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The present study was performed to establish an improved, efficient and modified form of non-enzy... more The present study was performed to establish an improved, efficient and modified form of non-enzymatic salting out method of genomic DNA isolation from human blood. The human blood samples were collected. DNA extraction from blood samples was performed from the original non-enzymatic salting out method as well as a modified form of this method. The reproducibility and sensitivity of modified form of non-enzymatic salting out method were also analyzed by suitable experimental design. The modified form of non-enzymatic salting out DNA extraction method from blood has provided the 2.8 fold more yield of DNA as compared to the original non-enzymatic salting out method. The purity of DNA (A260/A280 > 1.8) was approximately same in modified form of non-enzymatic salting out method of genomic DNA isolation as compared to the original method of non-enzymatic salting out method of genomic DNA isolation. The quantity of extracted DNA has showed the reproducibility. The least volume of bloo...

Journal of …, 2006

■ Abstract An open controlled trial of 0.75 mg/Kg/day predniso-lone was conducted at a stage when... more ■ Abstract An open controlled trial of 0.75 mg/Kg/day predniso-lone was conducted at a stage when the patients had started falling several times in a day and stopped on their attaining a chair bound stage, thus minimising the total period of steroid therapy. Out of the 67 DMD ...

Nmr in Biomedicine, 2008

Owing to the small quantity of tissue available in human biopsy specimens, aqueous and lipid comp... more Owing to the small quantity of tissue available in human biopsy specimens, aqueous and lipid components often have to be determined in the same tissue sample. Perchloric acid (PCA) used for the extraction of aqueous metabolites has a deleterious effect on lipid components; the severity of the damage is not known. In this study, human muscle tissue was first treated with PCA to extract aqueous metabolites, and the residue was then used for lipid extraction by conventional methods, i.e. the methods of Folch and Bligh & Dyer and a standardised one using methanol/chloroform (1:3, v/v) used in our laboratory. A 1H-NMR spectrum was obtained for each lipid extract. Lipid was quantified by measuring the integral area of N+-(CH3)3 signals of phospholipids (PLs). Triacylglycerol (TG) and cholesterol (CHOL) were quantified using the -CH2- signals of glycerol and the C18 methyl signal, respectively. This study shows that prior use of PCA caused marked attenuation of TG, PL, and CHOL. This was confirmed by recovery experiments and observation of the direct effect of PCA on the standard lipid components. On the basis of the quantity of lipid lost in each case, three novel equations (with respect to TG, PL, and CHOL) were derived. Application of these equations to lipid quantities estimated in different pathological tissues after PCA pre-treatment produced values equivalent to those estimated without PCA use. This study conclusively shows that PCA pre-treatment damages all three lipid moieties, TG, PL, and CHOL. When PCA is used in a fixed ratio to the tissue, the lipid damage is also proportional and correctable by statistically derived equations. These equations will be useful in human biopsy specimens where aqueous and lipid components have to be studied using the same tissue sample because of the small quantity available. Copyright © 2007 John Wiley & Sons, Ltd.

Annals of Neurosciences, 2005

Metabolic myopathies are inborn diseases of carbohydrate and lipid metabolism that takes place in... more Metabolic myopathies are inborn diseases of carbohydrate and lipid metabolism that takes place in cytoplasm and mitochondria. These characteristically result from lack of activity of one or more specific enzymes or their failure in the transportation of proteins. In this article, we present ...

Analytical Letters, 2006

Page 1. BIOANALYTICAL An Improved, Single Step Standardized Method of Lipid Extraction from Human... more Page 1. BIOANALYTICAL An Improved, Single Step Standardized Method of Lipid Extraction from Human Skeletal Muscle Tissue Niraj Kumar Srivastava and Sunil Pradhan Department of Neurology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India ...

Nmr in Biomedicine, 2009

In vitro, high-resolution 1H and 31P NMR based qualitative and quantitative analyses of the lipid... more In vitro, high-resolution 1H and 31P NMR based qualitative and quantitative analyses of the lipid components of the tissue, serum, and CSF of patients with primary brain tumors were performed. Proton NMR spectra of the lipid extract of serum (blood specimen collected before the surgical procedure) and surgically discarded tissue showed that the total cholesterol (T.CHOL) and choline containing phospholipids (PL) were significantly higher in quantity in medulloblastoma and glioblastoma multiforme as compared to normal subjects. Serum lipid extracts of grade II/ III gliomas showed a higher quantity of PL than normal subjects. Cholestrol esters (CHOLest) were detectable in the tissue lipid extract of the patients with tumors and absent in normal tissue. There was a reduction in the quantity of CHOLest in the serum lipid extract of the tumor patients as compared to normal subjects. Ratio of PL to T.CHOL in serum lipid extract showed a significant difference between different grades of tumors versus normal subjects, while, a significant difference was observed only in medulloblastoma versus normal subjects in tissue lipid extract. Ratio of CHOL to CHOLest distinguishes the different grades of tumors versus normal subjects as well as between different grades of tumors (except medulloblastoma versus glioblastoma). The ratio of the Ph (total phospholipids except phosphatidylcholine) to PC (phosphatidylcholine) in 31P NMR based study showed a significant difference in all grades of tumors (except medulloblastoma) in normal subjects in tissue lipid extract as well as between different grades of tumors. Medulloblastoma could be differentiated from glioblastoma as well as from normal subjects in serum lipid extract by the ratio of the Ph to PC. Proton NMR spectra of the lipid extract of CSF showed that the CHOL, CHOLest, and PL were present in the patients with tumors, although these were absent in the patients with meningitis, motor neuron disease, and mitochondrial myopathies as well as in normal subjects. PL and T.CHOL provided discrimination between different grades of tumors (except glioblastoma versus medulloblastoma) in the lipid extract of the CSF. This study suggests the role of lipid estimation in CSF and serum as a complementary diagnostic tool for the evaluation of brain tumors preoperatively. NMR-based lipid estimation of post-surgical tumor tissue may also contribute to differentiating the tumor types. Copyright © 2009 John Wiley & Sons, Ltd.

Nmr in Biomedicine, 2010

Proton NMR spectroscopic investigations on the lipid extract of the serum of 41 Duchenne muscular... more Proton NMR spectroscopic investigations on the lipid extract of the serum of 41 Duchenne muscular dystrophy (DMD) (age, mean ± SD; 8.0 ± 3.0 years) patients and 22 healthy subjects (age, mean ± SD; 9.0 ± 4.0 years) were performed in the northern Indian population. The concentration of triglycerides, phospholipids, free cholesterol, cholesterol esters and total cholesterol was significantly higher in DMD patients as compared to healthy subjects. Ratio of free-cholesterol to cholesterol-esters was also significantly higher in DMD patients. Among the individual lipids, concentration of phospholipids was found to be consistently higher in DMD patients compared to healthy subjects, with a discriminatory index of 87.5%. The highest discriminatory index of 92% was found along with the ratio of PL (phospholipids) to CHOL (cholesterol), i.e. PL/CHOL. No significant quantitative difference was observed in the serum lipid constituents of positive and negative gene deletion cases of DMD. The inferences drawn from this study may provide the possibility of the diagnostic importance for DMD, especially in cases where genetic analysis fails to provide the diagnosis. Copyright © 2009 John Wiley & Sons, Ltd.

Journal of Neurology, 2006

An open controlled trial of 0.75 mg/Kg/day prednisolone was conducted at a stage when the patient... more An open controlled trial of 0.75 mg/Kg/day prednisolone was conducted at a stage when the patients had started falling several times in a day and stopped on their attaining a chair bound stage, thus minimising the total period of steroid therapy. Out of the 67 DMD patients enrolled in this study, 44 were put on prednisolone therapy and 23 served as controls. All patients were followed-up at two-monthly intervals for two years and thereafter they continued to take their respective medications till their chair-bound stage; then the drug was gradually withdrawn. In the treatment group 24 patients could not continue the trial because of adverse effects – 14 due to excessive obesity, 3 due to measles, 4 due to pulmonary tuberculosis, 2 due to recurrent throat and chest infection and 1 due to an unexplained high leukocyte count. Of the remaining 20 patients in the treatment group, steroid therapy was stopped in 5 patients as there was no improvement in power in six months. Fifteen patients in the treatment group and 19 patients in the control group could be followed regularly for 2 years and then up to chair-bound stage. Outcome parameters included fall frequency, peak expiratory flow rate, limb muscle power, ability to lift weights, time taken in getting up from squatting position, walking 9 metres and climbing 13 stairs. Maximum improvement was noted between 2 and 4 months while mild improvement in some parameters continued up to six months. All parameters remained stabilised for 1 year or so, after which there was slight deterioration. Deterioration at 2 years was, however, less than the natural course of events noted in control patients. Prednisolone treated patients and controls became chair bound at the mean age of 169 ± 9 and 132 ± 8 months respectively. Till the ideal stage of the disease and the type or dosage of starting steroid therapy is defined by specially designed studies, 0.75 mg/Kg/day prednisolone therapy may be started in DMD patients at the stage of frequent falls ( > 10 / day) on walking or increased get-up time ( > 10 s) as observed while testing Gowers’ sign; this improves muscle power and timing of motor performance within 2–4 months of onset of therapy in about 75% of those who tolerate this therapy, with a possible gain of approximately 3 years in terms of independent walking.

d Proton NMR spectroscopic investigations on the lipid extract of the serum of 41 Duchenne muscul... more d Proton NMR spectroscopic investigations on the lipid extract of the serum of 41 Duchenne muscular dystrophy (DMD) (age, mean W SD; 8.0 W 3.0 years) patients and 22 healthy subjects (age, mean W SD; 9.0 W 4.0 years) were performed in the northern Indian population. The concentration of triglycerides, phospholipids, free cholesterol, cholesterol esters and total cholesterol was significantly higher in DMD patients as compared to healthy subjects. Ratio of freecholesterol to cholesterol-esters was also significantly higher in DMD patients. Among the individual lipids, concentration of phospholipids was found to be consistently higher in DMD patients compared to healthy subjects, with a discriminatory index of 87.5%. The highest discriminatory index of 92% was found along with the ratio of PL (phospholipids) to CHOL (cholesterol), i.e. PL/CHOL. No significant quantitative difference was observed in the serum lipid constituents of positive and negative gene deletion cases of DMD. The inferences drawn from this study may provide the possibility of the diagnostic importance for DMD, especially in cases where genetic analysis fails to provide the diagnosis.

The present study was performed to establish an improved, efficient and modified form of non-enzy... more The present study was performed to establish an improved, efficient and modified form of non-enzymatic salting out method of genomic DNA isolation from human blood. The human blood samples were collected. DNA extraction from blood samples was performed from the original non-enzymatic salting out method as well as a modified form of this method. The reproducibility and sensitivity of modified form of non-enzymatic salting out method were also analyzed by suitable experimental design. The modified form of non-enzymatic salting out DNA extraction method from blood has provided the 2.8 fold more yield of DNA as compared to the original non-enzymatic salting out method. The purity of DNA (A260/A280 > 1.8) was approximately same in modified form of non-enzymatic salting out method of genomic DNA isolation as compared to the original method of non-enzymatic salting out method of genomic DNA isolation. The quantity of extracted DNA has showed the reproducibility. The least volume of bloo...

Journal of …, 2006

■ Abstract An open controlled trial of 0.75 mg/Kg/day predniso-lone was conducted at a stage when... more ■ Abstract An open controlled trial of 0.75 mg/Kg/day predniso-lone was conducted at a stage when the patients had started falling several times in a day and stopped on their attaining a chair bound stage, thus minimising the total period of steroid therapy. Out of the 67 DMD ...

Nmr in Biomedicine, 2008

Owing to the small quantity of tissue available in human biopsy specimens, aqueous and lipid comp... more Owing to the small quantity of tissue available in human biopsy specimens, aqueous and lipid components often have to be determined in the same tissue sample. Perchloric acid (PCA) used for the extraction of aqueous metabolites has a deleterious effect on lipid components; the severity of the damage is not known. In this study, human muscle tissue was first treated with PCA to extract aqueous metabolites, and the residue was then used for lipid extraction by conventional methods, i.e. the methods of Folch and Bligh & Dyer and a standardised one using methanol/chloroform (1:3, v/v) used in our laboratory. A 1H-NMR spectrum was obtained for each lipid extract. Lipid was quantified by measuring the integral area of N+-(CH3)3 signals of phospholipids (PLs). Triacylglycerol (TG) and cholesterol (CHOL) were quantified using the -CH2- signals of glycerol and the C18 methyl signal, respectively. This study shows that prior use of PCA caused marked attenuation of TG, PL, and CHOL. This was confirmed by recovery experiments and observation of the direct effect of PCA on the standard lipid components. On the basis of the quantity of lipid lost in each case, three novel equations (with respect to TG, PL, and CHOL) were derived. Application of these equations to lipid quantities estimated in different pathological tissues after PCA pre-treatment produced values equivalent to those estimated without PCA use. This study conclusively shows that PCA pre-treatment damages all three lipid moieties, TG, PL, and CHOL. When PCA is used in a fixed ratio to the tissue, the lipid damage is also proportional and correctable by statistically derived equations. These equations will be useful in human biopsy specimens where aqueous and lipid components have to be studied using the same tissue sample because of the small quantity available. Copyright © 2007 John Wiley & Sons, Ltd.

Annals of Neurosciences, 2005

Metabolic myopathies are inborn diseases of carbohydrate and lipid metabolism that takes place in... more Metabolic myopathies are inborn diseases of carbohydrate and lipid metabolism that takes place in cytoplasm and mitochondria. These characteristically result from lack of activity of one or more specific enzymes or their failure in the transportation of proteins. In this article, we present ...

Analytical Letters, 2006

Page 1. BIOANALYTICAL An Improved, Single Step Standardized Method of Lipid Extraction from Human... more Page 1. BIOANALYTICAL An Improved, Single Step Standardized Method of Lipid Extraction from Human Skeletal Muscle Tissue Niraj Kumar Srivastava and Sunil Pradhan Department of Neurology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India ...

Nmr in Biomedicine, 2009

In vitro, high-resolution 1H and 31P NMR based qualitative and quantitative analyses of the lipid... more In vitro, high-resolution 1H and 31P NMR based qualitative and quantitative analyses of the lipid components of the tissue, serum, and CSF of patients with primary brain tumors were performed. Proton NMR spectra of the lipid extract of serum (blood specimen collected before the surgical procedure) and surgically discarded tissue showed that the total cholesterol (T.CHOL) and choline containing phospholipids (PL) were significantly higher in quantity in medulloblastoma and glioblastoma multiforme as compared to normal subjects. Serum lipid extracts of grade II/ III gliomas showed a higher quantity of PL than normal subjects. Cholestrol esters (CHOLest) were detectable in the tissue lipid extract of the patients with tumors and absent in normal tissue. There was a reduction in the quantity of CHOLest in the serum lipid extract of the tumor patients as compared to normal subjects. Ratio of PL to T.CHOL in serum lipid extract showed a significant difference between different grades of tumors versus normal subjects, while, a significant difference was observed only in medulloblastoma versus normal subjects in tissue lipid extract. Ratio of CHOL to CHOLest distinguishes the different grades of tumors versus normal subjects as well as between different grades of tumors (except medulloblastoma versus glioblastoma). The ratio of the Ph (total phospholipids except phosphatidylcholine) to PC (phosphatidylcholine) in 31P NMR based study showed a significant difference in all grades of tumors (except medulloblastoma) in normal subjects in tissue lipid extract as well as between different grades of tumors. Medulloblastoma could be differentiated from glioblastoma as well as from normal subjects in serum lipid extract by the ratio of the Ph to PC. Proton NMR spectra of the lipid extract of CSF showed that the CHOL, CHOLest, and PL were present in the patients with tumors, although these were absent in the patients with meningitis, motor neuron disease, and mitochondrial myopathies as well as in normal subjects. PL and T.CHOL provided discrimination between different grades of tumors (except glioblastoma versus medulloblastoma) in the lipid extract of the CSF. This study suggests the role of lipid estimation in CSF and serum as a complementary diagnostic tool for the evaluation of brain tumors preoperatively. NMR-based lipid estimation of post-surgical tumor tissue may also contribute to differentiating the tumor types. Copyright © 2009 John Wiley & Sons, Ltd.

Nmr in Biomedicine, 2010

Proton NMR spectroscopic investigations on the lipid extract of the serum of 41 Duchenne muscular... more Proton NMR spectroscopic investigations on the lipid extract of the serum of 41 Duchenne muscular dystrophy (DMD) (age, mean ± SD; 8.0 ± 3.0 years) patients and 22 healthy subjects (age, mean ± SD; 9.0 ± 4.0 years) were performed in the northern Indian population. The concentration of triglycerides, phospholipids, free cholesterol, cholesterol esters and total cholesterol was significantly higher in DMD patients as compared to healthy subjects. Ratio of free-cholesterol to cholesterol-esters was also significantly higher in DMD patients. Among the individual lipids, concentration of phospholipids was found to be consistently higher in DMD patients compared to healthy subjects, with a discriminatory index of 87.5%. The highest discriminatory index of 92% was found along with the ratio of PL (phospholipids) to CHOL (cholesterol), i.e. PL/CHOL. No significant quantitative difference was observed in the serum lipid constituents of positive and negative gene deletion cases of DMD. The inferences drawn from this study may provide the possibility of the diagnostic importance for DMD, especially in cases where genetic analysis fails to provide the diagnosis. Copyright © 2009 John Wiley & Sons, Ltd.

Journal of Neurology, 2006

An open controlled trial of 0.75 mg/Kg/day prednisolone was conducted at a stage when the patient... more An open controlled trial of 0.75 mg/Kg/day prednisolone was conducted at a stage when the patients had started falling several times in a day and stopped on their attaining a chair bound stage, thus minimising the total period of steroid therapy. Out of the 67 DMD patients enrolled in this study, 44 were put on prednisolone therapy and 23 served as controls. All patients were followed-up at two-monthly intervals for two years and thereafter they continued to take their respective medications till their chair-bound stage; then the drug was gradually withdrawn. In the treatment group 24 patients could not continue the trial because of adverse effects – 14 due to excessive obesity, 3 due to measles, 4 due to pulmonary tuberculosis, 2 due to recurrent throat and chest infection and 1 due to an unexplained high leukocyte count. Of the remaining 20 patients in the treatment group, steroid therapy was stopped in 5 patients as there was no improvement in power in six months. Fifteen patients in the treatment group and 19 patients in the control group could be followed regularly for 2 years and then up to chair-bound stage. Outcome parameters included fall frequency, peak expiratory flow rate, limb muscle power, ability to lift weights, time taken in getting up from squatting position, walking 9 metres and climbing 13 stairs. Maximum improvement was noted between 2 and 4 months while mild improvement in some parameters continued up to six months. All parameters remained stabilised for 1 year or so, after which there was slight deterioration. Deterioration at 2 years was, however, less than the natural course of events noted in control patients. Prednisolone treated patients and controls became chair bound at the mean age of 169 ± 9 and 132 ± 8 months respectively. Till the ideal stage of the disease and the type or dosage of starting steroid therapy is defined by specially designed studies, 0.75 mg/Kg/day prednisolone therapy may be started in DMD patients at the stage of frequent falls ( > 10 / day) on walking or increased get-up time ( > 10 s) as observed while testing Gowers’ sign; this improves muscle power and timing of motor performance within 2–4 months of onset of therapy in about 75% of those who tolerate this therapy, with a possible gain of approximately 3 years in terms of independent walking.

d Proton NMR spectroscopic investigations on the lipid extract of the serum of 41 Duchenne muscul... more d Proton NMR spectroscopic investigations on the lipid extract of the serum of 41 Duchenne muscular dystrophy (DMD) (age, mean W SD; 8.0 W 3.0 years) patients and 22 healthy subjects (age, mean W SD; 9.0 W 4.0 years) were performed in the northern Indian population. The concentration of triglycerides, phospholipids, free cholesterol, cholesterol esters and total cholesterol was significantly higher in DMD patients as compared to healthy subjects. Ratio of freecholesterol to cholesterol-esters was also significantly higher in DMD patients. Among the individual lipids, concentration of phospholipids was found to be consistently higher in DMD patients compared to healthy subjects, with a discriminatory index of 87.5%. The highest discriminatory index of 92% was found along with the ratio of PL (phospholipids) to CHOL (cholesterol), i.e. PL/CHOL. No significant quantitative difference was observed in the serum lipid constituents of positive and negative gene deletion cases of DMD. The inferences drawn from this study may provide the possibility of the diagnostic importance for DMD, especially in cases where genetic analysis fails to provide the diagnosis.