Development and validation of a liquid chromatography-mass spectrometry method for the quantitation of naltrexone and 6beta-naltrexol in guinea pig plasma

A quantitative liquid chromatographic-electrospray ionization mass spectrometry method for the determination of naltrexone and 6beta-naltrexol in guinea pig plasma has been developed and validated using naloxone as an internal standard. A single step precipitation-extraction technique was carried out to extract the plasma samples using acetonitrile:ethyl acetate (1:1, v/v). The chromatographic separation was performed on a C(18) column using a mobile phase consisting of 35:65 (v/v) acetonitrile:2 mM ammonium acetate with 0.01 mM ammonium citrate at a flow rate of 0.25 mL/min. The analyte was detected after positive electrospray ionization using selected ion monitoring (SIM) mode. The mean recoveries for naltrexone, naltrexol, and naloxone were 91.7, 89.3, and 99.0%, respectively. The lower limit of quantification (LLOQ) for naltrexone and 6beta-naltrexol was 1.25 ng/mL, and the limit of detection (LOD) was 0.75 ng/mL. The method was applied to a pharmacokinetic study in order to assess the drug disposition of naltrexone in guinea pigs.     

Double bond in the side chain of 1alpha,25-dihydroxy-22-ene-vitamin D(3) is reduced during its metabolism: studies in chronic myeloid leukemia (RWLeu-4) cells and rat kidney

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is mainly metabolized via the C-24 oxidation pathway and undergoes several side chain modifications which include C-24 hydroxylation, C-24 ketonization, C-23 hydroxylation and side chain cleavage between C-23 and C-24 to form the final product, calcitroic acid. In a recent study we reported that 1alpha,25-dihydroxyvitamin D(2) [1alpha,25(OH)(2)D(2)] like 1alpha,25(OH)(2)D(3), is also converted into the same final product, calcitroic acid. This finding indicated that 1alpha,25(OH)(2)D(2) also undergoes side chain cleavage between C-23 and C-24. As the side chain of 1alpha,25(OH)(2)D(2) when compared to the side chain of 1alpha,25(OH)(2)D(3), has a double bond between C-22 and C-23 and an extra methyl group at C-24 position, it opens the possibility for both (a) double bond reduction and (b) demethylation to occur during the metabolism of 1alpha,25(OH)(2)D(2). We undertook the present study to establish firmly the possibility of double bond reduction in the metabolism of vitamin D(2) related compounds. We compared the metabolism of 1alpha,25-dihydroxy-22-ene-vitamin D(3) [1alpha,25(OH)(2)-22-ene-D(3)], a synthetic vitamin D analog whose side chain differs from that of 1alpha,25(OH)(2)D(3) only through a single modification namely the presence of a double bond between C-22 and C-23. Metabolism studies were performed in the chronic myeloid leukemic cell line (RWLeu-4) and in the isolated perfused rat kidney. Our results indicate that both 1alpha,25(OH)(2)-22-ene-D(3) and 1alpha,25(OH)(2)D(3) are converted into common metabolites namely, 1alpha,24(R),25-trihydroxyvitamin D(3) [1alpha,24(R),25(OH)(3)D(3)], 1alpha,25-dihydroxy-24-oxovitamin D(3) [1alpha,25(OH)(2)-24-oxo-D(3)], 1alpha,23(S),25-trihydroxy-24-oxovitamin D(3) and 1alpha,23-dihydroxy-24,25,26,27-tetranorvitamin D(3). This finding indicates that the double bond in the side chain of 1alpha,25(OH)(2)-22-ene-D(3) is reduced during its metabolism. Along with the aforementioned metabolites, 1alpha,25(OH)(2)-22-ene-D(3) is also converted into two additional metabolites namely, 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3). Furthermore, we did not observe direct conversion of 1alpha,25(OH)(2)-22-ene-D(3) into 1alpha,25(OH)(2)D(3). These findings indicate that 1alpha,25(OH)(2)-22-ene-D(3) is first converted into 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3). Then the double bonds in the side chains of 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3) undergo reduction to form 1alpha,24(R),25(OH)(3)D(3) and 1alpha,25(OH)(2)-24-oxo-D(3), respectively. Thus, our study indicates that the double bond in 1alpha,25(OH)(2)-22-ene-D(3) is reduced during its metabolism. Furthermore, it appears that the double bond reduction occurs only during the second or the third step of 1alpha,25(OH)(2)-22-ene-D(3) metabolism indicating that prior C-24 hydroxylation of 1alpha,25(OH)(2)-22-ene-D(3) is required for the double bond reduction to occur.     

Heat and PAHs Emissions in Indoor Kitchen Air and Its Impact on Kidney Dysfunctions among Kitchen Workers in Lucknow,North India

Indoor air quality and heat exposure have become an important occupational health and safety concern in several workplaces including kitchens of hotels. This study investigated the heat, particulate matter (PM), total volatile organic compounds (TVOCs) and polycyclic aromatic hydrocarbons (PAHs) emissions in indoor air of commercial kitchen and its association with kidney dysfunctions among kitchen workers. A cross sectional study was conducted on 94 kitchen workers employed at commercial kitchen in Lucknow city, North India. A questionnaire-based survey was conducted to collect the personal and occupational history of the kitchen workers. The urine analysis for specific gravity and microalbuminuria was conducted among the study subjects. Indoor air temperature, humidity, wet/ dry bulb temperature and humidex heat stress was monitored during cooking activities at the kitchen. Particulate matter (PM) for 1 and 2.5 microns were monitored in kitchen during working hours using Hazdust. PAH_S in indoor air was analysed using UHPLC. Urinary hydroxy-PAHs in kitchen workers were measured using GC/MS-MS. Higher indoor air temperature, relative humidity, PM₁ and PM_2.5 (p<0.001) was observed in the kitchen due to cooking process. Indoor air PAHs identified are Napthalene, fluorine, acenaphthene, phenanthrene, pyrene, chrysene and indeno [1,2,3-cd) pyrene. Concentrations of all PAHs identified in kitchen were above the permissible OSHA norms for indoor air. Specific gravity of urine was significantly higher among the kitchen workers (p<0.001) as compared to the control group. Also, the prevalence of microalbuminuria was higher (p<0.001) among kitchen workers. Urinary PAH metabolites detected among kitchen workers were 1-NAP, 9-HF, 3-HF, 9-PHN and 1-OHP. Continuous heat exposure in kitchens due to cooking can alter kidney functions viz., high specific gravity of urine in kitchen workers. Exposure to PM, VOCs and PAHs in indoor air and presence of urinary PAHs metabolites may lead to inflammation, which can cause microalbuminuria in kitchen workers, as observed in the present study.     

Patient and Provider Reported Reasons for Lost to Follow Up in MDRTB Treatment: A Qualitative Study from a Drug Resistant TB Centre in India

Introduction

Multidrug-resistant Tuberculosis (MDR TB) is emerging public health concern globally. Lost to follow-up (LTFU) is one of the key challenge in MDRTB treatment. In 2013, 18% of MDR TB patients were reported LTFU in India. A qualitative study was conducted to obtain better understanding of both patient and provider related factors for LTFU among MDR TB treatment.

Methods

Qualitative semi-structured personal interviews were conducted with 20 MDRTB patients reported as LTFU and 10 treatment providers in seven districts linked to Nagpur Drug resistant TB Centre (DRTBC) during August 2012–February 2013. Interviews were transcribed and inductive content analysis was performed to derive emergent themes.

Results

We found multiple factors influencing MDR TB treatment adherence. Barriers to treatment adherence included drug side effects, a perceived lack of provider support, patient financial constraints, conflicts with the timing of treatment services, alcoholism and social stigma.

Conclusions

Patient adherence to treatment is multi-factorial and involves individual patient factors, provider factors, and community factors. Addressing issue of LTFU during MDRTB treatment requires enhanced efforts towards resolving medical problems like adverse drug effects, developing short duration treatment regimens, reducing pill burden, motivational counselling, flexible timings for DOT services, social, family support for patients & improving awareness about disease.     

Microbial acid-stable α-amylases: Characteristics,genetic engineering and applications

Among the wide variety of amylolytic enzymes synthesized by microorganisms, α-amylases are the most widely used biocatalysts in starch saccharification, baking industries and textile desizing. These enzymes randomly cleave the α-1,4-glycosidic linkages in starch, generating maltose and malto-oligosaccharides. The commercially available α-amylases have certain limitations, such as limited activity at low pH and Ca²⁺-dependence, and therefore, the search for novel acid-stable and thermostable amylases from extremophilic microorganisms and the engineering of the already available enzymes have been the major areas of research in this field over the years. Several attempts have been made to find suitable microbial sources of acid-stable and thermostable α-amylases. Acid-stable α-amylases have been reported in fungi, bacteria and archaea. α-Amylases that are active at elevated temperatures have been reported in bacteria as well as in archaea. α-Amylases that possess both characteristics, to the extent required for their various applications are very scarce. The developments that have been made in molecular biology, directed evolution and structural conformation studies of α-amylases for improving their properties to suit various industrial applications are discussed in this review.     

Biochemical and thermodynamic characteristics of thermo-alkali-stable xylanase from a novel polyextremophilic Bacillus halodurans TSEV1

The purified extracellular xylanase of polyextremophilic Bacillus halodurans TSEV1 has been visualized as a single band on SDS-PAGE and eluted as single peak by gel filtration, with a molecular mass of 40 kDa. The peptide finger print and cloned xylanase gene sequence analyses indicate that this enzyme belongs to GH family 10. The active site carboxyl residues are mainly involved in catalysis, while tryptophan residues are involved in substrate binding. The enzyme is optimally active at 80 °C and pH 9.0, and stable in the pH range of 7.0–12.0 with T _1/2 of 35 min at 80 °C (pH 9.0). Activation energy for birch wood xylan hydrolysis is 30.51 kJ mol^?1. The K _m, V _max and k _cat (birchwood xylan) are 2.05 mg ml^?1, 333.33 μmol mg^?1 min^?1 and 3.33 × 10⁴ min^?1, respectively. The pKa₁ and pKa₂ of ionizable groups of the active site that influence V _max are 8.51 and 11.0. The analysis of thermodynamic parameters for xylan hydrolysis suggests this as a spontaneous process. The enzyme is resistant to chemical denaturants like urea and guanidinium-HCl. The site-directed mutagenesis of catalytic glutamic acid residues (E196 and E301) resulted in a complete loss of activity. The birch wood xylan hydrolyzate contained xylobiose and xylotriose as the main products without any trace of xylose, and the enzyme hydrolyzes xylotetraose and xylopentaose rapidly to xylobiose. Thermo-alkali-stability, resistance to various chemical denaturants and mode of action make it a useful biocatalyst for generating xylo-oligosaccharides from agro-residues and bleaching of pulp in paper industries.     

Assessing forest products usage and local residents' perception of environmental changes in peri-urban and rural mangroves of Cameroon,Central Africa

Background

Deforestation is one of the most ubiquitous forms of land degradation worldwide. Although remote sensing and aerial photographs can supply valuable information on land/use cover changes, they may not regularly be available for some tropical coasts (e.g., Cameroon estuary) where cloud cover is frequent. With respect to mangroves, researchers are now employing local knowledge as an alternative means of understanding forest disturbances. This paper was primarily aimed at assessing the mangrove forest products usage, along with the local people's perceptions on environmental changes, between Littoral (Cameroon estuary) and Southern (mouth of the Nyong River and Mpalla village) regions of Cameroon.

Methods

The data from both locations were obtained through conducting household interviews and field observations.

Results

In the Cameroon estuary (Littoral region), 69.23% of respondents (mostly elders) could distinguish two to four mangrove plants, whereas the informants (65.45%) in the mouth of the Nyong River and Mpalla village (mostly young people interviewed from the Southern region) are familiar with only one or two commonly found mangroves. Also, more respondents from the Cameroon estuary are depending on mangroves for fuelwood (Rhizophora spp.) and housing (Rhizophora spp., Avicennia germinans (L.) Stearn and Nypa fruticans (Thumb.) Wurmb.) purposes, in contrast to Nyong River mouth and Mpalla village. Although local people perceived wood extraction as a greater disruptive factor, there are several causes for mangrove depletion in the Cameroon estuary. Among others, over-harvesting, clear-felled corridors, sand extraction and housing were found important. Furthermore, a decline in mangrove fauna composition (in terms of fishery products) was recorded in the Littoral as well as Southern regions. However, the causes of such perceived negative changes were not similar in both cases.

Conclusions

Findings of this study highlight the need to improve sustainable management of the mangrove ecosystems through afforestation (in large impacted areas), selective removal of senescent tree stems and branches (in little damage stands), regulating sand extraction and housing activities, and creating awareness and law enforcement.

     

Tuberculosis contact screening and isoniazid preventive therapy in a South Indian district: operational issues for programmatic consideration

Background

Under India''s Revised National Tuberculosis Control Programme (RNTCP), all household contacts of sputum smear positive Pulmonary Tuberculosis (PTB) patients are screened for TB. In the absence of active TB disease, household contacts aged <6 years are eligible for Isoniazid Preventive Therapy (IPT) (5 milligrams/kilogram body weight/day) for 6 months.

Objectives

To estimate the number of household contacts aged <6 years, of sputum smear positive PTB patients registered for treatment under RNTCP from April to June''2008 in Krishna District, to assess the extent to which they are screened for TB disease and in its absence initiated on IPT.

Methods

A cross sectional study was conducted. Households of all smear positive PTB cases (n = 848) registered for treatment from April to June''2008 were included. Data on the number of household contacts aged <6 years, the extent to which they were screened for TB disease, and the status of initiation of IPT, was collected.

Results

Households of 825 (97%) patients were visited, and 172 household contacts aged <6 years were identified. Of them, 116 (67%) were evaluated for TB disease; none were found to be TB diseased and 97 (84%) contacts were initiated on IPT and 19 (16%) contacts were not initiated on IPT due to shortage of INH tablets in peripheral health centers. The reasons for non-evaluation of the remaining eligible children (n = 56, 33%) include no home visit by the health staff in 25 contacts, home visit done but not evaluated in 31 contacts. House-hold contacts in rural areas were less likely to be evaluated and initiated on IPT [risk ratio 6.65 (95% CI; 3.06–14.42)].

Conclusion

Contact screening and IPT implementation under routine programmatic conditions is sub-optimal. There is an urgent need to sensitize all concerned programme staff on its importance and establishment of mechanisms for rigorous monitoring.     

Activity of antioxidant enzymes and secondary metabolites during in vitro regeneration of Sterculia urens

The changes in the activities of antioxidant enzymes and amounts of proteins, phenols, and flavonoids in regenerating and non-regenerating calli during organogenesis of Sterculia urens were monitored. Maximum growth of calli and the most efficient regeneration of shoots occurred on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^?3 benzylaminopurine (BAP) + 2 or 4 mg dm^?3 naphtalene acetic acid (NAA). Peroxidase (POD), catalase, and superoxide dismutase activities increased in the regenerating calli but decreased in the non-regenerating calli. Six POD isoenzymes were detected. Protein content decreased in the non-regenerating calli and increased significantly during regeneration of shoots from callus. Total phenols and flavonoids increased in the non regenerating calli. SDS-PAGE analysis revealed a role of many proteins in organogenesis.     

What Are the Reasons for Poor Uptake of HIV Testing among Patients with TB in an Eastern India District?

Background

National policy in India recommends HIV testing of all patients with TB. In West Bengal state, only 28% of patients with TB were tested for HIV between April-June, 2010. We conducted a cross-sectional survey to understand patient, provider and health system related factors associated with low uptake of HIV testing among patients with TB.

Methods

We reviewed TB and HIV program records to assess the HIV testing status of patients registered for anti-TB treatment from July-September 2010 in South-24-Parganas district, West Bengal, assessed availability of HIV testing kits and interviewed a random sample of patients with TB and providers.

Results

Among 1633 patients with TB with unknown HIV status at the time of diagnosis, 435 (26%) were tested for HIV within the intensive phase of TB treatment. Patients diagnosed with and treated for TB at facilities with co-located HIV testing services were more likely to get tested for HIV than at facilities without [RR = 1.27, (95% CI 1.20–3.35)]. Among 169 patients interviewed, 67 reported they were referred for HIV testing, among whom 47 were tested. During interviews, providers attributed the low proportion of patients with TB being referred and tested for HIV to inadequate knowledge among providers about the national policy, belief that patients will not test for HIV even if they are referred, shortage of HIV testing kits, and inadequate supervision by both programs.

Discussion

In West Bengal, poor uptake of HIV testing among patients with TB was associated with absence of HIV testing services at sites providing TB care services and to poor referral practices among providers. Comprehensive strategies to change providers’ beliefs and practices, decentralization of HIV testing to all TB care centers, and improved HIV test kit supply chain management may increase the proportion of patients with TB who are tested for HIV.