Biomonitoring Study of Urinary Bisphenol A Levels and Impact of Bottle-Feeding Practices Among Infants and Children From Northern India.

OBJECTIVES: To compare the urinary bisphenol A (BPA) levels in bottle-fed and never bottle-fed infants and under-five children and to determine the impact of bottle-feeding practices and sociodemographic factors on urinary BPA levels. METHODS: A community-based cross-sectional study was carried out on children aged between 2 to 60 months attending the Anganwadi centres in Chandigarh. RESULTS: Urine samples were collected from 184 children, out of which 94.56% (n = 174) children had detectable urinary BPA levels. The mean (SD) BPA level was 2.74 (2.60) ng/ml and BPA was detected in 93.9% of 'ever' bottle-fed children (n = 93/99) and 95.3% of 'never' bottle-fed children (n = 81/85) (P = 0.69). On multivariate regression analysis, there were no significant predictors for high (≥ 75th percentile) urinary BPA levels. Still, the odds of urinary BPA levels ≥75th percentile showed higher trend for significance among children from middle/higher socioeconomic background in reference to lower socioeconomic stratum (adjusted OR 7.02; 95% CI 1.24, 133.25; P = 0.07) and among children whose feeding bottles were brushed once or twice daily in reference to group with no daily brushing (adjusted OR 3.92, 95% CI 0.95, 20.56; P = 0.07). CONCLUSIONS: Although feeding with plastic bottle did not emerge as a statistically significant risk factor for BPA exposure, yet detection of BPA levels among majority of study children signals urgent need for unmasking exposure to other sources given the potential long-term toxicity of BPA among infants and young children.

A randomised controlled trial to evaluate the effectiveness of a culture and disease-specific, patient-centric multi-component tobacco cessation intervention package for the patients attending non-communicable disease clinics in Punjab, India.

BACKGROUND: Developing an infrastructure to support tobacco cessation through existing systems and resources is crucial for ensuring the greatest possible access to cessation services. The present study aims to evaluate the effectiveness of a newly developed multi-component cessation among tobacco users in Non- Communicable Disease (NCD) clinics, functioning under the National Programme for Prevention & Control of Cancer, Diabetes, Cardiovascular Diseases, & Stroke (NPCDCS) of the Government of India. METHODS: The intervention package consisting of culture- and disease-specific four face-to-face counselling sessions, pamphlets, and short text messages (bilingual) with follow-ups at 3rd, 6th, and 9th months with an endline assessment at 12th months was delivered to the intervention arm of the two-arm- parallel group randomised controlled trial at two selected NCD clinics. Self-reported seven-day abstinence, frequency of use, expenditure in seven days at each follow-up, FTND score, stage of change and plasma cotinine values were assessed at baseline, follow-ups, and endline (using Liquid Chromatography -Mass Spectrometry), as applicable. RESULTS: The intervention arm reported a significantly more reduction in self-reported frequency of tobacco use at 6 months (mean: 13.6, 95% CI (7.8-19.4)), 9 months (mean: 20.3, 95% CI (12.2-28.4)) and 12 months (mean: 18.7, 95% CI (8.7-28.7)). The plasma cotinine concentration at endline in the intervention arm was statistically significantly lower than the baseline concentration. CONCLUSION: Strengthening existing health systems is crucial for offering cessation support in the resource-restraint setting of LMICs to assist in quitting sustainably.

Rat BM-MSCs secretome alone and in combination with stiripentol and ISRIB, ameliorated microglial activation and apoptosis in experimental stroke.

BACKGROUND: Stroke, a devastating neurological emergency, is the leading cause of worldwide mortality and functional disability. Combining novel neuroprotective drugs offers a way to improve the stroke intervention outcomes. In the present era, the combination therapy has been proposed as a plausible strategy to target multiple mechanisms and enhance the treatment efficacy to rescue stroke induced behavioral abnormalities and neuropathological damage. In the current study, we have investigated the neuroprotective effect of stiripentol (STP) and trans integrated stress response inhibitor (ISRIB) alone and in combination with rat bone marrow derived mesenchymal stem cells (BM-MSCs) secretome in an experimental model of stroke. MATERIALS & METHODS: Stroke was induced in male Wistar rats (n = 92) by temporary middle cerebral artery occlusion (MCAO). Three investigational agents were selected including STP (350 mg/kg; i.p.), trans ISRIB (2.5 mg/kg; i.p.) and rat BM-MSCs secretome (100 µg/kg; i.v). Treatment was administered at 3 hrs post MCAO, in four doses with a 12 hrs inter-dose interval. Post MCAO, neurological deficits, brain infarct, brain edema, BBB permeability, motor functional and memory deficits were assessed. Molecular parameters: oxidative stress, pro inflammatory cytokines, synaptic protein markers, apoptotic protein markers and histopathological damage were assessed. RESULTS: STP and trans ISRIB, alone and in combination with rat BM-MSCs secretome, significantly improved neurological, motor function and memory deficits along with significant reduction in pyknotic neurons in the brain of post MCAO rats. These results were correlating with significant reduction in pro-inflammatory cytokines, microglial activation and apoptotic markers in the brain of drug treated post MCAO rats. CONCLUSION: STP and trans ISRIB, alone and in combination with rat BM-MSCs secretome, might be considered as potential neuroprotective agents in the acute ischemic stroke (AIS) management.

Detection of IEMs by Mass Spectrometry Techniques in High-Risk Children: A Pilot Study.

OBJECTIVES: To determine the incidence and types of inborn errors of metabolism (IEMs) in high-risk children using mass spectrometry techniques. METHODS: Children considered high-risk for IEM were screened for metabolic diseases during a 3-y period. Dried blood spots and urine samples were analyzed by tandem mass spectrometry (LC-MS/MS) and gas chromatograph-mass spectrometry (GCMS). Samples with abnormal amino acids were confirmed by high-performance liquid chromatography (HPLC). RESULTS: Eight hundred and twenty-two suspected cases were evaluated; of which, 87 possible cases of IEMs were identified. Homocystinuria (n = 51) was the most common IEM detected followed by biotinidase deficiency (n = 7), glutaric aciduria type 1 (n = 7), and carnitine uptake defect (n = 6). Overall, there were 45 (51.7%) cases of organic acidemia, 31 cases (35.6%) of amino acid defect, 9 (10.3%) cases of fatty-acid oxidation disorders, and 2 (2.3%) cases of probable mitochondrial disorder. CONCLUSION: IEMs are common in India, with a hospital-based incidence of 1 in approximately 6642 among high-risk children. Screening of high-risk children by mass spectrometry techniques is a valuable strategy for early diagnosis of IEMs where universal newborn screening is not yet available.

Cyclophosphamide, hydroxycyclophosphamide and carboxyethyl phosphoramide mustard quantification with liquid chromatography mass spectrometry in a single run human plasma samples: A rapid and sensitive method development.

A novel method for simultaneous quantification of cyclophosphamide along with its two major metabolites namely 4-hydroxycyclophosphamide (HCy) and carboxyethyl phosphoramide mustard (CEPM) in a single sample run was demonstrated in the present study. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument was used for analysis. Semicarbazide was used as a stabilizing agent for HCy whereas ifosfamide, hexamethyl phosphoramide mustard and deuterated CEPM were the internal standards for quantification of Cy, HCy and CEPM respectively. Chromatographic separation was achieved by Chromsystems C18 reverse-phase column (50 mm × 4.6 mm, particle size 3.2 µm). The mobile phase was composed of eluent A (2 mM ammonium acetate in water with 2% formic acid) and eluent B (100 % acetonitrile). The flow rate was 1 ml/min. Linearity of the assay was assured in the range of 19.53 ng/ml to 10,000 ng/ml concentration in human plasma, which is adequate for pharmacokinetic studies of any dose Cy used clinically. The quality control(QC) accuracy was between 99.58% and 101.62%, 97.85% to 103.53% and 99.64% to 100.10% for Cy, HCy and CEPM respectively. Precision limits for QC samples were between 3.9% and 9.4%, 5.2% to 8.9% and 1.8% to 9.2% respectively. The analytical method was validated in ten leukaemia patients undergoing haploidentical hematopoietic cell transplantation.

Plasma and Urinary Amino Acid Profile in Children with Infantile Tremor Syndrome.

BACKGROUND: Infantile Tremor Syndrome (ITS) is a disorder of infancy, and characterized by developmental delay and/or regression, pallor, skin hyperpigmentation and hypopigmented hair. It is commonly seen in infants in whom exclusive breastfeeding is given inappropriately for longer durations than recommended. ITS is predominantly reported from the Indian subcontinent and in children from a lower socioeconomic background. It is a clinical diagnosis and vitamin B12 deficiency is the most commonly accepted etiology of this entity. OBJECTIVES: The primary objectives of study were to compare the plasma and urine amino acid levels among children with ITS spectrum with those of healthy children. The secondary objectives were to compare the plasma and urine amino acid levels among children with ITS and Pre-ITS. STUDY DESIGN: This cross-sectional, observational study was carried out at a tertiary care hospital in North India. PARTICIPANTS: A total of 50 children aged < 36 months with ITS/Pre-ITS were enrolled. Children with Pre-ITS and ITS were compared with healthy age-matched study subjects. RESULTS: Thirty-nine (78%) cases and twelve (24%) healthy children had low serum vitamin B12 levels. Folate levels were normal in all the controls, while only one case had folate deficiency. There were significant differences (p < 0.05) in the values of 32 amino acids in plasma. Among 44 urinary amino acids, levels of 30 amino acids were significantly different in the cases compared with the controls (p < 0.05). CONCLUSIONS: Several changes in amino acids in the children suffering from ITS were observed. These changes may be a reflection of the metabolic derangements in ITS.

Bone density and bone health alteration in boys with Duchenne Muscular Dystrophy: a prospective observational study.

OBJECTIVES: Boys with Duchenne Muscular Dystrophy (DMD) are at increased risk for compromised bone health, manifesting as low-impact trauma long bone fractures and vertebral compression fractures. METHODS: In a prospective observational study, we studied bone health parameters in North Indian boys with DMD. We consecutively enrolled ambulatory boys with DMD on glucocorticoid therapy. Bone health was evaluated with X-ray spine, Dual-energy X-ray absorptiometry (DXA), serum calcium, vitamin D3 (25[OH]D), 1,25-dihyroxyvitamin D3 (1,25[OH]2D3), serum osteocalcin, osteopontin, and N terminal telopeptide of type 1 collagen (Ntx) levels. RESULTS: A total of 76 boys with DMD were enrolled. The median age was 8.5 (interquartile range [IQR] 7.04-10.77) years. Among these, seven (9.2%) boys had long bone fractures, and four (5.3%) had vertebral compression fractures. Fifty-four (71%) boys underwent DXA scan, and among these 31 (57%) had low bone mineral density (BMD, ≤-2 z-score) at the lumbar spine. The mean BMD z-score at the lumbar spine was -2.3 (95% confidence interval [CI] = -1.8, -2.8), and at the femoral neck was -2.5 (95% CI = -2, -2.9). 25(OH)D levels were deficient in 68 (89.5%, n=76) boys, and 1,25(OH)2D3 levels were deficient in all. Mean serum osteocalcin levels were 0.68 ± 0.38 ng/mL (n=54), serum osteopontin levels were 8.6 ± 4.6 pg/mL (n=54) and serum Ntx levels were 891 ± 476 nmol/L (n=54). Boys with low BMD received glucocorticoids for longer duration, in comparison to those with normal BMD (median, IQR [16.9 (6-34) months vs. 7.8 (4.8-13.4) months]; p=0.04). CONCLUSIONS: Bone health is compromised in North Indian boys with DMD. BMD at the lumbar spine is reduced in more than half of boys with DMD and nearly all had vitamin D deficiency on regular vitamin D supplements. Longer duration of glucocorticoid therapy is a risk factor for low BMD in our cohort.

Dietary Vitamin A Intake, Coverage of Vitamin A Megadose Supplementation, and Prevalence of Vitamin A Deficiency among Marginalized Children 6-59 Months in Anganwadis of Chandigarh: A Multistage Cluster Sampling Survey.

BACKGROUND: Lack of recent local data regarding Vitamin A deficiency, dietary intake, and coverage of biannual high-dose Vitamin A prophylaxis among marginalized children poses challenges for policy change. OBJECTIVE: Our study aimed to evaluate the burden of serum Vitamin A deficiency, dietary Vitamin A intake, and current Vitamin A supplementation coverage. METHODOLOGY: We enrolled 216 children aged 6-60 months from 30 Anganwadis of Chandigarh using two-stage cluster sampling. RESULTS: Dietary intake of Vitamin A-rich foods and vegetables in the last 24 h was observed only in 20% of enrolled children. Although 79.21% (160/202) of children aged ≥9 months reported receiving Vitamin A supplementation, only 38.52% had documented record of age-appropriate complete supplementation. None of the children showed ocular manifestations of Vitamin A deficiency. For the subjects in which C-reactive protein levels were ≤5 mg/l (n = 148), 35.14% (52/148) had serum retinol ≤0.7 µmol/L and 2.03% (3/148) had serum retinol ≤0.3 µmol/L. The multivariate odds ratios (ORs) of Vitamin A deficiency (serum retinol <0.7) were 2.23 times higher in subjects with inappropriate complementary feeding (OR: 2.23; 95% confidence interval [CI]: 1.07-4.80; P = 0.035) whereas odds were significantly lower for middle (OR: 0.27; 95% CI: 0.09-0.76; P = 0.015) and higher income (OR: 0.14; 95% CI: 0.03-0.53; P = 0.005) families in reference to low-income group. CONCLUSIONS: Subclinical Vitamin A deficiency is still a public health crisis with suboptimal dietary intake of Vitamin A-rich foods and compromised food diversity necessitating targeted approach for marginalized children.

Thiamine Status in Children with Septic Shock from a Developing Country: A Prospective Case-Control Study.

OBJECTIVE: Mitochondrial dysfunction is central to sepsis-induced multi-organ dysfunction. Thiamine deficiency may contribute to mitochondrial dysfunction and thus high mortality. Study was planned to assess thiamine status in children with septic shock in comparison to healthy controls from a developing country and to study the effect of thiamine levels on its outcome. METHODS: A prospective case-control study (April 2017 to May 2018) enrolling consecutive children with septic shock as 'cases' (n = 76), their healthy siblings (n = 51) and apparently healthy children from immunization clinic (n = 35) as 'controls'. Whole blood total thiamine (WBTT) level was measured on days 1, 10 and 1-month post-discharge. Outcome parameters were acute care area free days on days 14 and 28, and mortality. RESULTS: WBTT [nMol/l; median (interquartile range, IQR)] was significantly lower on day 1 in cases compared with sibling controls [23.1 (21.8-26.3) vs. 36.9 (33.6-40.5); p < 0.001]. It fell further on day 10 [20.8 (18.1-21.1); p < 0.02]. Levels rose significantly 1-month post-discharge [35.5 (31.2-36.6)] and became comparable to sibling controls (p = 0.4). Immunization clinic controls also had lower WBTT [42.3 (40.1-45.9)], but was significantly higher than sibling controls and cases at 1-month post-discharge (p < 0.001). Survivors and non-survivors of septic shock were similar. WBTT levels did not correlate with any of the severity indicators of septic shock or its outcomes. CONCLUSIONS: WBTT was significantly low in all children, and fell further during septic shock. Observed severe deficiency might have precluded any further association of thiamine levels with severity of septic shock and its outcome. Data obtained may inform trials on metabolic resuscitation in paediatric septic shock in developing countries. Lay summaryThiamine deficiency may contribute to high mortality in paediatric septic shock as thiamine is an essential factor for functioning of mitochondria, the powerhouse of the cells. This prospective case-control study was conducted to assess thiamine status in children with septic shock in comparison with healthy controls in a developing country. Consecutive children with fluid-refractory septic shock were enrolled as 'cases'. Their apparently healthy siblings, and apparently healthy children from immunization clinic, were enrolled as 'controls'. The whole blood total thiamine (WBTT) level was measured on days 1, 10 and 1 month after hospital discharge. Seventy-six children were enrolled as cases, 51 children as sibling controls and 35 children as immunization clinic controls. WBTT was significantly lower on day 1 in cases as compared with their sibling controls. It fell further on day 10. The level rose significantly after a month of discharge and became comparable to sibling controls. Immunization clinic controls also had lower WBTT but was significantly higher compared with sibling controls and cases at 1-month post-discharge. Survivors and non-survivors of septic shock had similar WBTT levels. Observed severe deficiency might have precluded any further association of thiamine levels with septic shock outcome.

Aneurysmal Subarachnoid Hemorrhage: Impact on Phenytoin Permeability across the Blood-Brain Barrier.

BACKGROUND: Phenytoin (PHT) is a routinely prescribed prophylactic antiepileptic following aneurysmal subarachnoid hemorrhage (aSAH). However, its prophylactic use in aSAH is controversial as emerging evidence suggests worsening of the neurological and functional outcomes. In addition, there is profound damage to the blood-brain barrier (BBB) in aSAH, posing uncertainty about the permeability of PHT across BBB in these patients. This pilot study was designed to evaluate the alteration in PHT permeability across BBB in aSAH patients. MATERIALS AND METHODS: For conducting the study, 20 patients (control n = 10; aSAH (grade 3 or 4) n = 10) were recruited from a tertiary care hospital. The patients undergoing cranial surgery for pathology with intracerebral mass lesions on MRI were chosen as control for aSAH group. Both groups were administered PHT loading dose (20 mg/kg), infused in 5% dextrose, at a rate not more than 50 mg/min, followed by a maintenance dose (5 mg/kg). Quantification of PHT concentration was performed in brain tissue, plasma, and cerebrospinal fluid (CSF) by LC-MS/MS. RESULTS: The median PHT concentration in brain was found to be significantly decreased (64.8%) in aSAH group (3.78 µg/g) as compared to control (10.73 µg/g), P = 0.010. Similarly, median PHT brain concentration as fraction of plasma was significantly decreased in aSAH group (36.72%) compared to that of control (89.55%), P = 0.003. There was no significant difference in PHT concentration in plasma, CSF, and CSF as a fraction of plasma between both the groups. CONCLUSION: There is a definite decrease in the penetration of PHT to the brain in patients with grade 3 and 4 aSAH.

Real-life Bioequivalence of Tacrolimus in Patients With Living-Related Donor De Novo Renal Transplantation: An Observational Study.

Tacrolimus is a narrow therapeutic index drug. As a result, regulatory agencies worldwide recommend stringent bioequivalence evaluation criteria for approval of generics. Despite this, the professional transplantation societies have raised concerns over the safety and efficacy of generic substitutions. We conducted this pragmatic real-life bioequivalence study to assess the effect of generic substitutions of tacrolimus. This was an observational study including recipients of renal transplantation who were considered for generic medication substitution. Transplanted organs were from living-related donors and were performed at least 1 month before the study. Time of administration of the drug, time of dosing with respect to meals, and time of blood sample collection were controlled; however, the lot number of the generic drugs was not controlled. The participants were allowed to use their usual supplies irrespective of the lot number. Concentration (C0) was quantified by liquid chromatography with tandem mass spectrometry after the generic substitution from ABC brand to XYZ brand. The average C0 ± SD with generic ABC was 11.09 ± 4.26 ng/mL and generic ABC was 9.7 ± 4.12 ng/mL. Though there was no statistically significant difference observed between the concentrations, when the individual patient data was examined, 2 patients were found to have a very high concentration of tacrolimus and at least 7 patients fell below the therapeutic range. These derangements called for retitration with the new generic tacrolimus (40%). The results of our study suggest that generic-to-generic substitutions should be carried out very carefully in a closely observed setting in patients with renal transplants. The strength of our study is that it matched the real clinical practice setting as much as possible unlike a bioequivalence study. Therefore, we recommend repeating C0 at least 3 times over a period of 7 to 10 days with a generic substitution to prevent untoward consequences.

A mechanistic approach to explore the neuroprotective potential of zonisamide in seizures.

BACKGROUND: Epilepsy, a disease of the brain, is one of the most common serious neurological conditions. It is associated with a group of processes which alter energy metabolism, interrupt cellular ionic homeostasis, cause receptor dysfunction, activate inflammatory cascade, alter neurotransmitter uptake and result in neuronal damage. The increasing knowledge and understanding about the basis of neuronal changes in epilepsy lead to investigate the mechanistic pathway of neuroprotective agents in epilepsy. With this background, the present study is designed to reveal the molecular and biochemical mechanisms involved in the neuroprotective potential of zonisamide in epilepsy. METHODS: Seizure-induced neuronal damage was produced by maximal electroshock seizures in animals. The oxidative stress and neuroinflammatory and apoptotic markers were assessed in the brain tissue of animals. RESULTS AND DISCUSSION: The present findings revealed that zonisamide treatment prevented the development of seizures in animals. Seizures-induced free radicals production and neuroinflammation were markedly ameliorated by zonisamide administration. In conclusion, the present study demonstrated the mechanisms behind the strong neuroprotective potential of zonisamide against seizures by attenuating the oxidative stress, inflammatory cascade and neuronal death associated with progression of seizures. It can be further developed as a neuroprotective agent for epilepsy and other neurodegenerative disorders.

Ocular Pharmacokinetics of 25-Hydroxyvitamin D3 After Weekly Supplementation in Rabbits Using Ultra Performance Liquid Chromatography-Tandem Mass Spectrometer.

BACKGROUND AND OBJECTIVES: The protective role of vitamin D supplementation has recently been shown to be present in various ocular inflammatory diseases. The oral supplementation of vitamin D may take time to achieve adequate levels in intraocular fluids. Therefore, the present study was performed to understand the ocular pharmacokinetics of 25-hydroxyvitamin D3 (25D3) in aqueous humor after weekly supplementation of 25D3 in rabbits. METHODS: A total of 21 rabbits were fed orally with 25D3 (7.22 µg/kg/week) for 8 weeks and 9th dose was given at the end of 8 weeks. The blood and aqueous humor samples were collected from ear vein and though anterior chamber paracentesis, respectively. The serum and aqueous humor samples were spiked with deuterium labeled internal standard and were extracted using liquid extraction method. Furthermore, the samples were derivatized and 25D3 estimation was performed using ultra performance liquid chromatography-tandem mass spectrometer (UHPLC-MS/MS). RESULTS: The 25D3 supplementation significantly increased the 25D3 levels in serum (78.5 ± 21.6 ng/ml) (mean ± SD) (p < 0.0001) and in aqueous humor (991.3 ± 180.6 pg/ml) (mean ± SD) (p < 0.0001) compared to baseline levels. The maximum concentration was achieved in serum after the 10th hour of supplementation of 1st and 9th dose, while the same was observed at the 24th hour in aqueous humor. CONCLUSION: The oral supplementation of 25D3 was found to significantly increase 25D3 levels in aqueous humor; however, the time required to achieve 25D3 concentration in aqueous humor was higher as compared to that in serum. Therefore, weekly oral supplementation of 25D3 may have a beneficial role in ocular diseases.

Vitamin D deficiency in critically ill children with sepsis.

BACKGROUND: Data on the prevalence of vitamin D deficiency (VDD) in critically ill children with sepsis and its association with illness severity and outcome are limited. AIM: To investigate the prevalence of VDD in critically ill children with sepsis. METHODS: One hundred and twenty-four critically ill children with sepsis aged 1-12 years were prospectively enrolled in a paediatric intensive care unit (PICU) in North India over a 1-year period. Demographic data, clinical signs and risk factors for VDD, Paediatric Index of Mortality III (PRISM III) score, and sequential organ failure assessment (SOFA) score were recorded. Plasma 25-hydroxy vitamin D [25(OH)D] levels were measured by ELISA within 24 hours of admission. The occurrence of septic shock, multiple organ dysfunction syndrome (MODS) and healthcare-associated infection (HCAI), need for mechanical ventilation and catecholamines, length of PICU stay and mortality were also recorded. Cases were compared with 338 apparently healthy children for baseline variables and vitamin D status. RESULTS: Prevalence of VDD [25(OH)D level < 50 nmol/L] was higher among critically ill children with sepsis compared to healthy controls (50.8% vs 40.2%, P = 0.04). VDD was not associated with any significant difference in baseline demographic variables or risk factors for VDD. Although there was a trend toward increased PRISM III score, septic shock, MODS, HCAI, need for mechanical ventilation and catecholamines, length of PICU stay, and mortality, the difference was not statistically significant. CONCLUSION: A high prevalence of VDD in critically ill children with sepsis was found but it was not associated with greater severity of illness or other clinical outcomes.