Analysis of calpain-3 protein in muscle biopsies of different muscular dystrophies from India.

Background & objectives: Calpain-3, a Ca2+-dependent protease has been implicated in the pathology of neuromuscular disorders (NMDs). The current study aimed to analyze calpain-3 expression in cases diagnosed as muscular dystrophy from the Indian population. Methods: Calpain-3 Western blot analysis in muscle biopsies of immunohistochemically confirmed cases of Duchenne muscular dystrophy (DMD) (n=10), dysferlinopathy (n=30) and sarcoglycanopathy (n=8) was carried out. Calpain-3 Western blotting was also used in a blinded study to identify cases of calpain-3 deficiency in 28 NMD patients with potential muscular dystrophy. Results: Calpain-3 appeared as a full length 94 kDa band with an autolytic product (~60 kDa) on Western blots with antibody NCL-CALP-12A2 (Ab-2). Eight of the 10 DMD samples showed absence of 94 kDa band but presence of 60 kDa band while one case of sarcoglycanopathy showed absence of both. Twenty one of the 30 dysferlinopathy samples showed both bands while six showed only the 60 kDa band and three showed absence of both. In the blinded study, five NMD cases with potential muscular dystrophy that showed complete absence of both bands in retrospect exhibited clinical features of limb girdle muscular dystrophy 2A (LGMD2A). Interpretation & conclusions: While the study revealed a consistent pattern of calpain-3 in DMD, one sarcoglycanopathy and three dysferlinopathy samples exhibited secondary reduction in calpain-3. It was recognized that both calpain-3 bands should be considered to confirm calpain deficiency. Further, western blot offers an economical and fast preliminary screening method for LGMD2A especially in cases of complete absence of calpain-3 prior to conclusive diagnosis by genetic testing.

Qualitative high performance thin layer chromatography (HPTLC) analysis of cannabinoids in urine samples of Cannabis abusers.

Background & objectives: Cannabis is one of the most commonly abused drugs worldwide. There is a distinct clinical correlation between cannabis abuse and mental disorders. However, it is essential to establish cannabis intake in the abusers in order to establish causality between cannabis and psychiatric illness. The limitations of current detection methods using commercial cassettes prompted us to standardize the method of extraction and detection of cannabinoids in the urine samples of cannabis abusers attending a de-addiction centre in south India. Methods: In this study, diagnostic tests on 102 male patients suspected with cannabis abuse were done. Liquid-liquid extraction of cannabinoids from urine was done and screened by Duquenois-Levine, fast blue B salt and p-dimethylaminobenzaldehyde (p-DMAB) tests. All the results were confirmed by high performance thin layer chromatography (HPTLC). Samples were considered positive for cannabis based on the positive indication in colour test and by detection of 11-nor-Δ9 tetrahydrocannabinol-9-carboxylic acid (THC-COOH) on HPTLC. Results: Based on the colour tests and HPTLC, cannabis abuse was detected in 64 of 102 patients tested. HPTLC method was found to be sensitive for detection and possible quantitation of THC-COOH. Interpretation & conclusion: We report the standardization and utility of cannabinoid extraction, screening and detection by HPTLC in the urine samples of cannabis abusers. The HPTLC method was found to be high throughput, sensitive, reproducible and cost-effective compared to commercial kits.