosis was provided by an ultrasound abdomen, giving the final diagnosis of acetaminophen poisoning causing drug-induced liver injury. Key Words: Acetaminophen poisoning, Drug-induced liver injury, Fulminant hepatic failure, N-acetylcysteine, N-acetyl-p-benzoquinone imine (NAPQI).

Dyskinesia in a Child: A Concern for a Rare Neuro-Metabolic Disorder.

A 3-year child is discussed who presented with dyskinesia, large head size, developmental delay, and recurrent infections necessitating multiple hospital admissions. The diagnosis was not made at initial presentation or even after multiple hospital admissions. An organic acidemia was suspected, based on raised ammonia and lactate levels and metabolic acidosis and the diagnosis of glutaric aciduria Type 1 was established by finding markedly elevated levels of glutaric acid and its specific metabolites on urine organic acids analysis by gas chromatography-mass spectrometry, in the setting of specific clinical features. Further supporting evidence was provided by CT scan brain showing subdural hygroma along left cerebral hemisphere causing gyral flattening and widening of sylvian fissure.

Bilirubin Interference in Plasma Amino Acid Analysis by Ion Exchange Chromatography.

OBJECTIVE: To evaluate the effect of bilirubin interference on plasma amino acid analysis by Ion Exchange Chromatography (IEC). STUDY DESIGN: Cross-sectional (method validation) study. PLACE AND DURATION OF STUDY: Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi from August 2016 to July 2017. METHODOLOGY: Twenty non-icteric samples from paediatric patients were collected in lithium heparin tubes and analysed for amino acids on IEC-based Biochrome® 30+ Analyzer (Harvard Biosciences UK). Baseline bilirubin levels were noted. Samples were spiked with neonatal bilirubin standard with concentration of 488.4 mol/L (Spinreact®-Spain) at final concentrations of 50, 150 and 230 mol/L and re-analysed for amino acids at these three concentrations. RESULTS: Among the 20 selected patients with normal amino acid profiles, 12 (60%) were males. Majority (55%) were in age group of 1-5 years. Significant difference was observed for Arginine (p = 0.01), Histidine (p = 0.001), Isoleucine (p = 0.01), Leucine (p = 0.007), Lysine (p = 0.005), Ornithine (p = 0.03) and Phenylalanine (p = 0.02). Mean rank of these amino acids showed decreasing trend with the increase of bilirubin concentration, and pronounced interference was identified at bilirubin level of 50 mol/L. No difference was observed for alanine, citrulline, glutamic acid, glycine, methionine, proline, threonine, tyrosine, asparagine, aspartic acid, cystine, valine and tryptophan. CONCLUSION: Bilirubin significantly interferes with certain amino acid levels when analysis is carried out by ion exchange chromatography. A close follow-up of such patients with other biochemical tests and a repeat amino acid analysis, after jaundice is settled, is recommended to confidently rule out any possible inherited metabolic disorder in these patients.

Classical Homocystinuria in a Juvenile Patient.

Classical homocystinuria, also known as cystathionine beta synthase deficiency, is a rare disorder of methionine metabolism, leading to an abnormal accumulation of homocysteine and its metabolites in blood and urine. A young child with homocystinuria is discussed, who presented with behavioral abnormalities, involuntary movement, mental retardation, and decreased vision since birth. The diagnosis of homocystinuria was not made at initial presentation. Subtle phenotypic features with developmental delay and MRI brain finding of bilateral medially dislocated lens, eventually provided the first indication at five years of age. Laboratory screening with plasma amino acid profile by ion exchange chromatography (IEC) showed elevated homocystine and methionine, and low cystine in plasma in the absence of vitamin B12, and folate deficiency; giving the diagnosis of classical homocysteinuria.

Cost-effectiveness of Screening and Confirmatory Tests for Multiple Myeloma in Pakistani Population: An Audit Report.

OBJECTIVE: To find out the use of screening and confirmatory tests for diagnosis of multiple myeloma as ordered by clinicians. STUDY DESIGN: An Audit. PLACE AND DURATION OF STUDY: Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi, from January 2012 to January 2017. METHODOLOGY: Data retrieved from Laboratory Information Management system (LIMs) by selecting serum protein electrophoresis (SPE) as screening test and immunoelectophoresis (IE) and immunofixation (IF) as confirmatory tests. RESULTS: There were 3,108 tests of serum protein electrophoesis and 1,329 tests of immunoelectophoresis had been performed in last five year. Cost-effective clinical audit of SPE tests showed that only 17.1% tests of SPE were either used for proper diagnosis of multiple myeloma (totally justified tests 13%) or useful for diagnosis of other important diseases whose early diagnosis were helpful for patients management (partially justified tests 4.1%). The cost related to these tests were justified while 82.8% tests of SPE were either normal (total unjustified tests 24.4%), or diagnosed irrevalent and nonspecific diseases (partially unjustified 58.4%). IE and IF audit revealed that only 26.6% tests were properly utilised for diagnosis and differentiation of multiple myeloma and its subtypes and cost attributable to these tests were justified while 73.4% of these confirmatory tests were normal and cost related to them was not justified. CONCLUSION: Overutilisation of laboratory tests for diagnosis of multiple myeloma can be minimised by proper clinical scrutiny of request forms.

Accuracy of Non-Fasting Lipid Profile for the Assessment of Lipoprotein Coronary Risk.

OBJECTIVE: To determine the diagnostic accuracy of non-fasting lipid profile in the diagnosis of hyperlipidemia, taking fasting lipid profile as gold standard, in adult population. STUDY DESIGN: Cross-sectional validation study. PLACE AND DURATION OF STUDY: Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi, from July to December 2014. METHODOLOGY: One hundred seventy-five adult patients coming for fasting lipid profile were included; their non-fasting samples were taken on the next day. Patients on anti-cholesterol treatment and indoor patients were excluded. Total cholesterol (TC), high density lipoprotein-cholestrol (HDL-C), and triglycerides were measured by direct enzymatic colorimetric method by Modular p-800®. Low density lipoprotein-cholesterol (LDL-C) was calculated by Friedewald's formula, but when triglyceride was greater than 4.5 mmol/l, then LDL-C was measured directly by homogenous enzymatic colorimetric method. Non-HDL-C was calculated by simple equation, i.e. TC-HDL-C. RESULTS: Non-fasting lipid profile had 93% specificity , 51% sensitivity, 94% positive predictive value and 49% negative predictive value; and 65% accuracy with 7.28 positive likelihood ratio and 0.52 negative likelihood ratio. Non-fasting TC and non-HDL-C were significantly higher than fasting TC and non-HDL-C by mean difference of 0.2 mmol/l each with p=0.001 and p=0.004, respectively. Fasting and non fasting HDL-C are comparable to each other with mean difference of 0.01 mmol/l (p=0.745). Receiver operating curve (ROC) of non-fasting non-HDL-C showed 0.804 (95%CI (0.738-0.870), (p=0.000) area under the curve (AUC) indicating that it was a significant test for ruling out hyperlipidemia. Bland-Altmann plot showed a significant difference between non-fasting, non-HDL-C and fasting LDL-C and non-fasting, non-HDL-C -0.087540 with bias -0.00109; therefore, these cannot be alternative to each other. CONCLUSION: Diagnostic accuracy of non-fasting lipid profile was found significantly higher than fasting lipid profile (p=0.004) for the assessment of lipoprotein coronary risk on the basis of non-HDL-C, which seemed to be significant test for ruling out hyperlipidemia.