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Papers by ravinder singh

Research paper thumbnail of Advances in metanephrine testing for the diagnosis of pheochromocytoma

Clinics in Laboratory Medicine, 2004

Research paper thumbnail of Assessment of proteins of the immune system

Clinical Immunology, 2008

Research paper thumbnail of Unpleasant Spells

Tietz's Applied Laboratory Medicine, 2007

Research paper thumbnail of Estradiol assays – The path ahead

Steroids, 2014

Estradiol quantitation is useful in the clinical assessment of diseases like hypogonadism, hirsut... more Estradiol quantitation is useful in the clinical assessment of diseases like hypogonadism, hirsutism, polycystic ovary syndrome (PCOS), amenorrhea, ovarian tumors and for monitoring response in women receiving aromatase inhibitor therapy. Physiologically relevant serum estradiol concentration in women can span across four orders of magnitude. For example, in women undergoing ovulation induction serum estradiol concentration can range between 250-2000pg/mL whereas aromatase inhibitor therapy can decrease serum estradiol concentration to <5pg/mL. While high-through-put automated un-extracted (direct) immunoassays accommodate the growing clinical need for estradiol quantitation, are amenable to implementation by most hospital clinical laboratories, they display a significant loss of specificity and accuracy at low concentrations. Most clinical scenarios (example: estradiol monitoring in fertility treatments) place a modest demand on accuracy and precision of the assay in use but accurate quantitation of estradiol in certain clinical scenarios (pediatric and male patients and for monitoring aromatase inhibitor therapy) can be challenging using currently available immunoassays since the direct immunoassays are prone to issues with sub-optimal accuracy and specificity due to cross reactivity with estradiol conjugates and metabolites. In this review we discuss the bases for the evolution of estradiol assays from extracted (indirect) radio-immunoassays to direct immunoassays to liquid-chromatography tandem mass spectrometry (LC-MS/MS) based assays, discuss technical factors relevant for development and optimization of a LC-MS/MS assay for estradiol and present the details and performance characteristics of an ultra-sensitive LC-MS/MS estradiol assay with a limit of quantitation of 0.2pg/mL.

Research paper thumbnail of SIX-MONTH STABILITY OF BEVACIZUMAB (AVASTIN) BINDING TO VASCULAR ENDOTHELIAL GROWTH FACTOR AFTER WITHDRAWAL INTO A SYRINGE AND REFRIGERATION OR FREEZING

Retina-the Journal of Retinal and Vitreous Diseases, 2006

To determine the change in anti-vascular endothelial growth factor (VEGF) activity of bevacizumab... more To determine the change in anti-vascular endothelial growth factor (VEGF) activity of bevacizumab (Avastin, Genentech, Inc., San Francisco, CA) after refrigeration or freezing.

Research paper thumbnail of Validation of a High-Throughput Liquid Chromatography-Tandem Mass Spectrometry Method for Urinary Cortisol and Cortisone

Urinary free cortisol and cortisone measurements are useful in evaluation of Cushing syndrome, ap... more Urinary free cortisol and cortisone measurements are useful in evaluation of Cushing syndrome, apparent mineralocorticoid excess, congenital adrenal hyperplasia, and adrenal insufficiency. To reduce analytical interference, improve accuracy, and shorten the analysis time, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for urinary cortisol and cortisone. We added 190 pmol (70 ng) of stable isotope cortisol-9,11,12,12-d(4) to 0.5 mL of urine as an internal standard before extraction. The urine was extracted with 4.5 mL of methylene chloride, washed, and dried, and 10 microL of the reconstituted extract was injected onto a reversed-phase C(18) column and analyzed using a tandem mass spectrometer operating in the positive mode. Multiple calibration curves for urinary cortisol and cortisone exhibited consistent linearity and reproducibility in the range 7-828 nmol/L (0.25-30 microg/dL). Interassay CVs were 7.3-16% for mean concentrations of 6-726 nmol/L (0.2-26.3 microg/dL) for cortisol and cortisone. The detection limit was 6 nmol/L (0.2 microg/dL). Recovery of cortisol and cortisone added to urine was 97-123%. The regression equation for the LC-MS/MS (y) and HPLC (x) method for cortisol was: y = 1.11x + 0.03 microg cortisol/24 h (r(2) = 0.992; n = 99). The regression equation for the LC-MS/MS (y) and immunoassay (x) methods for cortisol was: y = 0.66x - 12.1 microg cortisol/24 h (r(2) = 0.67; n = 99). The sensitivity and specificity of the LC-MS/MS method for urinary free cortisol and cortisone offer advantages over routine immunoassays or chromatographic methods because of elimination of drug interferences, high throughput, and short chromatographic run time.

Research paper thumbnail of A Comparison of Biochemical Tests for Pheochromocytoma: Measurement of Fractionated Plasma Metanephrines Compared with the Combination of 24Hour Urinary Metanephrines and Catecholamines

We compared the diagnostic efficacy of fractionated plasma metanephrine measurements to measureme... more We compared the diagnostic efficacy of fractionated plasma metanephrine measurements to measurements of 24-h urinary total metanephrines and catecholamines in outpatients tested for pheochromocytoma at Mayo Clinic Rochester from January 1, 1999, until November 27, 2000. Catecholaminesecreting tumors were histologically proven. The sensitivity of fractionated plasma metanephrines was 97% (30 of 31 patients), compared with a sensitivity of 90% (28 of 31) for urinary total metanephrines and catecholamines (P = 0.63). The specificity of fractionated plasma metanephrines was 85% (221 of 261), compared with 98% (257 of 261; P < 0.001) for urinary measurements. The likelihood ratios for positive tests were 6.3 (95% confidence interval, 4.7 to 8.5) for fractionated plasma metanephrines and 58.9 (95% confidence interval, 22.1 to 156.9) for urinary total metanephrines and catecholamines. An adrenal pheochromocytoma was missed by urinary testing in two patients with familial syndromes and one asymptomatic patient with an incidentally discovered adrenal mass. An extra-adrenal paraganglioma was missed by plasma testing in one patient. In conclusion, measurements of 24-h urinary total metanephrines and catecholamines yield fewer false-positive results, an attribute preferred for testing low-risk patients, but fractionated plasma metanephrine measurements may be preferred in high-risk patients with familial endocrine syndromes.

Research paper thumbnail of Atorvastatin Inhibits Hypercholesterolemia-Induced Cellular Proliferation and Bone Matrix Production in the Rabbit Aortic Valve

Background-Despite the common occurrence of aortic stenosis, the cellular causes of the disorder ... more Background-Despite the common occurrence of aortic stenosis, the cellular causes of the disorder are unknown, in part because of the absence of experimental models. We hypothesized that atherosclerosis and early bone matrix expression in the aortic valve occurs secondary to experimental hypercholesterolemia and that treatment with atorvastatin modifies this transformation. Methods and Results-To test this hypothesis, we developed an experimental hypercholesterolemic rabbit model. New

Research paper thumbnail of The role of glutathione in the transport and catabolism of nitric oxide

Febs Letters, 1996

Nitric oxide acts as a neuronal and vascular messenger implying diffusion through intracellular e... more Nitric oxide acts as a neuronal and vascular messenger implying diffusion through intracellular environments containing 5–10 mM glutathione. Nitric oxide reacts with glutathione under aerobic conditions generating S-nitrosoglutathione (GSNO). GSNO reacts with glutathione (k = 8.3 × 10−3 M−1 · s−1) to generate nitrous oxide and glutathione disulfide (GSSG). Anaerobically, glutathione reacts with nitric oxide generating nitrous oxide and GSSG (k = 4.8 × 10−4 s−1 at 5 mM GSH). In both aerobic and anaerobic situations the nitroxyl anion may be an intermediate in the synthesis of nitrous oxide and, under aerobic conditions, nitroxyl anion may generate peroxynitrite. We present a hypothesis for the intracellular interaction between nitric oxide and glutathione.

Research paper thumbnail of Vitamin D (25OHD) Serum Seasonality in the United States

PLoS ONE, 2013

Background: Vitamin D is an important micronutrient for health. Hypovitaminosis D is thought to p... more Background: Vitamin D is an important micronutrient for health. Hypovitaminosis D is thought to play a role in the seasonality of a number of diseases and adverse health conditions. To refine hypotheses about the links between vitamin D and seasonal diseases, good estimates of the cyclicality of serum vitamin D are necessary.

Research paper thumbnail of PHOTODYNAMIC ACTION OF MEROCYANINE 540 IN ARTIFICIAL BILAYERS AND NATURAL MEMBRANES: ACTION SPECTRA AND QUANTUM YIELDS

Photochemistry and Photobiology, 1991

The action spectra and quantum yields for singlet oxygen (1O2) generation by merocyanine 540 (MC5... more The action spectra and quantum yields for singlet oxygen (1O2) generation by merocyanine 540 (MC540) in liposomes and isolated erythrocyte membranes were obtained using electron spin resonance techniques. Oxygen consumption was measured by spin label oximetry in the presence of histidine for fully-saturated dimyristoylphosphatidylcholine vesicles, mono-unsaturated 1-palmitoyl-2-oleoylphosphatidylcholine vesicles and erythrocyte membranes. The quantum yield for the photogeneration of 1O2 by membrane-bound MC540 in aqueous buffer was determined to be 0.065 ± 0.005, which is approx. 1/10 of the value determined for Rose Bengal under similar conditions. Using unilamellar liposomes and isolated erythrocyte membranes containing MC540 at different monomer/dimer ratios, we have observed that the action spectra of 1O2 generation closely overlap the absorption spectra of the monomeric dye in these systems. It is likely that factors which affect the monomer-dimer equilibrium of MC540 will influence the production of 1O2. These findings have important implications for the phototherapeutic efficacy of MC540.

Research paper thumbnail of Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photospray ionization tandem mass spectrometry

Clinica Chimica Acta, 2006

Serum steroid assays play an important role in the clinical evaluation of a number of common endo... more Serum steroid assays play an important role in the clinical evaluation of a number of common endocrine disorders. Among various assays, tandem mass spectrometry (MS/MS) has being increasingly applied in clinical laboratories for its high sensitivity, specificity, and simultaneous multi-analyte quantitation capability. Our first generation isotope dilution steroid profile assay by HPLC-tandem MS/MS with a C-18 column allowed for the measurement of 9 steroids in 18 min employing a sample volume of 760 ul serum. We describe our second generation steroid profile assay which allows for the quantitation of 12 steroids simultaneously employing HPLC-MS/MS and isotope dilution tandem MS in 11 min. This method requires a sample volume of 200 μl.An API-5000 triple-quadrupole mass spectrometer (Sciex, Concord, Canada) coupled with the PhotoSpray source and Shimadzu HPLC system (Shimadzu Scientific Instruments, Columbia, MD) was used employing isotope dilution with deuterium labeled internal standard (IS) for each analyte. Two hundred microliters of serum were deproteinized by adding 300 μl of acetonitrile containing internal standards. After centrifugation, 450 μl of supernatant were diluted with 900 μl of water and 1000 μl aliquot were injected onto a C-8 column. After a 3 min wash the valve was activated to initiate the gradient elution program which eluted the steroids. Quantitation by MRM analysis was performed both in positive ion mode for 11 analytes and in negative ion mode for aldosterone. Within-day and between-day precision, reliability and accuracy of this method were assessed by correlation with other MS/MS and immunoassay methods and by recovery study.Within-day CVs were < 11.5% for all analytes tested and between-day CVs ranged from 3.5% to 12.2%. The results of the comparison study yield r values ranging between 0.908 and 0.999. Recovery ranged from 90% to 110%.This method can simultaneously measure 12 steroids in serum within 11 min with minimal sample preparation. It can be routinely employed in a clinical environment and is attractive because of its simplicity in sample processing and high throughput.

Research paper thumbnail of INTRAVITREAL CONCENTRATION AND CLEARANCE OF TRIAMCINOLONE ACETONIDE IN NONVITRECTOMIZED HUMAN EYES

Retina-the Journal of Retinal and Vitreous Diseases, 2004

To determine the intravitreal concentration and clearance of triamcinolone acetonide at various i... more To determine the intravitreal concentration and clearance of triamcinolone acetonide at various intervals after intravitreal injection into nonvitrectomized eyes. Six participants were administered 4 mg (0.1 cc) of triamcinolone acetonide ophthalmic suspension. All six eyes underwent therapeutic pars plana vitrectomy with membranectomy at various post injection intervals ranging from 1.25 to 5 months from the intravitreal injection. Undiluted specimens of vitreous overlying the macula and of aqueous humor were submitted for analysis. Vitreous and aqueous humor concentrations of triamcinolone were measured by high performance liquid chromatography. Four eyes demonstrated detectable intravitreal concentrations of triamcinolone acetonide between 1.25 and 2.75 months after a single injection. Two eyes had an undetectable level of triamcinolone in both the vitreous and aqueous at 3 and 5 months post single injection. The intravitreal concentration of triamcinolone acetonide is detectable up to 2.75 months post a single 4 mg injection in nonvitrectomized eyes. A reinjection interval of 3 months may be needed to achieve sustained measurable levels of triamcinolone in nonvitrectomized patients.

Research paper thumbnail of The Peroxynitrite Generator, SIN1, Becomes a Nitric Oxide Donor in the Presence of Electron Acceptors

Archives of Biochemistry and Biophysics, 1999

SIN-1 has been used,in vitro, to simultaneously generate nitric oxide (•NO) and superoxide (O•−2)... more SIN-1 has been used,in vitro, to simultaneously generate nitric oxide (•NO) and superoxide (O•−2). However, the pharmacological activity of SIN-1 resembles that of a•NO donor. SIN-1 decays by a three-step mechanism. After initial isomerization to an open ring form, SIN-1A reduces oxygen by a one-electron transfer reaction to give O•−2and the SIN-1 cation radical, which decomposes to form SIN-1C and•NO. Here we report that one-electron oxidizing agents, in addition to oxygen, can oxidize SIN-1A, resulting in the release of•NO without the concomitant formation of O•−2. We demonstrate that easily reducible nitroxides, such as the nitronyl and imino nitroxides, are able to oxidize SIN-1. Biological oxidizing agents such as ferricytochromecalso stimulate•NO production from SIN-1. In addition, decomposition of SIN-1 by human plasma or by the homogenate of rat liver, kidney, and heart tissues results in the formation of•NO. Our findings suggest that SIN-1 may react with heme proteins and other electron acceptors in biological systems to produce•NO. Thus, at the relatively lowin vivooxygen concentrations, SIN-1 is likely to behave more like an•NO donor than a peroxynitrite donor. The relevance of this reaction to myocardial protection afforded by SIN-1 in ischemia/reperfusion-induced injury is discussed.

Research paper thumbnail of Intraocular concentration and pharmacokinetics of triamcinolone acetonide after a single intravitreal injection

Ophthalmology, 2003

To describe the pharmacokinetics occurring after the direct injection of triamcinolone acetonide ... more To describe the pharmacokinetics occurring after the direct injection of triamcinolone acetonide into the vitreous humor of humans.Interventional case series.Five patients who received a single 4-mg intravitreal injection of triamcinolone acetonide.An aqueous humor sample was obtained from 5 eyes via an anterior chamber paracentesis at days 1, 3, 10, 17, and 31 after injection. At each visit, visual acuity and intraocular pressure were measured and indirect ophthalmoscopy was performed. A fluorescein angiogram was carried out at day 10. Concentrations were determined using high performance liquid chromatography; pharmacokinetic analysis was carried out using PK Analyst, an iterative, nonlinear, weighted, least-squares regression program.Intraocular concentrations of triamcinolone were measured and population pharmacokinetic parameters were calculated.Pharmacokinetic data followed a two-compartment model. Peak aqueous humor concentrations ranged from 2151 to 7202 ng/ml, half-lives from 76 to 635 hours, and the integral of the area under the concentration-time curve (AUC0-t) from 231 to 1911 ng/h per milliliter. After a single intravitreal injection of triamcinolone, the mean elimination half-life was 18.6 days in nonvitrectomized patients. The half-life in a patient who had undergone a vitrectomy was shorter at 3.2 days.There was considerable intrasubject variation among peak concentration, AUC0-t values, and elimination half-lives. After intravitreal injection, measurable concentrations of triamcinolone would be expected to last for approximately 3 months (93 ± 28 days) in the absence of a vitrectomy. Because triamcinolone pharmacokinetics were characterized only in elderly patients with macular edema, the results cannot be extrapolated to other patient populations.

Research paper thumbnail of Pharmacokinetics of Intravitreal Ranibizumab (Lucentis)

Ophthalmology, 2007

To describe the pharmacokinetics of 0.5 mg of intravitreal ranibizumab (Lucentis) and to compare ... more To describe the pharmacokinetics of 0.5 mg of intravitreal ranibizumab (Lucentis) and to compare it with that of 1.25 mg of intravitreal bevacizumab (Avastin), using the same rabbit model.Experimental animal study.Twenty-eight Dutch-belted rabbits.One eye of each of 20 rabbits was injected with 0.5 mg of intravitreal ranibizumab. Both eyes of each of 4 rabbits were enucleated at days 1, 3, 8, 15, and 29. Ranibizumab concentrations were measured in aqueous fluid, whole vitreous, and serum. A further 8 rabbits were used to measure serum and fellow ranibizumab at additional time points of 3 and 8 hours.Ranibizumab concentrations in the aqueous, vitreous, and serum.Although vitreous concentrations of ranibizumab declined in a monoexponential fashion with a half-life of 2.88 days, concentrations of >0.1 μg/ml ranibizumab were maintained in the vitreous humor for 29 days. Ranibizumab concentrations in the aqueous humor of the injected eye reached a peak concentration of 17.9 μg/ml, 3 days after drug administration. Elimination of ranibizumab from the aqueous humor paralleled that found in the vitreous humor, with a half-life value of 2.84 days. No ranibizumab was detected in the serum or the fellow eye.In the rabbit, the vitreous half-life of 0.5-mg intravitreal ranibizumab is 2.88 days, shorter than the half-life of 1.25-mg intravitreal bevacizumab of 4.32 days. No ranibizumab was detected in the serum or the fellow uninjected eye; whereas small amounts of intravitreal bevacizumab have been detected in the serum and fellow uninjected eye.

Research paper thumbnail of Pharmacokinetics of Intravitreal Bevacizumab (Avastin)

Ophthalmology, 2007

To describe the pharmacokinetics of 1.25 mg of intravitreal bevacizumab (Avastin).Experimental an... more To describe the pharmacokinetics of 1.25 mg of intravitreal bevacizumab (Avastin).Experimental animal study.Twenty Dutch-belted rabbits.One eye of each of 20 rabbits was injected with 1.25 mg of intravitreal bevacizumab. Both eyes of each of 4 rabbits were enucleated at days 1, 3, 8, 15, and 29. Bevacizumab concentrations were measured in aqueous fluid, whole vitreous, and serum.Bevacizumab concentrations in the aqueous, vitreous, and serum.Whereas vitreous concentrations of bevacizumab declined in a monoexponential fashion with a half-life of 4.32 days, concentrations of >10μg/ml bevacizumab were maintained in the vitreous humor for 30 days. Bevacizumab concentrations in the aqueous humor of the injected eye reached a peak concentration of 37.7 μg/ml 3 days after drug administration. A maximum serum concentration of 3.3 μg/ml was achieved 8 days after intravitreal injection and the concentration fell below 1 μg/ml 29 days after injection. Elimination of bevacizumab from the aqueous humor and serum paralleled that found in the vitreous humor, with half-life values of 4.88 days and 6.86 days, respectively. Very low concentrations of bevacizumab were detected in the fellow uninjected eye. Concentrations of bevacizumab in the vitreous of the fellow eye varied incrementally, from 0.35 ng/ml at 1 day to 11.17 ng/ml at 4 weeks. Concentrations of bevacizumab in the aqueous humor of the fellow eye reached their peak at 1 week, at 29.4 ng/ml, and declined to 4.56 ng/ml at 4 weeks.The vitreous half-life of 1.25 mg intravitreal bevacizumab is 4.32 days in a rabbit eye. Very small amounts of bevacizumab were detected in the serum and in the fellow uninjected eye.

Research paper thumbnail of Morphometry of landslides in Garhwal Himalaya, India

Disaster Prevention and Management, 2011

Purpose -The purpose of this paper is to introduce a simple and convenient method for the landsli... more Purpose -The purpose of this paper is to introduce a simple and convenient method for the landslides study. Design/methodology/approach -This study is based upon the individual case study of three past landslides which may probably prove fatal in the near future. It is useful for the measurement of the horizontal length, longitudinal length, altitude at different points and area of the landslide. These measurements are very helpful in calculating the valley side gradient, longitudinal gradient of the landslide scarp and the volume of the debris generated due to mass movement. Findings -Reactivation of slope instability problems were found along these landslides spots, which will create havoc for the people and a problem for the administration in the near future. Originality/value -The implication in the paper is that structural and non-structural mitigation and management strategies are needed.

Research paper thumbnail of Advances in metanephrine testing for the diagnosis of pheochromocytoma

Clinics in Laboratory Medicine, 2004

Research paper thumbnail of Assessment of proteins of the immune system

Clinical Immunology, 2008

Research paper thumbnail of Unpleasant Spells

Tietz's Applied Laboratory Medicine, 2007

Research paper thumbnail of Estradiol assays – The path ahead

Steroids, 2014

Estradiol quantitation is useful in the clinical assessment of diseases like hypogonadism, hirsut... more Estradiol quantitation is useful in the clinical assessment of diseases like hypogonadism, hirsutism, polycystic ovary syndrome (PCOS), amenorrhea, ovarian tumors and for monitoring response in women receiving aromatase inhibitor therapy. Physiologically relevant serum estradiol concentration in women can span across four orders of magnitude. For example, in women undergoing ovulation induction serum estradiol concentration can range between 250-2000pg/mL whereas aromatase inhibitor therapy can decrease serum estradiol concentration to &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;5pg/mL. While high-through-put automated un-extracted (direct) immunoassays accommodate the growing clinical need for estradiol quantitation, are amenable to implementation by most hospital clinical laboratories, they display a significant loss of specificity and accuracy at low concentrations. Most clinical scenarios (example: estradiol monitoring in fertility treatments) place a modest demand on accuracy and precision of the assay in use but accurate quantitation of estradiol in certain clinical scenarios (pediatric and male patients and for monitoring aromatase inhibitor therapy) can be challenging using currently available immunoassays since the direct immunoassays are prone to issues with sub-optimal accuracy and specificity due to cross reactivity with estradiol conjugates and metabolites. In this review we discuss the bases for the evolution of estradiol assays from extracted (indirect) radio-immunoassays to direct immunoassays to liquid-chromatography tandem mass spectrometry (LC-MS/MS) based assays, discuss technical factors relevant for development and optimization of a LC-MS/MS assay for estradiol and present the details and performance characteristics of an ultra-sensitive LC-MS/MS estradiol assay with a limit of quantitation of 0.2pg/mL.

Research paper thumbnail of SIX-MONTH STABILITY OF BEVACIZUMAB (AVASTIN) BINDING TO VASCULAR ENDOTHELIAL GROWTH FACTOR AFTER WITHDRAWAL INTO A SYRINGE AND REFRIGERATION OR FREEZING

Retina-the Journal of Retinal and Vitreous Diseases, 2006

To determine the change in anti-vascular endothelial growth factor (VEGF) activity of bevacizumab... more To determine the change in anti-vascular endothelial growth factor (VEGF) activity of bevacizumab (Avastin, Genentech, Inc., San Francisco, CA) after refrigeration or freezing.

Research paper thumbnail of Validation of a High-Throughput Liquid Chromatography-Tandem Mass Spectrometry Method for Urinary Cortisol and Cortisone

Urinary free cortisol and cortisone measurements are useful in evaluation of Cushing syndrome, ap... more Urinary free cortisol and cortisone measurements are useful in evaluation of Cushing syndrome, apparent mineralocorticoid excess, congenital adrenal hyperplasia, and adrenal insufficiency. To reduce analytical interference, improve accuracy, and shorten the analysis time, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for urinary cortisol and cortisone. We added 190 pmol (70 ng) of stable isotope cortisol-9,11,12,12-d(4) to 0.5 mL of urine as an internal standard before extraction. The urine was extracted with 4.5 mL of methylene chloride, washed, and dried, and 10 microL of the reconstituted extract was injected onto a reversed-phase C(18) column and analyzed using a tandem mass spectrometer operating in the positive mode. Multiple calibration curves for urinary cortisol and cortisone exhibited consistent linearity and reproducibility in the range 7-828 nmol/L (0.25-30 microg/dL). Interassay CVs were 7.3-16% for mean concentrations of 6-726 nmol/L (0.2-26.3 microg/dL) for cortisol and cortisone. The detection limit was 6 nmol/L (0.2 microg/dL). Recovery of cortisol and cortisone added to urine was 97-123%. The regression equation for the LC-MS/MS (y) and HPLC (x) method for cortisol was: y = 1.11x + 0.03 microg cortisol/24 h (r(2) = 0.992; n = 99). The regression equation for the LC-MS/MS (y) and immunoassay (x) methods for cortisol was: y = 0.66x - 12.1 microg cortisol/24 h (r(2) = 0.67; n = 99). The sensitivity and specificity of the LC-MS/MS method for urinary free cortisol and cortisone offer advantages over routine immunoassays or chromatographic methods because of elimination of drug interferences, high throughput, and short chromatographic run time.

Research paper thumbnail of A Comparison of Biochemical Tests for Pheochromocytoma: Measurement of Fractionated Plasma Metanephrines Compared with the Combination of 24Hour Urinary Metanephrines and Catecholamines

We compared the diagnostic efficacy of fractionated plasma metanephrine measurements to measureme... more We compared the diagnostic efficacy of fractionated plasma metanephrine measurements to measurements of 24-h urinary total metanephrines and catecholamines in outpatients tested for pheochromocytoma at Mayo Clinic Rochester from January 1, 1999, until November 27, 2000. Catecholaminesecreting tumors were histologically proven. The sensitivity of fractionated plasma metanephrines was 97% (30 of 31 patients), compared with a sensitivity of 90% (28 of 31) for urinary total metanephrines and catecholamines (P = 0.63). The specificity of fractionated plasma metanephrines was 85% (221 of 261), compared with 98% (257 of 261; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001) for urinary measurements. The likelihood ratios for positive tests were 6.3 (95% confidence interval, 4.7 to 8.5) for fractionated plasma metanephrines and 58.9 (95% confidence interval, 22.1 to 156.9) for urinary total metanephrines and catecholamines. An adrenal pheochromocytoma was missed by urinary testing in two patients with familial syndromes and one asymptomatic patient with an incidentally discovered adrenal mass. An extra-adrenal paraganglioma was missed by plasma testing in one patient. In conclusion, measurements of 24-h urinary total metanephrines and catecholamines yield fewer false-positive results, an attribute preferred for testing low-risk patients, but fractionated plasma metanephrine measurements may be preferred in high-risk patients with familial endocrine syndromes.

Research paper thumbnail of Atorvastatin Inhibits Hypercholesterolemia-Induced Cellular Proliferation and Bone Matrix Production in the Rabbit Aortic Valve

Background-Despite the common occurrence of aortic stenosis, the cellular causes of the disorder ... more Background-Despite the common occurrence of aortic stenosis, the cellular causes of the disorder are unknown, in part because of the absence of experimental models. We hypothesized that atherosclerosis and early bone matrix expression in the aortic valve occurs secondary to experimental hypercholesterolemia and that treatment with atorvastatin modifies this transformation. Methods and Results-To test this hypothesis, we developed an experimental hypercholesterolemic rabbit model. New

Research paper thumbnail of The role of glutathione in the transport and catabolism of nitric oxide

Febs Letters, 1996

Nitric oxide acts as a neuronal and vascular messenger implying diffusion through intracellular e... more Nitric oxide acts as a neuronal and vascular messenger implying diffusion through intracellular environments containing 5–10 mM glutathione. Nitric oxide reacts with glutathione under aerobic conditions generating S-nitrosoglutathione (GSNO). GSNO reacts with glutathione (k = 8.3 × 10−3 M−1 · s−1) to generate nitrous oxide and glutathione disulfide (GSSG). Anaerobically, glutathione reacts with nitric oxide generating nitrous oxide and GSSG (k = 4.8 × 10−4 s−1 at 5 mM GSH). In both aerobic and anaerobic situations the nitroxyl anion may be an intermediate in the synthesis of nitrous oxide and, under aerobic conditions, nitroxyl anion may generate peroxynitrite. We present a hypothesis for the intracellular interaction between nitric oxide and glutathione.

Research paper thumbnail of Vitamin D (25OHD) Serum Seasonality in the United States

PLoS ONE, 2013

Background: Vitamin D is an important micronutrient for health. Hypovitaminosis D is thought to p... more Background: Vitamin D is an important micronutrient for health. Hypovitaminosis D is thought to play a role in the seasonality of a number of diseases and adverse health conditions. To refine hypotheses about the links between vitamin D and seasonal diseases, good estimates of the cyclicality of serum vitamin D are necessary.

Research paper thumbnail of PHOTODYNAMIC ACTION OF MEROCYANINE 540 IN ARTIFICIAL BILAYERS AND NATURAL MEMBRANES: ACTION SPECTRA AND QUANTUM YIELDS

Photochemistry and Photobiology, 1991

The action spectra and quantum yields for singlet oxygen (1O2) generation by merocyanine 540 (MC5... more The action spectra and quantum yields for singlet oxygen (1O2) generation by merocyanine 540 (MC540) in liposomes and isolated erythrocyte membranes were obtained using electron spin resonance techniques. Oxygen consumption was measured by spin label oximetry in the presence of histidine for fully-saturated dimyristoylphosphatidylcholine vesicles, mono-unsaturated 1-palmitoyl-2-oleoylphosphatidylcholine vesicles and erythrocyte membranes. The quantum yield for the photogeneration of 1O2 by membrane-bound MC540 in aqueous buffer was determined to be 0.065 ± 0.005, which is approx. 1/10 of the value determined for Rose Bengal under similar conditions. Using unilamellar liposomes and isolated erythrocyte membranes containing MC540 at different monomer/dimer ratios, we have observed that the action spectra of 1O2 generation closely overlap the absorption spectra of the monomeric dye in these systems. It is likely that factors which affect the monomer-dimer equilibrium of MC540 will influence the production of 1O2. These findings have important implications for the phototherapeutic efficacy of MC540.

Research paper thumbnail of Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photospray ionization tandem mass spectrometry

Clinica Chimica Acta, 2006

Serum steroid assays play an important role in the clinical evaluation of a number of common endo... more Serum steroid assays play an important role in the clinical evaluation of a number of common endocrine disorders. Among various assays, tandem mass spectrometry (MS/MS) has being increasingly applied in clinical laboratories for its high sensitivity, specificity, and simultaneous multi-analyte quantitation capability. Our first generation isotope dilution steroid profile assay by HPLC-tandem MS/MS with a C-18 column allowed for the measurement of 9 steroids in 18 min employing a sample volume of 760 ul serum. We describe our second generation steroid profile assay which allows for the quantitation of 12 steroids simultaneously employing HPLC-MS/MS and isotope dilution tandem MS in 11 min. This method requires a sample volume of 200 μl.An API-5000 triple-quadrupole mass spectrometer (Sciex, Concord, Canada) coupled with the PhotoSpray source and Shimadzu HPLC system (Shimadzu Scientific Instruments, Columbia, MD) was used employing isotope dilution with deuterium labeled internal standard (IS) for each analyte. Two hundred microliters of serum were deproteinized by adding 300 μl of acetonitrile containing internal standards. After centrifugation, 450 μl of supernatant were diluted with 900 μl of water and 1000 μl aliquot were injected onto a C-8 column. After a 3 min wash the valve was activated to initiate the gradient elution program which eluted the steroids. Quantitation by MRM analysis was performed both in positive ion mode for 11 analytes and in negative ion mode for aldosterone. Within-day and between-day precision, reliability and accuracy of this method were assessed by correlation with other MS/MS and immunoassay methods and by recovery study.Within-day CVs were < 11.5% for all analytes tested and between-day CVs ranged from 3.5% to 12.2%. The results of the comparison study yield r values ranging between 0.908 and 0.999. Recovery ranged from 90% to 110%.This method can simultaneously measure 12 steroids in serum within 11 min with minimal sample preparation. It can be routinely employed in a clinical environment and is attractive because of its simplicity in sample processing and high throughput.

Research paper thumbnail of INTRAVITREAL CONCENTRATION AND CLEARANCE OF TRIAMCINOLONE ACETONIDE IN NONVITRECTOMIZED HUMAN EYES

Retina-the Journal of Retinal and Vitreous Diseases, 2004

To determine the intravitreal concentration and clearance of triamcinolone acetonide at various i... more To determine the intravitreal concentration and clearance of triamcinolone acetonide at various intervals after intravitreal injection into nonvitrectomized eyes. Six participants were administered 4 mg (0.1 cc) of triamcinolone acetonide ophthalmic suspension. All six eyes underwent therapeutic pars plana vitrectomy with membranectomy at various post injection intervals ranging from 1.25 to 5 months from the intravitreal injection. Undiluted specimens of vitreous overlying the macula and of aqueous humor were submitted for analysis. Vitreous and aqueous humor concentrations of triamcinolone were measured by high performance liquid chromatography. Four eyes demonstrated detectable intravitreal concentrations of triamcinolone acetonide between 1.25 and 2.75 months after a single injection. Two eyes had an undetectable level of triamcinolone in both the vitreous and aqueous at 3 and 5 months post single injection. The intravitreal concentration of triamcinolone acetonide is detectable up to 2.75 months post a single 4 mg injection in nonvitrectomized eyes. A reinjection interval of 3 months may be needed to achieve sustained measurable levels of triamcinolone in nonvitrectomized patients.

Research paper thumbnail of The Peroxynitrite Generator, SIN1, Becomes a Nitric Oxide Donor in the Presence of Electron Acceptors

Archives of Biochemistry and Biophysics, 1999

SIN-1 has been used,in vitro, to simultaneously generate nitric oxide (•NO) and superoxide (O•−2)... more SIN-1 has been used,in vitro, to simultaneously generate nitric oxide (•NO) and superoxide (O•−2). However, the pharmacological activity of SIN-1 resembles that of a•NO donor. SIN-1 decays by a three-step mechanism. After initial isomerization to an open ring form, SIN-1A reduces oxygen by a one-electron transfer reaction to give O•−2and the SIN-1 cation radical, which decomposes to form SIN-1C and•NO. Here we report that one-electron oxidizing agents, in addition to oxygen, can oxidize SIN-1A, resulting in the release of•NO without the concomitant formation of O•−2. We demonstrate that easily reducible nitroxides, such as the nitronyl and imino nitroxides, are able to oxidize SIN-1. Biological oxidizing agents such as ferricytochromecalso stimulate•NO production from SIN-1. In addition, decomposition of SIN-1 by human plasma or by the homogenate of rat liver, kidney, and heart tissues results in the formation of•NO. Our findings suggest that SIN-1 may react with heme proteins and other electron acceptors in biological systems to produce•NO. Thus, at the relatively lowin vivooxygen concentrations, SIN-1 is likely to behave more like an•NO donor than a peroxynitrite donor. The relevance of this reaction to myocardial protection afforded by SIN-1 in ischemia/reperfusion-induced injury is discussed.

Research paper thumbnail of Intraocular concentration and pharmacokinetics of triamcinolone acetonide after a single intravitreal injection

Ophthalmology, 2003

To describe the pharmacokinetics occurring after the direct injection of triamcinolone acetonide ... more To describe the pharmacokinetics occurring after the direct injection of triamcinolone acetonide into the vitreous humor of humans.Interventional case series.Five patients who received a single 4-mg intravitreal injection of triamcinolone acetonide.An aqueous humor sample was obtained from 5 eyes via an anterior chamber paracentesis at days 1, 3, 10, 17, and 31 after injection. At each visit, visual acuity and intraocular pressure were measured and indirect ophthalmoscopy was performed. A fluorescein angiogram was carried out at day 10. Concentrations were determined using high performance liquid chromatography; pharmacokinetic analysis was carried out using PK Analyst, an iterative, nonlinear, weighted, least-squares regression program.Intraocular concentrations of triamcinolone were measured and population pharmacokinetic parameters were calculated.Pharmacokinetic data followed a two-compartment model. Peak aqueous humor concentrations ranged from 2151 to 7202 ng/ml, half-lives from 76 to 635 hours, and the integral of the area under the concentration-time curve (AUC0-t) from 231 to 1911 ng/h per milliliter. After a single intravitreal injection of triamcinolone, the mean elimination half-life was 18.6 days in nonvitrectomized patients. The half-life in a patient who had undergone a vitrectomy was shorter at 3.2 days.There was considerable intrasubject variation among peak concentration, AUC0-t values, and elimination half-lives. After intravitreal injection, measurable concentrations of triamcinolone would be expected to last for approximately 3 months (93 ± 28 days) in the absence of a vitrectomy. Because triamcinolone pharmacokinetics were characterized only in elderly patients with macular edema, the results cannot be extrapolated to other patient populations.

Research paper thumbnail of Pharmacokinetics of Intravitreal Ranibizumab (Lucentis)

Ophthalmology, 2007

To describe the pharmacokinetics of 0.5 mg of intravitreal ranibizumab (Lucentis) and to compare ... more To describe the pharmacokinetics of 0.5 mg of intravitreal ranibizumab (Lucentis) and to compare it with that of 1.25 mg of intravitreal bevacizumab (Avastin), using the same rabbit model.Experimental animal study.Twenty-eight Dutch-belted rabbits.One eye of each of 20 rabbits was injected with 0.5 mg of intravitreal ranibizumab. Both eyes of each of 4 rabbits were enucleated at days 1, 3, 8, 15, and 29. Ranibizumab concentrations were measured in aqueous fluid, whole vitreous, and serum. A further 8 rabbits were used to measure serum and fellow ranibizumab at additional time points of 3 and 8 hours.Ranibizumab concentrations in the aqueous, vitreous, and serum.Although vitreous concentrations of ranibizumab declined in a monoexponential fashion with a half-life of 2.88 days, concentrations of >0.1 μg/ml ranibizumab were maintained in the vitreous humor for 29 days. Ranibizumab concentrations in the aqueous humor of the injected eye reached a peak concentration of 17.9 μg/ml, 3 days after drug administration. Elimination of ranibizumab from the aqueous humor paralleled that found in the vitreous humor, with a half-life value of 2.84 days. No ranibizumab was detected in the serum or the fellow eye.In the rabbit, the vitreous half-life of 0.5-mg intravitreal ranibizumab is 2.88 days, shorter than the half-life of 1.25-mg intravitreal bevacizumab of 4.32 days. No ranibizumab was detected in the serum or the fellow uninjected eye; whereas small amounts of intravitreal bevacizumab have been detected in the serum and fellow uninjected eye.

Research paper thumbnail of Pharmacokinetics of Intravitreal Bevacizumab (Avastin)

Ophthalmology, 2007

To describe the pharmacokinetics of 1.25 mg of intravitreal bevacizumab (Avastin).Experimental an... more To describe the pharmacokinetics of 1.25 mg of intravitreal bevacizumab (Avastin).Experimental animal study.Twenty Dutch-belted rabbits.One eye of each of 20 rabbits was injected with 1.25 mg of intravitreal bevacizumab. Both eyes of each of 4 rabbits were enucleated at days 1, 3, 8, 15, and 29. Bevacizumab concentrations were measured in aqueous fluid, whole vitreous, and serum.Bevacizumab concentrations in the aqueous, vitreous, and serum.Whereas vitreous concentrations of bevacizumab declined in a monoexponential fashion with a half-life of 4.32 days, concentrations of >10μg/ml bevacizumab were maintained in the vitreous humor for 30 days. Bevacizumab concentrations in the aqueous humor of the injected eye reached a peak concentration of 37.7 μg/ml 3 days after drug administration. A maximum serum concentration of 3.3 μg/ml was achieved 8 days after intravitreal injection and the concentration fell below 1 μg/ml 29 days after injection. Elimination of bevacizumab from the aqueous humor and serum paralleled that found in the vitreous humor, with half-life values of 4.88 days and 6.86 days, respectively. Very low concentrations of bevacizumab were detected in the fellow uninjected eye. Concentrations of bevacizumab in the vitreous of the fellow eye varied incrementally, from 0.35 ng/ml at 1 day to 11.17 ng/ml at 4 weeks. Concentrations of bevacizumab in the aqueous humor of the fellow eye reached their peak at 1 week, at 29.4 ng/ml, and declined to 4.56 ng/ml at 4 weeks.The vitreous half-life of 1.25 mg intravitreal bevacizumab is 4.32 days in a rabbit eye. Very small amounts of bevacizumab were detected in the serum and in the fellow uninjected eye.

Research paper thumbnail of Morphometry of landslides in Garhwal Himalaya, India

Disaster Prevention and Management, 2011

Purpose -The purpose of this paper is to introduce a simple and convenient method for the landsli... more Purpose -The purpose of this paper is to introduce a simple and convenient method for the landslides study. Design/methodology/approach -This study is based upon the individual case study of three past landslides which may probably prove fatal in the near future. It is useful for the measurement of the horizontal length, longitudinal length, altitude at different points and area of the landslide. These measurements are very helpful in calculating the valley side gradient, longitudinal gradient of the landslide scarp and the volume of the debris generated due to mass movement. Findings -Reactivation of slope instability problems were found along these landslides spots, which will create havoc for the people and a problem for the administration in the near future. Originality/value -The implication in the paper is that structural and non-structural mitigation and management strategies are needed.