Electrocardiographic and biochemical analysis of anthracycline induced cardiotoxicity in breast cancer patients from Southern Sri Lanka.

BACKGROUND: The clinical application of anthracycline chemotherapy is hindered due to the cumulative dose-dependent cardiotoxicity followed by the oxidative stress initiated during the mechanism of action of anthracyclines. Due to a lack of prevalence data regarding anthracycline-induced cardiotoxicity in Sri Lanka, this study was conducted to determine the prevalence of cardiotoxicity among breast cancer patients in Southern Sri Lanka in terms of electrocardiographic and cardiac biomarker investigations. METHODS: A cross-sectional study with longitudinal follow-up was conducted among 196 cancer patients at the Teaching Hospital, Karapitiya, Sri Lanka to determine the incidence of acute and early-onset chronic cardiotoxicity. Data on electrocardiography and cardiac biomarkers were collected from each patient, one day before anthracycline (doxorubicin and epirubicin) chemotherapy, one day after the first dose, one day and six months after the last dose of anthracycline chemotherapy. RESULTS: Prevalence of sub-clinical anthracycline-induced cardiotoxicity six months after the completion of anthracycline chemotherapy was significantly higher (p < 0.05) and there were strong, significant (p < 0.05) associations among echocardiography, electrocardiography measurements and cardiac biomarkers including troponin I and N-terminal pro-brain natriuretic peptides. The cumulative anthracycline dose, > 350 mg/m2 was the most significant risk factor associated with the sub-clinical cardiotoxicity in breast cancer patients under study. CONCLUSION: Since these results confirmed the unavoidable cardiotoxic changes following anthracycline chemotherapy, it is recommended to carry out long-term follow-ups in all patients who were treated with anthracycline therapy to increase their quality of life as cancer survivors.

The accuracy of fatty liver index for the screening of overweight and obese children for non-alcoholic fatty liver disease in resource limited settings.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) has become the most common liver disease among children with a higher prevalence among obese. Fatty liver index (FLI), an algorithm derived to screen NAFLD using the waist circumference, body mass index, triglyceride and gamma glutamyl-transferase concentration was rarely investigated in Asian paediatric population. Since each component is routinely measured in clinical practice, it is feasible to measure FLI even in resource limited settings. Hence, we determined the accuracy of FLI for the screening of NAFLD in obese children in Southern Sri Lanka. METHODS: A descriptive cross sectional study was conducted on ninety five children (56 boys) aged 5-15 years with BMI ≥ 85th percentile for age and gender based on CDC 2000 growth charts recruited from the nutrition clinic at the Teaching Hospital, Karapitiya, Sri Lanka. NAFLD was diagnosed by ultrasonography. Factors associated with ultrasonographic fatty liver such as biochemical parameters and fatty liver index in either sex or the whole population were determined by a multivariate analysis. The ability of FLI to screen NAFLD was determined by the analysis of area under the receiver operator characteristic curve (AUROC) and the maximum Youden index analysis. RESULTS: Overweight and obese children with ultrasonographic fatty liver had a significantly higher FLI than those without fatty liver according to the multivariate analysis performed (Odds ratio 3.524; 95% CI 1.104-11.256, P = 0.033). AUROC of FLI for NAFLD was 0.692 (95% CI; 0.565-0.786) and the optimal cut off value for the screening of NAFLD was 30 (Maximum Youden index 0.2782, Sensitivity, 58.33%; Specificity, 69.49%). CONCLUSION: FLI could accurately be used in resource limited community settings and in epidemiological studies to screen overweight and obese children for NAFLD.

Antiurolithiatic Potential of Three Sri Lankan Medicinal Plants by the Inhibition of Nucleation, Growth, and Aggregation of Calcium Oxalate Crystals In Vitro.

Background: Deposition and formation of stones in any part of the urinary system is called urolithiasis. CaOx is the predominant component of most stones, and the formation of these stones is a multistep process that includes supersaturation, nucleation, aggregation, growth, and retention. In ayurvedic medicine, medicinal plants are used for the management of kidney stones. The objective of this study was to determine the effect of aqueous, ethanol, and hexane extracts of Drymoglossum piloselloides leaves, Kalanchoe laciniata leaves, and Aegle marmelos flowers against CaOx urolithiasis in vitro. Methods: The crystallization of CaOx monohydrate (COM) and dihydrate (COD) was induced in a synthetic urine system. The nucleation, growth, and aggregation of crystals were measured using spectrophotometric methods. The results were compared against the polyherbal drug, Cystone, under identical concentrations. Crystals generated in the urine were also observed under light microscopy. Statistical differences and percentage inhibitions were calculated using standard formulae and compared. A preliminary phytochemical screening was also performed to detect active phytoconstituents present in the three plants used in the study. Results: The results obtained clearly demonstrated that Kalanchoe laciniata, Aegle marmelos, and Drymoglossum piloselloides have the capacity to inhibit the nucleation, growth, and aggregation of CaOx crystals. Microscopic examination of crystals revealed the presence of more COM than COD crystals but a dose-dependent reduction in crystals was observed in the presence of plant extracts. Hexane, ethanol, and aqueous extracts of all three plants had different capabilities to inhibit nucleation, growth, and aggregation of CaOx crystals but their activities were different at different concentrations. Preliminary phytochemical screening revealed the presence of reducing sugars, proteins, flavonoids, tannins, and polyphenol compound in Kalanchoe laciniata and Drymoglossum piloselloides and reducing sugars, proteins, anthracene glycosides, and saponins in Aegle marmelos. Conclusions: This study provided evidence that Kalanchoe laciniata, Aegle marmelos, and Drymoglossum piloselloides have the potential to be developed as inhibitors of nucleation, growth, and aggregation of CaOx crystals in the treatment of urolithiasis.

Nauclea orientalis (L.) Bark Extract Protects Rat Cardiomyocytes from Doxorubicin-Induced Oxidative Stress, Inflammation, Apoptosis, and DNA Fragmentation.

The therapeutic efficacy of anthracycline antibiotic, doxorubicin (Dox), is hampered due to the dose-dependent cardiotoxicity. The objective of the study was to explore the counteraction of aqueous bark extract of Nauclea orientalis in Dox-induced cardiotoxicity in Wistar rats. The acute and subchronic toxicity study performed with 2.0 g/kg of the plant extract revealed biochemical and haematological parameters to be within the physiological range, and no histological alterations were observed in any organs isolated. Screening of plant extract for the protection of the myocardium from Dox-induced oxidative stress, inflammation, and apoptosis was performed on five groups of rats: control, plant extract control, Dox control (distilled water (D.H2O) 2 weeks + on the 11th day single injection of Dox, 18 mg/kg), plant + Dox (2.0 g/kg plant extract 2 weeks + on the 11th day Dox, 18 mg/kg), and positive control, dexrazoxane. A significant increase in cardiac biomarkers and lipid peroxidation (p < 0.001) and a significant decrease in antioxidant parameters (p < 0.001) were observed in the Dox control group. All these parameters were reversed significantly (p < 0.05) in the plant-pretreated group. The histopathological assessment of myocardial damage provided supportive evidence for the biochemical results obtained. Inflammatory markers, myeloperoxidase, expression of TNFα and caspase-3, and DNA fragmentation (TUNEL positive nuclei) were significantly elevated (p < 0.05), and expression of Bcl-2 was significantly decreased (p < 0.05) in the Dox control; however, all these parameters were significantly reversed in the plant extract-treated group. In conclusion, the aqueous bark extract of Nauclea orientalis (2.0 g/kg) has the ability to attenuate the Dox-induced oxidative stress, inflammation, apoptosis, and DNA fragmentation in Wistar rats.

Cinnamomum zeylanicum Blume (Ceylon cinnamon) bark extract attenuates doxorubicin induced cardiotoxicity in Wistar rats.

Anti-tumour efficacy of doxorubicin is hindered by the cumulative dose-dependent cardiotoxicity induced by reactive oxygen species during its metabolism. As Cinnamomum zeylanicum has proven antioxidant potential, objective of this study was to investigate the cardioprotective activity of Cinnamomum bark extract against doxorubicin induced cardiotoxicity in Wistar rats. Physicochemical and phytochemical analysis was carried out and dose response effect and the cardioprotective activity of Cinnamomum were determined in vivo. 180 mg/kg dexrazoxane was used as the positive control. Plant extracts were free of heavy metals and toxic phytoconstituents. In vivo study carried out in Wistar rats revealed a significant increase (p < 0.05) in cardiac troponin I, NT-pro brain natriuretic peptide, AST and LDH concentrations in the doxorubicin control group (18 mg/kg) compared to the normal control. Rats pre-treated with the optimum dosage of Cinnmamomum (2.0 g/kg) showed a significant reduction (p < 0.05) in all above parameters compared to the doxorubicin control. A significant reduction was observed in the total antioxidant capacity, reduced glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase activity while the lipid peroxidation and myeloperoxidase activity were significantly increased in the doxorubicin control group compared to the normal control (p < 0.05). Pre-treatment with Cinnamomum bark showed a significant decrease in lipid peroxidation, myeloperoxidase activity and significant increase in rest of the parameters compared to the doxorubicin control (p < 0.05). Histopathological analysis revealed a preserved appearance of the myocardium and lesser degree of cellular changes of necrosis in rats pre-treated with Cinnamomum extract. In conclusion, Cinnamomum bark extract has the potential to significantly reduce doxorubicin induced oxidative stress and inflammation in Wistar rats.

Chemical Composition and Antimicrobial Activity of Two Sri Lankan Lichens, Parmotrema rampoddense, and Parmotrema tinctorum against Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus.

INTRODUCTION: Medicinal utility of lichens is ascribed to the presence of various secondary metabolites of low molecular weight and they have been used in traditional medicine including Ayurveda in the treatment of wounds and skin disorders. Despite the urgent need to effectively address the antibiotic resistance worldwide, the discovery of new antibacterial drugs has declined in the recent past. This emphasizes the increasing importance of investigating and developing new classes of antibiotics that can withstand antibiotic resistance. Aims of the study. The present study was conducted to investigate the chemical composition and the antibacterial activity of hexane, ethanol, and aqueous extracts of Parmotrema rampoddense and Parmotrema tinctorum, two lichens collected from Belihuloya, Sri Lanka, against Gram-negative and Gram-positive bacteria including twenty clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). Materials and methods. Phytochemical analysis, thin layer chromatography (TLC), and Gas Chromatography Mass Spectrometry (GC-MS) were performed to determine the chemical composition of the two lichens. Hexane, ethanol, and aqueous extracts of both lichens were tested against clinical isolate of Gram-negative and Gram-positive bacteria including twenty clinical isolates of MRSA. Bacterial susceptibility was tested using a disc diffusion assay. Minimum inhibitory concentration (MIC) was determined by a broth microdilution method. Vancomycin was used as the positive control. RESULTS: Alectorialic acid, atranorin, atraric acid, orcinol, and O-orsellinaldehyde were among the secondary metabolites identified by the TLC and GC-MS analysis. None of the lichen extracts were active against Gram-negative bacteria but both lichens showed a concentration-dependent activity against methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA. Ethanol extract of P. rampoddense showed the highest activity against MSSA with the MIC, 0.0192 mg/ml, but all MRSA isolates investigated showed MIC between 0.096 and 2.4 mg/ml for the same extract. CONCLUSION: Both lichens, P. rampoddense and P. tinctorum, represent potentially important sources of future antimicrobial drugs. Further investigation on the ethanol extract of P. rampoddense will enable us to determine the most active phytoconstituents responsible for the activity, their mechanism of action against bacterial pathogens, and also their cytotoxicity against normal cells.

Concordance between Body Composition Indices Measured with Dual-Energy X-Ray Absorptiometry and Bioelectrical Impedance Analysis in Obese Children in Sri Lanka.

Dual-energy X-ray absorptiometry (DXA) is the reference standard in the measurement of body composition indices. But, its utility is limited due to the high cost, expertise required, lack of portability, and restricted availability. Therefore, bioelectrical impedance analysis (BIA) has gained recognition in resource-limited settings for the measurement of body composition indices in the screening of children for childhood obesity. To determine whether BIA represents a viable alternative to DXA in the assessment of body composition in obese children in the community setting in Sri Lanka, the concordance between BIA and DXA was determined. Fat mass (FM), percentage fat mass (%FM), and fat-free mass (FFM) were measured in 97 obese children using DXA and BIA, and the concordance between the methods was analyzed using independent sample t-test, regression analysis, and Bland-Altman plots. Significant mean differences were observed between DXA and BIA in measuring FM and FFM. However, high correlations were seen in DXA- and BMI-derived FM and FFM measurements (FM r = 0.92 and FFM 0.83, P < 0.001 for both). Compared to DXA, BIA overestimated FM and %FM and underestimated FFM. When compared with DXA-derived measurements, the accuracy errors (SEE) of BIA for FM, FFM, and %FM were relatively higher in boys (3.56 kg, 4.49 kg, and 5.46%, respectively) than in girls (2.44 kg, 3.72 kg, and 3.5%), respectively. BA plots showed a systematic error in the measurements of FM, FFM, and %FM in both sexes. Despite the limitations inherited, BIA is a viable alternative to DXA for the measurement of body composition in obese children of 5-15 yrs. The accuracy errors observed, however, need to be taken into consideration when interpreting results at the individual level.

Anthracycline-Induced Cardiotoxicity in Breast Cancer Patients from Southern Sri Lanka: An Echocardiographic Analysis.

Anthracycline-induced cardiotoxicity has never been investigated in Sri Lanka. Therefore, this study was conducted to determine the prevalence of anthracycline-induced cardiotoxicity in breast cancer patients using echocardiographic findings. A prospective cohort study was performed. All newly diagnosed breast cancer patients who were administered with anthracycline and cyclophosphamide (AC schedule) for the first time were enrolled in the study. In the hospital setting, anthracycline is administered only as a combination therapy, and only this combination was selected to limit the effect of other cardiotoxic chemotherapy agents. Records of echocardiography were obtained: one day before anthracycline chemotherapy (baseline), one day after the first chemotherapy dose, one day after the last chemotherapy dose, and six months after the completion of anthracycline chemotherapy. Following parameters were recorded from the echocardiography results: ejection fraction (EF, %), fractioning shortening (FS, %), posterior wall thickness, left ventricle (PWT, mm), the thickness of interventricular septum (IVS, mm), left ventricular end-diastolic diameter (LVEDD, mm), and left ventricular end-systolic diameter (LVESD, mm). Statistical analysis of the echocardiography results was performed using ANOVA at four stages. A p value <0.05 was considered significant. Subclinical cardiac dysfunction was defined as a fall of EF >10% during the follow-up echocardiography. There was no significant change (p > 0.05) between the baseline echocardiographic parameters and one day after the 1st anthracycline dose. However, significant differences (p < 0.05) were observed between the baseline echocardiographic parameters and one day after the last anthracycline dose and six months after the completion of anthracycline therapy with a gradual and progressive deterioration in functional parameters including EF, FS, PWT, and IVS over time. There were 65 patients out of 196 (33.16%) who developed subclinical cardiac dysfunction six months after the completion of anthracycline chemotherapy. The prevalence of subclinical anthracycline-induced cardiotoxicity was relatively higher in these patients. An equation was also developed based on left ventricular ejection fraction (LVEF) to predict the anthracycline-induced cardiotoxicity of a patient six months after the completion of anthracycline chemotherapy. We believe that this will help in the monitoring of patients who undergo anthracycline therapy for cardiotoxicity. It is recommended to carry out a long-term follow-up to detect early-onset chronic progressive cardiotoxicity in all patients who were treated with anthracycline therapy.

Comparison of In Vitro Antibacterial Activity of Epaltes divaricata and Vetiveria zizanioides against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital- and community-acquired infections worldwide. Therefore, this study was conducted to explore the antibacterial activity of the two medicinal plants Epaltes divaricata and Vetiveria zizanioides against strains of MRSA which were isolated from patients with skin and soft tissue infections. Hexane, ethanol, and water extracts of E. divaricata (whole plant) and V. zizanioides (roots) were prepared. Clinical isolates of MRSA strains (n = 20) were used for the study. Bacterial susceptibility was tested using a disc diffusion assay. Minimum inhibitory concentration (MIC) was determined by a broth microdilution method. Vancomycin was used as the positive control. Hexane, ethanol, and water extracts of E. divaricata showed inhibitory zones against MRSA. Except for water extract, both hexane and ethanol extracts of V. zizanioides showed inhibitory zones. MIC ranges of hexane, ethanol, and water extracts in E. divaricata were 0.012-0.32 mg/mL, 0.019-2.4 mg/mL, and 0.019-0.48 mg/mL, respectively. Respective MIC ranges of hexane and ethanol extracts of V. zizanioides were 0.003-0.032 mg/mL and 0.019-2.4 mg/mL. The hexane extract of V. zizanioides inhibited 55% of the selected MRSA strains at a relatively low MIC value of 0.012 mg/mL. The hexane extract of both plants demonstrated inhibition of 75% of MRSA strains at a MIC value of 0.064 mg/mL. Ethanol extract of V. zizanioides and E. divaricata, respectively, inhibited 70% and 45% of MRSA strains at the MIC of 0.096 mg/mL, whereas water extract of E. divaricata inhibited 80% of MRSA strains at the same MIC. Both E. divaricata and V. zizanioides were equally effective against MRSA at a MIC of 0.064 mg/mL. But V. zizanioides was more effective since the hexane extract inhibited more than 50% of MRSA strains at significantly a lower MIC value of 0.012 mg/mL. Fractionation, purification, and identification of active compounds will warrant further evaluation of the therapeutic potential of both plant extracts.

In vitro Evaluation of the Antimicrobial Activity of Leaf Extracts of Litsea iteodaphne Against a Selected Group of Bacteria Including Methicillin-Resistant Staphylococcus aureus.

BACKGROUND: The quest for scientific endorsement of new drugs from plants continues due to the rising antibiotic resistance against pathogenic bacteria. Litsea iteodaphne is used in Sri Lanka in the treatment of infectious diseases. Therefore, in vitro antibacterial activity of L. iteodaphne plant extracts were evaluated against selected human pathogenic bacteria.Materials and Methods:: Antibacterial activity of 400, 40, and 4 mg/ml concentrations of hexane, ethanol, and aqueous leaf extracts of L. iteodaphne were evaluated against Staphylococcus aureus, Psedomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and methicillin-resistant S. aureus (MRSA) clinical isolates using disc diffusion method. Minimum inhibitory concentration (MIC) was identified, and phytochemical screening was carried out. RESULTS: Significant zones of inhibition ranging from 5.7 mm to 8.1 mm, 7.1 mm to 8.0 mm, and 7.6 mm were obtained for ethanol, hexane, and aqueous extracts at 400 mg/ml, respectively, against above four bacteria. For MRSA clinical isolates, zones of inhibition ranging from 6.1 mm to 10.9 mm, 6.7 mm to 10.8 mm, and 6.4 mm to 8.6 mm were obtained for ethanol, hexane, and aqueous extracts at 400 mg/ml, respectively. Ethanol extract of L. iteodaphne showed the lowest MIC value (0.0256 mg/ml). Phytochemical screening revealed the presence of tannins, cardiac glycosides, reducing sugars, phenolic compounds, saponins, and flavonoids. CONCLUSIONS: L. iteodaphne crude leaf extracts showed promising antibacterial activity against Gram-positive and Gram-negative bacteria and clinical isolates of MRSA. Further investigations toward fractionation and the identification of an active compound will enhance the antimicrobial potential of L. iteodaphne.

Cardioprotective Potential of Murraya koenigii (L.) Spreng. Leaf Extract against Doxorubicin-Induced Cardiotoxicity in Rats.

Dose-dependent cardiotoxicity of doxorubicin may lead to irreversible congestive heart failure. Although multiple mechanisms are involved, generation of free radicals is the most commonly postulated mechanism. Therefore, free radical scavengers are considered as potential therapeutic agents. As Murraya koenigii leaves are a rich source of flavonoids and phenols, they have the ability to scavenge free radicals effectively. Therefore, the objective of this study was to investigate the cardioprotective potential of Murraya leaf extract against doxorubicin-induced cardiotoxicity in rats. Rats were randomly divided into five groups with 10 animals in each group. Doxorubicin was administered intraperitonially at 18 mg/kg while lyophilized plant extract was administered orally at 2 g/kg. Dexrazoxane, at 180 mg/kg, was used as the positive control. Cardiac damage of doxorubicin control was evident with a significant increase (p < 0.05) in cardiac troponin I, NT-pro BNP, AST, and LDH compared to the normal control. Plant-treated group showed cardioprotective effect by significantly reducing (p < 0.05) all of the above parameters compared to doxorubicin control (p < 0.05). Increased oxidative stress in doxorubicin control was evident with a significant reduction in reduced glutathione, glutathione reductase, glutathione peroxidase, total antioxidant capacity, superoxide dismutase, and catalase activity and a significant increase in lipid peroxidation compared to the control. Interestingly, treatment with Murraya leaf extract showed a significant increase in all of the above antioxidant parameters and a significant reduction in lipid peroxidation by showing an antioxidant effect. A significant increase in myeloperoxidase activity confirmed the increased inflammatory activity in doxorubicin control group whereas plant-treated group showed a significant reduction (p < 0.05) which expressed the anti-inflammatory effect of Murraya leaf extract. Doxorubicin-treated group showed histological evidence of extensive damage to the myocardium while plant-treated group showed a preserved myocardium with lesser degree of damage. Pretreatment with Murraya leaf extract may replenish cardiomyocytes with antioxidants and promote the defense against doxorubicin-induced cardiotoxicity.

Determination of In Vitro Antimicrobial Activity of Five Sri Lankan Medicinal Plants against Selected Human Pathogenic Bacteria.

INTRODUCTION: Antibiotic resistance is one of the greatest threats of the 21st century. Scientists search for potential antimicrobial sources that can cope with antibiotic resistance. Plants used in traditional medicine can be identified as potential candidates for the synthesis of novel drug compounds to act against antibiotic-resistant bacteria. OBJECTIVE: To determine the potential antimicrobial effects of ethanol, aqueous, and hexane extracts of five Sri Lankan medicinal plants against four human pathogens. METHODS: Asparagus falcatus (tubers), Asteracantha longifolia (whole plant), Vetiveria zizanioides (roots), Epaltes divaricata (whole plant), and Coriandrum sativum (seeds) were used in the study. Plant extracts were screened against four clinically important Gram-positive and Gram-negative bacterial strains, Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 700603). Antibacterial activity of plant extracts were monitored using the agar disc diffusion method. Eight concentrations of each positive plant extract were used to determine the minimum inhibitory concentration (MIC) by 5-fold dilution of plant extracts yielding a serial dilution of the original extract. RESULTS: Ethanol, aqueous, and hexane extracts of E. divaricata gave the maximum zones of inhibition of 16.3 mm, 7.4 mm, and 13.7 mm and MIC values of 0.48 mg/ml, 1.2 mg/ml, and 1.6 mg/ml, respectively, against S. aureus. Ethanol and hexane extracts of V. zizanioides gave the maximum zones of inhibition of 12.1 mm and 11.4 mm and MIC values 2.4 mg/ml and 0.003 mg/ml, respectively, against S. aureus. None of the other plants were effective against any microorganism used for the study. CONCLUSIONS: It can be concluded that E. divaricata and V. zizanioides crude ethanol, aqueous, and hexane extracts exhibited significant in vitro antibacterial activity against S. aureus, and the active compounds isolated from them can be potential sources for the synthesis of antibacterial drugs.

Cord Leptin, C-Peptide and Insulin Levels in Large for Gestational Age Newborns in Sri Lanka.

BACKGROUND: Large for gestational age (LGA) infants are more prone to be obese and are at a higher risk of metabolic complications later in life. It is established that Asians have lower skeletal muscle mass and excess body fat for a given body mass index. Thus, objective of this study was to determine the relationship between leptin, insulin, C-peptide in cord blood on the birth weight of newborns and to determine whether these parameters are deviated from data already published from other populations. METHODS: Umbilical cord blood was collected from 90 newborns (male 50, gestational age 38-42 weeks) which comprise of 43 LGA and 47 appropriate for gestational age (AGA) newborns. Serum leptin, insulin and C-peptide levels were measured and anthropometric parameters of the newborn and maternal characteristics were recorded. RESULTS: Significantly higher (P < 0.001) concentrations of leptin, insulin and C-peptide levels (12.670 ± 2.345 ng/mL, 18.725 ± 0.644 µIU/mL, 9.318 ± 0.772 ng/mL) were observed in the LGA group compared to AGA group (7.108 ± 0.906 ng/mL, 13.081 ± 0.428 µIU/mL, 5.439 ± 0.192 ng/mL) and all three parameters showed positive and significant correlations with anthropometric parameters of the newborn and maternal characteristics. CONCLUSION: Although increased leptin, insulin and C-peptide levels may be involved in insulin resistance, increased adiposity and macrosomia, they were not significantly deviated from published data from other populations. Other factors may contribute to higher fat mass found in Asian populations and finding this relationship during neonatal period is useful to predict risk factors for childhood obesity.

The GSTM2 C-Terminal Domain Depresses Contractility and Ca2+ Transients in Neonatal Rat Ventricular Cardiomyocytes.

The cardiac ryanodine receptor (RyR2) is an intracellular ion channel that regulates Ca2+ release from the sarcoplasmic reticulum (SR) during excitation-contraction coupling in the heart. The glutathione transferases (GSTs) are a family of phase II detoxification enzymes with additional functions including the selective inhibition of RyR2, with therapeutic implications. The C-terminal half of GSTM2 (GSTM2C) is essential for RyR2 inhibition, and mutations F157A and Y160A within GSTM2C prevent the inhibitory action. Our objective in this investigation was to determine whether GSTM2C can enter cultured rat neonatal ventricular cardiomyocytes and influence contractility. We show that oregon green-tagged GSTM2C (at 1 µM) is internalized into the myocytes and it reduces spontaneous contraction frequency and myocyte shortening. Field stimulation of myocytes evoked contraction in the same percentage of myocytes treated either with media alone or media plus 15 µM GSTM2C. Myocyte shortening during contraction was significantly reduced by exposure to 15 µM GSTM2C, but not 5 and 10 µM GSTM2C and was unaffected by exposure to 15 µM of the mutants Y160A or F157A. The amplitude of the Ca2+ transient in the 15 µM GSTM2C - treated myocytes was significantly decreased, the rise time was significantly longer and the decay time was significantly shorter than in control myocytes. The Ca2+ transient was not altered by exposure to Y160A or F157A. The results are consistent with GSTM2C entering the myocytes and inhibiting RyR2, in a manner that indicates a possible therapeutic potential for treatment of arrhythmia in the neonatal heart.

The inhibitory glutathione transferase M2-2 binding site is located in divergent region 3 of the cardiac ryanodine receptor.

The muscle-specific glutathione transferase GSTM2-2 modulates the activity of ryanodine receptor (RyR) calcium release channels: it inhibits the activity of cardiac RyR (RyR2) channels with high affinity and activates skeletal RyR (RyR1) channels with low affinity. The C terminal domain of GSTM2-2 (GSTM2C) alone physically binds to RyR2 and inhibits its activity, but it does not bind to RyR1. We have now used yeast two-hybrid analysis, chemical cross-linking, intrinsic tryptophan fluorescence and Ca(2+) release studies to determine that the binding site for GSTM2C is in divergent region 3 (D3) of RyR2. The D3 region encompasses residues 1855-1890 in RyR2. Specific mutagenesis shows the binding primarily involves electrostatic interactions with residues K1875, K1886, R1887 and K1889, all residues that are present in RyR2, but not in RyR1. The significant sequence differences between the D3 regions of RyR2 and RyR1 explain why GSTM2-2 specifically inhibits RyR2. This specific inhibition of RyR2 could modulate Ca cycling and be useful for the treatment of heart failure. RyR2 inhibition during diastole may improve filling of the SR with Ca(2+) and improve contractility.

Regulation of the cardiac muscle ryanodine receptor by glutathione transferases.

Glutathione transferases (GSTs) are generally recognized for their role in phase II detoxification reactions. However, it is becoming increasingly apparent that members of the GST family also have a diverse range of other functions that are, in general, unrelated to detoxification. One such action is a specific inhibition of the cardiac isoform of the ryanodine receptor (RyR2) intracellular Ca(2+) release channel. In this review, we compare functional and physical interactions between members of the GST family, including GSTO1-1, GSTA1-1, and GSTM2-2, with RyR2 and with the skeletal isoform of the ryanodine receptor (RyR1). The active part of the muscle-specific GSTM2-2 is localized to its nonenzymatic C-terminal α-helical bundle, centered around α-helix 6. The GSTM2-2 binding site is in divergent region 3 (DR3 region) of RyR2. The sequence differences between the DR3 regions of RyR1 and RyR2 explain the specificity of the GSTs for one isoform of the protein. GSTM2-2 is one of the few known endogenous inhibitors of the cardiac RyR and is likely to be important in maintaining low RyR2 activity during diastole. We discuss interactions between a nonenzymatic member of the GST structural family, the CLIC-2 (type 2 chloride intracellular channel) protein, which inhibits both RyR1 and RyR2. The possibility that the GST and CLIC2 proteins bind to different sites on the RyR, and that different structures within the GST and CLIC proteins bind to RyR channels, is discussed. We conclude that the C-terminal part of GSTM2-2 may provide the basis of a therapeutic compound for use in cardiac disorders.

The structure of the C-terminal helical bundle in glutathione transferase M2-2 determines its ability to inhibit the cardiac ryanodine receptor.

Ca(2+) release from the sarcoplasmic reticulum through cardiac ryanodine receptors (RyR2) is essential for heart function and is inhibited by the carboxy terminal domain of glutathione transferase M2-2 (GSTM2-C) and derivative fragments containing helix 6. Since a peptide encoding helix 6 alone does not fold into a helix and does not inhibit RyR2 Ca(2+) release, the importance of the structure of helix 6 and its role in stabilizing GSTM2-C was tested by inserting potentially destabilizing mutations into this helical segment. GSTM2-C preparations with D156A or L163A mutations were so insoluble that the protein could not be purified. Proteins with F157A and Y260A substitutions were soluble, but had lost their capacity to inhibit both RyR2 Ca(2+) release from vesicles and RyR2 channels in bilayers. Circular dichroism studies indicated that these mutated proteins retained their helical secondary structure, although changes in their endogenous tryptophan fluorescence indicated that the F157A and Y160A mutations caused changes in their folding. The single channel studies were conducted with 2mM ATP and 10microM Ca(2+) in the cytoplasmic solution, mimicking concentrations in the cytosol of cardiac myocytes. Wild type GSTM2-C inhibited RyR2 only at a potential of +40mV, which may develop during Ca(2+) efflux, but not at -40mV. Together, the results indicate that the structure of helix 6 in the C-terminal fold is critical to the inhibitory action of GSTM2-2 and suggest that therapeutics mimicking this structure may reduce excess Ca(2+) release during diastole, which can lead to fatal arrhythmia.

Dissection of the inhibition of cardiac ryanodine receptors by human glutathione transferase GSTM2-2.

The muscle specific glutathione transferase GSTM2-2 inhibits the activity of cardiac ryanodine receptor (RyR2) calcium release channels with high affinity and activates skeletal RyR (RyR1) channels with lower affinity. To determine which overall region of the GSTM2-2 molecule supports binding to RyR2, we examined the effects of truncating GSTM2-2 on its ability to alter Ca(2+) release from sarcoplasmic reticulum (SR) vesicles and RyR channel activity. The C-terminal half of GSTM2-2 which lacks the critical GSH binding site supported the inhibition of RyR2, but did not support activation of RyR1. Smaller fragments of GSTM2-2 indicated that the C-terminal helix 6 was crucial for the action of GSTM2-2 on RyR2. Only fragments containing the helix 6 sequence inhibited Ca(2+) release from cardiac SR. Single RyR2 channels were strongly inhibited by constructs containing the helix 6 sequence in combination with adjacent helices (helices 5-8 or 4-6). Fragments containing helices 5-6 or helix 6 sequences alone had less well-defined effects. Chemical cross-linking indicated that C-terminal helices 5-8 bound to RyR2, but not RyR1. Structural analysis with circular dichroism showed that the helical content was greater in the longer helix 6 containing constructs, while the helix 6 sequence alone had minimal helical structure. Therefore the active centre of GSTM2-2 for inhibition of cardiac RyR2 involves the helix 6 sequence and the helical nature of this region is essential for its efficacy. GSTM2-2 helices 5-8 may provide the basis for RyR2-specific compounds for experimental and therapeutic use.