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Biomedical subjects

P K Grover

Publications and source records attributed to P K Grover.

At least 37 records · Page 2Linked to original sources
PubMed1995

Evidence for the presence of abnormal proteins in the urine of recurrent stone formers.

Two-thirds of matrix of all urinary stones consists of proteins. Despite intense research, their relationship to calculogenesis remains controversial. In an attempt to study excretion of proteins in stone formers, their urinary profiles were analyzed and compared with those of healthy subjects. Two-dimensional gel electrophoresis was employed to obtain high resolution separation of proteins. The urine of patients with histories of idiopathic calcium oxalate (CaOx) calculi contained 7 unique proteins, and 2 others that appeared to be overexpressed. Except for alpha 1-acid glycoprotein, the remaining 8 proteins are previously unknown constituents of urine with molecular weights of approximately 43, 39.5, 29, 26, 25.5, 26.5, 27 and 18.5 kD. Their isoelectric points range from 5.5 to 8.0. Coelectrophoresis of pooled urinary proteins of male and female stone formers disclosed that all 9 proteins had identical charges and molecular weights, regardless of the donors' sex. Analyses of urines of idiopathic recurrent CaOx stone formers who had no radiologically detectable calculi also revealed the presence of these proteins. This excludes the possibility that the proteins might be a consequence of abrasion of urothelial lining by the developing stone(s). Recently defective Tamm-Horsfall mucoprotein (THM) has been implicated in urinary stone formation. Coelectrophoresis of pooled urinary proteins of healthy subjects and stone formers denoted that it had an identical charge and molecular weight in both groups. This suggested that stone formation could not be ascribed to a difference in composition of THM. Whether this is attributable to a dissimilar amino acid sequence of this mucoprotein, remains to be probed.

Calcium Oxalate↗
PubMed1994

Simultaneous liquid chromatographic determination of centchroman and its 7-demethylated metabolite in serum and milk.

A precise and sensitive high-performance liquid chromatographic assay was developed and validated for determination of centchroman (I) and its 7-demethylated metabolite (II) in human serum and milk. The serum, at alkaline pH, was extracted with diethyl ether. In the case of milk, after precipitation of the milk protein with acetonitrile, the supernatant was evaporated to dryness and then extracted with diethyl ether at alkaline pH. After solvent evaporation the residue was reconstituted in mobile phase. Separations were accomplished by reversed-phase liquid chromatography using a Spheri-5 cyano column. Recoveries of I and II were always > 95%. Excellent linear relationships (r > 0.999) were obtained between the measured and added concentration ratios of the corresponding serum and milk concentrations over a range of 1 to 1000 ng/ml and 2.5 to 1000 ng/ml for I and II, respectively.

Acetonitriles↗
PubMed1994

Quantification of 2,3-dihydro-7-methoxypyrrolo-[2,1-b]-quinazolin-9(1H)-one, a new antiallergic agent, by high-performance liquid chromatography in serum.

The CDRI compound 73/602 (I, 7-methoxydeoxyvasicinone) is an antiallergic agent presently undergoing Phase II clinical trials. A rapid, precise and sensitive high-performance liquid chromatographic assay has been developed and validated for the detection of I in rat serum. The method involves extraction of rat serum samples with diethyl ether after addition of 1 M potassium hydroxide. Separations were accomplished on a RP-18 column using acetonitrile-phosphate buffer (pH 7) (20:80, v/v) as the mobile phase. Recovery of I was always > 90%. An excellent linear relationship (r = 0.9999) was obtained between the quantified and added concentrations over the range of 2.5 to 500 ng/ml. The sensitivity of the assay for 0.5 ml serum was 2.5 ng/ml.

Animals↗
PubMed1994

Determination of ampicillin in serum by high-performance liquid chromatography with precolumn derivatization.

A high-performance liquid chromatographic assay method using precolumn derivatization and fluorescence detection has been developed and validated for the determination of ampicillin in serum. The presented method is simple and provides improved selectivity and sensitivity over other existing HPLC methods. It is linear over the concentration range of 100 to 10,000 ng/ml (method 1) and 2 to 1000 ng/ml (method 2) and the extraction recovery is more than 75%. The coefficient of variation is found to be less than 10% over the concentration ranges studied.

Ampicillin↗
PubMed1994

High resolution 2-dimensional electrophoretic analysis of proteins in saline bladder washings.

Human urine contains a spectrum of proteins derived from various organs in the body. This investigation was undertaken to identify a group of proteins secreted primarily by bladder urothelium. Saline bladder washouts were collected from 9 male and 4 female patients undergoing routine cystoscopic examination. Each sample was sieved, desalted, freeze-dried and solubilized in urea mix. identical amounts of proteins were pooled according to donor sex. All individual and pooled samples were then subjected to 2-dimensional gel analysis using the ISO-DALT system. Profiles of both groups of subjects were highly reproducible, comparable and resembled those of urine and plasma. Several hundred spots were visualized although most of them as yet remain unrecognized. The proteins identified in bladder washouts include albumin, transferrin, IgG gamma-heavy chain, Gc-globulin, alpha 1-antichymotrypsin, alpha 1-antitrypsin, alpha 1-acid glycoprotein, G4, IgG light chains, alpha 1-microglobulin, and low and high density lipoproteins. Most importantly, while some abundant urinary macromolecules such as Tamm-Horsfall mucoprotein and most acidic urinary proteins were undetectable, albumin and transferrin were relatively less expressed. The 2-dimensional profiles we developed can be used as a data base to study urothelium derived proteins in various disease conditions such as bladder cancer.

Cystoscopy↗
PubMed1994

Tamm-Horsfall mucoprotein reduces promotion of calcium oxalate crystal aggregation induced by urate in human urine in vitro.

1. Increasing the concentration of dissolved urate promotes calcium oxalate crystallization in urine from which Tamm-Horsfall mucoprotein, an inhibitor of calcium oxalate crystal aggregation, has almost completely been removed. This study aimed to determine whether the effect of urate could be reduced or abolished by a physiological concentration of Tamm-Horsfall mucoprotein. This was approached in two ways. 2. The effect of Tamm-Horsfall mucoprotein on calcium oxalate crystallization induced by urate was tested in ultrafiltered (10 kDa) urine samples from 10 healthy men. Tamm-Horsfall mucoprotein (35 mg/l) was added to half of each specimen, the urate concentration was increased by the addition of sodium urate solution and crystallization was induced by a standard load of oxalate. The remainder of each urine specimen was used as a control; these specimens were treated with an identical amount of urate solution, but contained no Tamm-Horsfall mucoprotein. Tamm-Horsfall mucoprotein had no effect on the urinary metastable limit or on the deposition of calcium oxalate, but significantly reduced the size of the particles precipitated. 3. The effect of increasing the urate concentration in the presence of Tamm-Horsfall mucoprotein was tested. Tamm-Horsfall mucoprotein (35 mg/l) was added to 10 ultrafiltered urine samples as before, the samples were divided, and the concentration of urate was increased in half of each specimen. Compared with the control to which no urate was added, urate significantly reduced the amount of oxalate required to induce spontaneous calcium oxalate nucleation and increased the median volume and the particle size of the material deposited.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium Oxalate↗
PubMed1994

Urate and calcium oxalate stones: from repute to rhetoric to reality.

A critical appraisal of the evidence commonly cited to support a link between high urate excretion and calcium oxalate (CaOx) urinary calculi is presented. Two theories have been invoked to provide a scientific explanation for urate's apparent promotory effect. The first proposes that urinary urate crystals promote CaOx precipitation by the phenomenon of epitaxy; the second hypothesis is that colloidal particles of urate reduce the inhibitory activity of urinary glycosaminoglycans (GAGs) which normally prevent the crystallization of CaOx. However, to the present, neither has been verified experimentally. More recent research from our group has revealed that at normal physiological pH values dissolved urate directly promotes CaOx precipitation by the classic 'salting-out' effect by enhancing nucleation, growth and aggregation of CaOx crystals. It is therefore suggested that the beneficial effect of allopurinol in reducing CaOx stone recurrences may be attributed to its lowering the urinary output of urate and thereby reducing the probability that CaOx will be salted out of urine, rather than to epitaxy or inactivation of urinary GAGs.

Allopurinol↗
PubMed1993

Simultaneous determination of a new antimalarial agent, CDRI compound 80/53, and its metabolite primaquine in serum by high-performance liquid chromatography.

Compound 80/53 (I) is a new substance being developed as an antimalarial agent. It is unstable in acidic conditions where it is converted into primaquine. A high-performance liquid chromatographic assay for simultaneous determination in serum of I and primaquine has been developed. Conditions were optimized to minimize the conversion of I into primaquine. The method includes extraction of the unchanged compound and primaquine from serum samples with hexane-2-propanol (pH > 8). Separation was accomplished by reversed-phase chromatography on a C18 column with acetonitrile-tetrahydrofuran-phosphate buffer. The recoveries of I and primaquine were always greater than 70%. No interference was observed in extracts obtained from drug-free serum. The detector response was linear with concentrations of I and the metabolite in the ranges 25-400 and 10-180 ng/ml, respectively, and the within-day precision (coefficient of variation) remained less than 13.7% for I and 12.5% for primaquine. The method is suitable for the determination of concentration-time profiles of I and primaquine in human serum.

Antimalarials↗
PubMed1993

Study of activities of arginase, hexosaminidase, and leucine aminopeptidase in prostate fluid.

Our previous studies demonstrated that extracts of the cancerous prostate tissues exhibit increased activities of arginase and hexosaminidase and decreased activity of leucine aminopeptidase. In an endeavor to devise a reliable, noninvasive and cost effective method for the diagnosis of prostate carcinoma, prostate fluids collected from 123 patients were analyzed for arginase, hexosaminidase, and leucine aminopeptidase. Based on the clinical diagnoses, the patients were divided into four groups: 13 men with chronic prostatitis, 29 with no recognized prostatic lesions, 63 with benign prostatic hyperplasia, and 18 with prostate cancer. The prostate fluids had no detectable arginase activity. The means of the total (units/mL) and the specific activities (units/mg of protein) of hexosaminidase and leucine aminopeptidase varied, albeit there were no significant differences in the four groups of patients recruited in this study. It was concluded that the profiles of enzymes in prostate fluids might differ from that in the secreting prostate tissues and that the results of enzyme analysis in prostate fluids for the diagnosis of cancer should be interpreted with caution.

Adolescent↗
PubMed1993

Two-dimensional analysis of proteins in unprocessed human urine using double stain.

In the present study, a revised two-dimensional (2-D) map of proteins in unprocessed human urine was established. Spot urines were obtained from ten healthy men and ten healthy women each in Dewar flasks over a mixture of protease inhibitors. The specimens were sieved, desalted and freeze-dried, and identical amounts were pooled according to sex. The resulting samples were dissolved in urea mix and subjected to 2-D gel analysis employing the ISO-DALT system: the electrophoretograms were developed by double stain. We visualized more protein spots than reported previously, though most of them as yet remain unidentified. With the exception of similar relative positions of major protein spots, the samples demonstrated different patterns: urine of women, in general, had more spots and a greater proportion of low molecular weight proteins. Besides alpha 1-acid glycoprotein and the most acidic urinary proteins (MAUP), which were discerned earlier only in the ACIDO-DALT system, the ones known to precipitate while processing the samples were also detected. The 2-D profiles we established could be used as a diagnostic aid to study the excretion of proteins under various disease conditions.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1993

Dissolved urate promotes calcium oxalate crystallization: epitaxy is not the cause.

1. Increasing the concentration of dissolved urate promotes the crystallization of calcium oxalate from urine. The possibility was investigated that this effect may be caused by heterogeneous nucleation of calcium oxalate by particles of crystalline urate. 2. Urine samples were collected from 10 healthy men, centrifuged and filtered, and a solution of sodium urate was added to increase the medium urate concentration from 2.2 to 5.6 mmol/l. Calcium oxalate crystallization was induced by the addition of oxalate, followed by incubation for 90 min in a shaking waterbath at 37 degrees C. The crystalline material was filtered out and the urate concentration was determined in the filtrate. 3. No difference in the urate concentration before and after induction of calcium oxalate crystallization was observed. These findings were confirmed by infra-red spectroscopy, X-ray powder diffraction and ultraviolet wet chemical analysis with detection limits of 5-10, 1.0 and 0.055%, respectively; urate was not detected in the calcium oxalate crystals. 4. In addition, three urine samples were collected and passed through 10 kDa ultrafiltration membranes to remove any colloidal particles which might have been present. The urate concentration was increased and an oxalate load was added as before, prior to incubation at 37 degrees C in a shaking water bath for 5 min, followed by passage through 10 kDa ultrafiltration membranes. Scanning electron microscopy revealed no particles on the membranes thereby indicating that colloidal or crystalline urate was not formed early in the crystallization experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

A highly specific heterologous enzyme-linked immunosorbent assay for measuring testosterone in plasma using antibody-coated immunoassay plates or polypropylene tubes.

A highly specific and sensitive enzyme-linked immunosorbent assay, using a heterologous combination of antiserum raised against testosterone-3-(O-carboxymethyl) oxime-bovine serum albumin and penicillinase-labeled testosterone-11 beta-carboxymethyl ether, was developed for measuring testosterone in human plasma. Immunoassay plates (96 wells) provided a sensitivity of 2.5 pg/well. This was achieved by maintaining the molar ratios of steroid to enzyme between 10 and 20. The assay was very specific for testosterone and did not show any cross-reaction with the related C19 steroids tested. Replacement of immunoassay plates with the locally available polypropylene tubes raised the detection limits to 25 pg/tube, but improved the range of doses of testosterone that could be measured up to 10,000 pg. The antiserum to testosterone derivative was linked to both immunoassay plates and polypropylene tubes through immunochemical bridges. Comparison of testosterone values of 52 plasma specimens obtained by both solid phase methods with those of radioimmunoassay showed good correlation.

Antibodies↗
PubMed1992

Calcium oxalate crystallization in urine: role of urate and glycosaminoglycans.

Increasing the concentration of urate promotes the crystallization of calcium oxalate in human urine. In this study the possibility that this effect might be attributable to the attenuation of the inhibitory activity of urinary glycosaminoglycans (GAGs) was explored. Urine sample were collected from 20 men with no history of urolithiasis and the intact GAGs removed by 10 kDa ultrafiltration. Ten of these specimens, designated type A, spontaneously precipitated calcium oxalate crystals when the median urate concentration was increased from 3.13 to 7.33 mmol/liter by the addition of a saturated solution of sodium urate. In the remaining more dilute urines, which were designated type B, spontaneous calcium oxalate crystallization did not occur when the median urate concentration was raised from 2.20 to 6.40 mmol/liter. In these samples crystallization was induced by a standard load of oxalate above the empirically determined metastable limit. Addition of urate significantly reduced the median metastable limit from the control value of 125 to 46 mumol oxalate, and the volume of calcium oxalate deposited was increased fourfold from 25,000 to 104,000 microns 3/microliters. The median size of the precipitated particles was also increased in the presence of urate from 12.06 microns to 14.3 microns; this was confirmed by scanning electron microscopy, which demonstrated that the crystals precipitated in the presence of added urate, though individually smaller, were markedly more numerous and more highly aggregated than those deposited in the control. Re-ultrafiltration of the urines to which urate had been added did not alter the urate concentration, and SEM examination of the ultrafiltration membranes did not reveal the presence of any particulate material.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Simultaneous determination of centbutindole and its hydroxy metabolite in serum by high-performance liquid chromatography.

A high-performance liquid chromatographic assay has been developed and validated for the determination of centbutindole and its hydroxy metabolite in serum. The method involves extraction of serum samples with diethyl ether at pH greater than 8, back-extraction into 0.5 M hydrochloric acid and finally again with diethyl ether after addition of 2 M potassium hydroxide. Separation was accomplished by reversed-phase high-performance liquid chromatography on a cyano column with an acetonitrile-phosphate buffer system. The recovery of centbutindole and its metabolite was always greater than 80%. Calibration curves were linear over the concentration range 0.25-5 ng/ml for centbutindole and 0.05-1 ng/ml for the hydroxy metabolite. Although the lower limit of detection was 0.1 ng/ml for centbuntindole and 0.02 ng/ml for the hydroxy metabolite, the reliable limits of quantitation were 0.25 and 0.05 ng/ml, respectively, using 4 ml of serum.

Antipsychotic Agents↗
PubMed1991

Urate and calcium stones--picking up a drop of mercury with one's fingers?

The evidence invariably cited to support the suspicion that urinary urate is a predisposing factor in calcium oxalate (CaOx) stone formation is critically reviewed. Analysis of the relevant literature shows that speculation is based on the clinical impression that CaOx stone-formers appear to excrete more urate than do normal subjects, and that allopurinol reduces the rate of CaOx stone recurrences. On balance, this is sufficient to suggest that a high urinary excretion of urate promotes CaOx stone formation. However, in the past, evidence to disclose the mechanism by which urate could exert this effect has been largely shrouded in confusion and controversy. The evidence for two theories that have dominated thinking in this area are reviewed and new findings are reported that indicate that neither can account for the purported effect of urate. It is concluded that dissolved urate in urine, at normal physiological pH values, directly provokes CaOx crystal nucleation by the phenomenon of salting-out. The possibility that urate promotes CaOx stone formation is further strengthened by its ability to increase significantly the amount of CaOx precipitated from solution and to cause the aggregation of individual crystals into large clusters. Future avenues of investigation that should assist in the formulation of diagnostic and therapeutic guidelines are presented.

Allopurinol↗
PubMed1990

Enzyme immunosorbant assay of oestradiol in unextracted plasma using penicillinase as label.

An enzyme-linked immunosorbant assay (ELISA) for measuring oestradiol directly in plasma without extraction utilizing antibodies raised against oestradiol-3-(O-carboxymethyl) ether-bovine serum albumin conjugate, and oestradiol-6-(O-carboxymethyl) oxime linked to penicillinase (EC 3.5.2.6) as a marker was developed. Polyvinyl 96-well microtitre plates were used for immobilization of anti-oestradiol IgG. Standards of oestradiol (92 to 9,190 pmol/l were prepared in oestradiol-free plasma and 8-anilino-1-naphthalene sulphonic acid (8-ANS, 5 mg/ml of 10 mmol/l PBS) was added to the microtitre plate wells to displace oestradiol from plasma binding proteins. The assay had a lower limit of detection of 92 pmol/l plasma and could be performed within 4 h. Comparison of oestradiol values of 51 plasma specimens obtained by ELISA with those of radioimmunoassay (RIA), in which oestradiol was extracted with diethyl ether, showed good correlation (y = 0.786x + 0.03; r = 0.900).

Enzyme-Linked Immunosorbent Assay↗
PubMed1990

Does Tamm-Horsfall mucoprotein inhibit or promote calcium oxalate crystallization in human urine?

Using two different experimental techniques, Tamm-Horsfall mucoprotein (THM) has been reported both to inhibit and to promote calcium oxalate (CaOx) crystallization in ultrafiltered human urine. In this study, these two techniques were used to compare the effects of THM on CaOx crystallization in the same ultrafiltered urine samples. Urine was collected from 10 healthy men and ultrafiltered (10,000 Da). Each sample was divided and to one half was added sufficient human THM to give a final concentration of 35 mg/L. CaOx crystallization was induced in the samples by addition of an oxalate load and by evaporation. Using the evaporation technique THM significantly increased the deposition of CaOx determined as 14C-oxalate, from 9,772 cpm to 43,652 cpm (P less than 0.01). Using the oxalate load method THM had no effect on the metastable limits of the urine with respect to CaOx, and significantly increased the volume of particulate material deposited from 26,000 to 39,995 microns 3/microliters - an increase of 54%. This increase was reduced to 21% when values were corrected for the volume of THM particles recorded in control samples to which no oxalate load was added. Using 14C-oxalate, it was shown that this increase in volume could not be attributed to an enhanced deposition of crystalline CaOx, but was probably the result of an increased polymerization of THM in the presence of CaOx crystals. Despite this, the average size of the particles precipitated in the presence of THM (6.5 microns) was significantly (P less than 0.01) less than that observed in the absence of THM (12.1 microns). It was concluded that the effect of THM on CaOx crystallization in urine depends upon the methodology used to assess it and that promotion would only be expected in vivo in cases of extreme dehydration. Under usual physiological conditions THM would be expected to inhibit CaOx crystal aggregation and to have little effect, if any, on the amount of crystalline material deposited.

Adult↗