Automated determination of hypoxanthine and xanthine in urine by high-performance liquid chromatography with column switching

We report a high-performance liquid chromatographic method with column switching for urinary hypoxanthine and xanthine. Analyses were carried out with both a reversed-phase column and an anion-exchange column connected by a column switch and controlled automatically by a computerized system controller. The relationships between standard concentrations and peak heights were linear in a concentration range of 1 to 1000 nmol/ml. The recovery of hypoxanthine added to urine was 101.1%, and that of xanthine was 98.1%. With our method urinary hypoxanthine and xanthine can be measured accurately without any sample preparation other than filtration.     

Simultaneous measurement of allantoin, uric acid, xanthine and hypoxanthine in blood by high-performance liquid chromatography

A high-performance liquid chromatographic method for determining catabolism products of nucleic acids and purines, such as oxypurines (i.e. uric acid, xanthine and hypoxanthine) and allantoin in the blood plasma of ruminants was developed. The plasma was deproteinized with 10% trichloroacetic acid. The method enabled determination of oxypurines without derivatization. Allantoin was determined after conversion with 2,4-dinitrophenylhydrazine to a hydrazone (GLX-DNPH). Separation of converted allantoin, uric acid, xanthine and hypoxanthine derivatives was carried out using two reversed-phase C18 columns. The combination of pre-column derivatization and gradient elution with monitoring of the effluent at 205, 254 and 360 nm provides a simple and selective analytical tool for studying oxypurines and allantoin in plasma. The total run time of the HPLC analysis was 60 min. The recovery of the purine derivatives (i.e. oxypurines and allantoin) added to the plasma was between 95 and 106%. Purine derivatives were stable when the processed samples were stored for 7 days at -10 degrees C. The low values of the intra-assay coefficient of variations (2.5-4.6%) and the low values of the detection limits (0.187-0.004 nmol) point to the satisfactory precision and sensitivity of the method.     

In vitro activity of guinea pig transfer factor released into plasma

Plasma fractions and plasma dialysate from 2,4-dinitrochlorobenzene- and tuberculin-sensitive guinea pigs that had been treated with either antilymphocytic serum or normal control serum were analyzed for their ability to transfer lymphocyte transformation, passive cutaneous anaphylaxis, and macrophage migration inhibition, as well as delayed hypersensitivity in vivo. Antilymphocytic serum caused rapid release of material, which has characteristics of transfer factor, into the plasma. It was dialyzable, migrated electrophoretically with the alpha globulins and albumin, possessed a 280/260 (nm) optical density ratio of 0.7, and caused in vitro lymphocyte transformation in the presence of the specific antigen. Passive cutaneous anaphylaxis antibodies were also present in the plasma of sensitive animals, but they were isolated in electrophoretic or dialysis fractions separate from those containing transfer activity.     

Desulphation of heparin by mice and guinea pig leukocytes

A series of 603 patients referred with atypical Papanicolaou smears was evaluated by repeat smears, colposcopically directed cervical biopsies, and endocervical curettage. These techniques as a unit can establish an accurate outpatient diagnosis superior to any of these modalities used alone and comparable with findings in conization and hysterectomy specimens. Endocervical curettage has made a unique contribution to the evaluation of such patients; these curettings have allowed examination of tissue fragments and are more reliable in diagnosing neoplasia than are endocervical smears. Invasive carcinoma and its precursors confined to the anatomic endocervical canal can be recognized by this technique, and conversely the absence of neoplastic epithelium in adequate endocervical curettings rules out occult carcinoma. Indications for conization of the cervix are discussed in reference to the other biopsy and cytologic findings, and guidelines are presented for patient management, stressing clinicopathologic correlation and cooperation.     

Bicarbonate secretion in the guinea pig duodenum: functional characterization of peptide hormone receptors in duodenal enterocytes

Human peripheral blood mononuclear cells from healthy donors were treated ex vivo with the proteolytic enzyme bromelain and studied by flow cytometry. Bromelain-treated lymphocytes exhibited 60-90% reduced cell surface staining for CD44 and CD62-L molecules. While the staining for molecules CD16, CD56 and CD49d was unaffected, a moderate increase (10-40%) in expression of the beta(2)-integrins CD11a-c was seen. This selective modulation of cell adhesion molecules (CAM) was seen on T cells and NK cells, as well. The selective modulation of CAM may help explain some of the clinical effects observed after bromelain treatment in patients suffering from chronic inflammatory disease, HIV and cancer.     

Impairment of neural nitric oxide-mediated relaxation after antigen exposure in guinea pig airways in vitro

A solid-phase erythrocyte adherence assay has been developed for the serological detection of reagin antibodies in syphilis. Capture-S (Immucor, Inc., Norcross, Ga.) is a nontreponemal, qualitative screening test for the detection of immunoglobulin G (IgG) and IgM antilipid antibodies in serum or plasma samples from blood donors. The Capture-S assay utilizes a modified Venereal Disease Research Laboratory antigen bound to microtitration wells and anti-IgG- plus anti-IgM-coated indicator erythrocytes as the detection system. The Capture-S assay was evaluated at six separate sites on 10,942 specimens. For patient samples of clinically diagnosed syphilis categories (n = 366), the Capture-S assay yielded a sensitivity of 80.7% versus 80.3% for the rapid plasma reagin (RPR) card test (Becton Dickinson Microbiology Systems, Cockeysville, Md.). In comparative experiments on patient and donor samples (n = 10,222), the Capture-S assay demonstrated a sensitivity of 94% compared to 91.2% for the RPR card test. The Capture-S and RPR card tests produced essentially equivalent specificities of 99.2% and 99.3%, respectively, for this sample population. For five test sites, the Capture-S and RPR card test demonstrated a 98.3% agreement (10,085 of 10,264) of test results. These evaluations indicate that the Capture-S compares favorably to the RPR card test in assay sensitivity and specificity, with the added benefits of ease of use, accommodation of high-volume testing, and potential for automation.     

Propagation by sporulation in the guinea pig symbiont Metabacterium polyspora

The Gram-positive bacterium Metabacterium polyspora is an uncultivated symbiont of the guinea pig gastrointestinal tract. Here we present evidence that in M. polyspora vegetative cell division has taken on a minor, and apparently dispensable, role in propagation. Instead, this unusual bacterium has evolved the capacity to produce progeny in the form of multiple endospores. Endospore formation is coordinated with transit of the bacterium through the gastrointestinal tract of the guinea pig. For the majority of cells, sporulation is initiated in the ileum, whereas later stages of development take place in the cecum. We show that multiple endospores are generated both by asymmetric division at both poles of the cell and by symmetric division of the endospores at an early stage of their development. Our findings suggest that M. polyspora represents an intermediate step in the evolution of a novel mode of cellular propagation that originates with endospore-forming Bacillus and Clostridium spp., which reproduce by binary fission, and extends to Epulopiscium spp., which create multiple viviparous offspring by a process of internal reproduction.     

Potentiation of purinergic neurotransmission in guinea pig urinary bladder by histamine

Patients suffering from the inflammatory condition of interstitial cystitis frequently exhibit an increased number of mast cells in the bladder. To determine whether mast cell mediators have the potential to influence the neurogenic contraction of the bladder smooth muscle and thereby possibly contribute to the symptoms of interstitial cystitis, we examined the effects of histamine, a major inflammatory mediator of mast cell origin, on nerve- and agonist-induced contractions of in vitro strips of guinea pig urinary bladder. Histamine (10 microM.) potentiated by more than 50% the nerve-induced contraction of bladder strips evoked by field stimulation with 0.5 msec. pulses at 4 Hz. Because the neurogenic contraction of the bladder is mediated by at least two neurotransmitters, acetylcholine (ACh) and ATP, we examined the effects of histamine on each of these transmitters. Histamine potentiated responses to the purinergic component of the neurogenic response (that part of the neurogenic response that remains after treatment with atropine) and potentiated responses to exogenously applied ATP. Histamine did not potentiate the response to the cholinergic component of the neurogenic response (that part of the neurogenic response that remains after desensitization of purinoceptors with alpha, beta-methylene ATP) nor responses to carbachol, a cholinergic agonist. These results indicate that histamine potentiates the neurogenic response of the bladder by influencing the purinergic component, apparently at postjunctional sites.     

Simultaneous determination of guanine, uric acid, hypoxanthine and xanthine in human plasma by reversed-phase high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method with amperometric detection is described for the separation and quantification of uric acid, guanine, hypoxanthine and xanthine. The isocratic separation of a standard mixture of the compounds was achieved in 5 min on a Spherisorb 5 C18 reversed-phase column, with a mobile phase of NaH2PO4 (300 mmol dm-3, pH 3.0)-methanol-acetonitrile-tetrahydrofuran (97.8 + 0.5 + 1.5 + 0.2). Uric acid, guanine, hypoxanthine and xanthine were completely separated, with detection limits in the range 2-20 pmol per injection. The effect of pH and the composition of the mobile phase on the separation are described. The hydrodynamic voltammograms of these compounds were recorded at a glassy carbon electrode. The linear range of the calibration graph for each compound was: uric acid, 1-5000 mumol dm-3; guanine, 0.5-2000 mumol dm-3; hypoxanthine, 0.1-500 mumol dm-3 and xanthine, 0.5-5000 mumol dm-3. The within- and between-day precision was good. The uric acid and hypoxanthine content in human plasma was measured using the proposed method. Good recoveries of uric acid (97.9-103%), hypoxanthine (98.0-99.2%), guanine (96.0-98.3%) and xanthine (96.0-102%) were obtained from human plasma. The results of electrochemical detection were in good agreement with those of UV detection.     

Transmural potential changes associated with the in vitro absorption of theanine in the guinea pig intestine

Theanine, L-N-ethylglutamine, is one of the major components of amino acids in Japanese green tea. To characterize the mode for intestinal absorption of theanine, the ionic dependency and kinetic properties of the theanine- and glutamine-evoked transmural electrical potential difference changes (delta PD) were investigated in vitro by using everted sacs prepared from the guinea pig ileum. Both theanine and glutamine applied to the luminal side induced dose-dependent increases in delta PD (increase in serosal positive value). The theanine- and glutamine-evoked delta PD values conformed to the Michaelis-Menten relationship, with delta PDmax not being different, whereas the half-saturation concentration was lower for glutamine (3.1 +/- 0.2 mM) than for theanine (21.4 +/- 0.6 mM). The theanine-evoked delta PD value was much smaller when theanine was applied in the presence of glutamine than when applied alone. The theanine- and glutamine-evoked delta PD values were both inhibited by removing Na+ from the luminal solution. These results suggest that the intestinal absorption of theanine and glutamine is mediated by a common Na(+)-coupled co-transporter in the brush-border membrane, the affinity of which is lower for theanine than for glutamine.     

In vitro oxidation of 6-MP and its metallo complexes by xanthine oxidase

Xanthine oxidase oxidized pure 6-MP, and its bismuth complex to 6-thiouric acid, in vitro. The enzyme did not oxidize the palladium complex of 6-MP under identical conditions.     

Signal transduction pathways in guinea pig sperm

Trifluoperazine (TFP), the antagonist of calmodulin (CaM), significantly stimulated the capacitation and acrosome reaction of guinea pig spermatozoa at the concentration of 10-100 mumol/L, independent of the external Ca2+. Forskolin, dbcAMP and caffeine evidently promoted the occurrence of acrosome reaction of spermatozoa at early capacitation stage (5 h) in nonsynchronous system but not in synchronous system. If the spermatozoa were capacitated for 15 h in synchronous system, the above three drugs significantly stimulated acrosome reaction in a Ca(2+)-independent manner. Protein kinase C activators, i.e. phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDB) did not influence the occurrence of acrosome reaction of spermatozoa at early capacitation stage, but significantly increased the acrosome reaction rate in capacitated spermatozoa in a Ca(2+)-independent manner. In contrast, PKC inhibitor staurosporine significantly inhibited the occurrence of acrosome reaction.     

Neurokinin receptors in the guinea pig ileum

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.     

Elastase in hyperpnea-induced guinea pig airway constriction

Aerosolized elastase has been shown to produce airway constriction in guinea pigs. In this study, we examined whether endogenous elastase plays a role in isocapnic hyperpnea-induced airway constriction using an elastase inhibitor, eglin-c. The study was divided into three experiments. In the first experiment, we used an elastase inhibitor, eglin-c, to suppress hyperpnea-induced bronchoconstriction. Twenty-two young male Hartley guinea pigs were divided into three groups: control (n=8), eglin-c(1) (a lower dose of eglin-c, n=7), and eglin-c(2) (a higher dose of eglin-c, n=7). In the second experiment, we tested whether eglin-c affects pulmonary function following 15 min of normal air ventilation in two groups of animals: control (n=8) and eglin-c (n=8). In the third experiment, animals were divided into two groups: control (n=7) and compound 48/80 (a mast cell degranulating agent, n=7). Airway function was examined in the anesthetized-paralyzed animal. In the first and third experiments, 15 min of isocapnic hyperpnea caused marked decreases in dynamic respiratory compliance, forced expiratory flow at 0.1 s and maximal expiratory flow at 50% total lung capacity, demonstrating hyperpnea-induced airway constriction. This bronchoconstriction was significantly attenuated by eglin-c and by pretreatment with compound 48/80. In the second experiment, eglin-c did not significantly affect bronchial function following normal air ventilation. These data suggest that elastase released from mast cells directly or indirectly induces hyperpnea-induced bronchoconstriction.     

Circularization and cleavage of guinea pig cytomegalovirus genomes

The mechanisms by which herpesvirus genome ends are fused to form circles after infection and are re-formed by cleavage from concatemeric DNA are unknown. We used the simple structure of guinea pig cytomegalovirus genomes, which have either one repeated DNA sequence at each end or one repeat at one end and no repeat at the other, to study these mechanisms. In circular DNA, two restriction fragments contained fused terminal sequences and had sizes consistent with the presence of single or double terminal repeats. This result implies a simple ligation of genomic ends and shows that circularization does not occur by annealing of single-stranded terminal repeats formed by exonuclease digestion. Cleavage to form the two genome types occurred at two sites, and homologies between these sites identified two potential cis elements that may be necessary for cleavage. One element coincided with the A-rich region of a pac2 sequence and had 9 of 11 bases identical between the two sites. The second element had six bases identical at both sites, in each case 7 bp from the termini. To confirm the presence of cis cleavage elements, a recombinant virus in which foreign sequences displaced the 6- and 11-bp elements 1 kb from the cleavage point was constructed. Cleavage at the disrupted site did not occur. In a second recombinant virus, restoration of 64 bases containing the 6- and 11-bp elements to the disrupted cleavage site restored cleavage. Therefore, cis cleavage elements exist within this 64-base region, and sequence conservation suggests that they are the 6- and 11-bp elements.     

Limited survey of genital infection by guinea pig inclusion conjunctivitis agent

Cervical or urethral scrapings were collected from 245 guinea pigs that had clinical signs of guinea pig inclusion conjunctivitis (GPIC) or were parents of newborn young having clinical signs of GPIC. Giemsa-stained smears were examined for cytoplasmic inclusion bodies, and samples were passaged in 6-day-old embryonating eggs. Complement-fixation tests were performed on 44 samples passaged through eggs in an effort to detect the presence of GPIC antigen. Unequivocal evidence of chlamydial infection of the genital tract was not found.     

Sulphated glycosaminoglycans in guinea pig eosinophils studied by means of cationic colloidal gold

Using bone marrow embedded in hydrophilic resin Lowicryl K4M and cationic colloidal gold pH 1.0 labelling, we studied sites of sulphation and sulphated glycosaminoglycans ultrastructurally in various maturational stages of both eosinophil granulocytes and eosinophil granules of guinea pig. Eosinophil granules reacted positively to cationic gold, the pattern of labelling varying according to the degree of cell maturation. The formation of eosinophil granules takes place throughout the myelocyte stage. Early eosinophil myelocytes contain a large Golgi apparatus with active granulogenesis, while late ones contain a small and less active Golgi apparatus. All the immature granules were labelled positively. However, mature granules with a central crystal bar lost their affinity towards colloidal gold. Interestingly, strong colloidal gold labelling was also observed in the trans to transmost Golgi apparatus, especially in immature eosinophil granulocytes. This indicates that sulphation of glycosaminoglycans occurs in the trans to transmost Golgi apparatus of eosinophil granulocytes. Prior absorption with poly-L-lysine prevented colloidal gold labelling of tissue sections. Methylation of sections at 37 degrees C did not alter the gold labelling, whereas the labelling disappeared after methylation at 60 degrees C. Prior treatment with chondroitinase ABC or heparinase I abolished the majority of colloidal gold labelling in immature eosinophil granules. Taking these results together, we conclude that immature eosinophil granules contain sulphated glycosaminoglycans including chondroitin sulphate or heparan sulphate or both.     

Changes in [K+]o evoked by baclofen in guinea pig hippocampus

OBJECTIVE: Studies using adult human subjects indicate that dietary protein and sodium chloride have negative effects on the retention of calcium by increasing urinary calcium excretion, while alkaline potassium improves calcium retention along with decreasing urinary calcium losses. This study investigated the effect of these dietary factors on acute urinary calcium excretion in 14 prepubescent girls age 6.7 to 10.0 years. METHODS: Subjects provided a fasting urine sample then consumed a meal containing one of five treatments: moderate protein (MP) providing 11.8 g protein, moderate protein plus 26 mmol sodium chloride (MP+Na), high protein (HP) providing 28.8 g protein, high protein plus 26 mmol sodium chloride (HP+Na), or high protein plus 32 mmol potassium as tripotassium citrate (HP+K). Urine was collected at 1.5 and 3.0 hours after the meal. Supplemental protein was given as 80:20 casein:lactalbumin. Test meals were isocaloric, and unless intentionally altered, components of interest except phosphate were equal between treatments. Each subject completed all five treatments. RESULTS: Urinary calcium excretion rose after the meal, peaking at 1.5 hours. There were no significant differences in calcium excretion between treatments at any time point. The high protein treatments did not result in a significant increase in either net acid or sulfate excretion at 1.5 hours compared to moderate protein. Dietary sodium chloride had no effect on urinary sodium or calcium excretion over the 3 hours. After the potassium treatment, sodium excretion increased (p< or =0.002) and net acid excretion decreased (p<0.001) compared to other treatments at 1.5 hours. CONCLUSIONS: In children, a simultaneous increase in protein and phosphorus due to increased milk protein intake did not increase acute urinary calcium excretion. An effect of dietary sodium chloride on acute urinary calcium excretion was not observed. Both these findings were similar to those of adult studies previously conducted in the same laboratory using similar format and treatments. Potassium citrate was not hypocalciuric in children, a response differing from that for adults, who have shown a decrease in acute urinary calcium excretion in response to alkaline potassium treatment. Further characterization of calciuric responses to dietary factors is required for children, who may differ from adults in many respects.