Small Urine Samples Research Articles

Whole-body cooling effectiveness of cold intravenous saline following exercise hyperthermia: a randomized trial

IntroductionIn some athletic, occupational, military and emergency settings, cold intravenous (IV) fluids are used to facilitate whole-body cooling in an effort to treat heat illness. This treatment has anecdotal support, but currently lacks evidence supporting it as a whole-body cooling modality. Other modalities may offer superior cooling rates, and thus, patient outcomes following treatment. We sought to evaluate cooling rates of cold-IV normal saline immediately following exercise-induced hyperthermia. MethodsEight healthy participants (3 females; 25 ± 2y; 72.9 ± 10.9 kg) completed 2 trials in random order. Prior to exercise, participants provided a small urine sample to confirm hydration status via urine specific gravity. Wet bulb globe temperature (WBGT) was assessed throughout trials. In both trials, participants exercised outdoors until rectal temperature (Tre) reached ∼38.9 °C, or volitional exhaustion, and then were cooled. In cooling, participants received either cold-IV (∼5 °C 0.9% NaCl fluids) or no treatment (sat in the shade; passive). Throughout exercise and treatment, Tre and heart rate (HR) were monitored. During exercise and every 10 min throughout cooling, participants were asked to assess thermal sensation. ResultsHydration status (P = .847) was not significantly different prior to exercise between trials. WBGT throughout was not different between trials (P = .426). Maximum Tre reached was not different between cold-IV (38.88 ± 0.30 °C) and passive cooling (38.76 ± 0.28 °C) trials (P = .184). Mean cooling rate for cold-IV (0.039 ± 0.005 °C·min−1) was significantly greater than for passive cooling (0.028 ± 0.005 °C·min−1; P = .002). Tre throughout cooling was not different between trials (P = .707), but did decrease throughout (P = .008), regardless of trial. HR was decreased over time (P < .001), but cold-IV and passive cooling were not different throughout HR recovery (P = .141). Thermal sensation decreased throughout cooling (P < .001), but was not different between trials (p = .278). ConclusionEmergency medical personnel should adopt treatment protocols that employ documented effective treatments for exertional heat stroke. In isolation, our data casts significant doubt for the use of cold-IV saline infusion for whole-body cooling of hyperthermic individuals.

Assessment of clinical performance of an ultrasensitive nanowire assay for detecting human papillomavirus DNA in urine

ObjectiveTo evaluate whether HPV DNA in urine has potential advantages as an alternative biomarker for HPV-based cervical cancer screening. MethodsAmong patients with Cobas HPV test results, a total of 67 HPV-positive (n = 42) and -negative (n = 25) women who agreed to participate in this study were willing to provide paired cervical and urine samples, and we observed concordance between sample types from each patient in identifying HPV genotypes using the nanowire assay. ResultsWe detected high-risk strains of HPV DNA in unprocessed urine specimens using polyethyleneimine-conjugated nanowires (PEI-NWs). Concordance for high-risk HPV (hrHPV) between paired urine and cervical samples was 90.4% (κ = 0.90; 95% CI: 0.80–100.00). The virological sensitivity and specificity for detection of HPV DNA from a small urine sample (200 μL) were 81.3% (κ = 0.83; 95% CI: 62.1–100.0) and 98.0% (κ = 0.83; 95% CI: 94.2–100.0) for HPV16 group, 100.0% (κ = 0.65; 95% CI: 100.0–100.0) and 95.3% (κ = 0.65; 95% CI: 90.1–100.0) for HPV18 group, and 96.4% (κ = 0.97; 95% CI: 89.6–100.0) and 100.0% (κ = 0.97; 95% CI: 100.0–100.0) for other hrHPV group, respectively. ConclusionsThe nanowire assay demonstrated excellent ability to identify HPV DNA from urine specimens. We observed an excellent agreement in the detection of high-risk HPV between paired urine and cervical samples, even with small urine sample volume.

SAT-144 AN ULTRASENSITIVE SURFACE ENHANCED RAMAN SCATTERING (SERS)-BASED ASSAY FOR KIDNEY INJURY MOLECULE-1 (KIM-1)

Kidney injury molecule-1 (KIM-1) is a biomarker of acute and chronic kidney injury. However, dilution of urine samples to minimize the impact of pH and solute on results can push the concentration levels to below the lower limits detection of conventional ELISAs. Additionally, small samples from infants, children or from non-human renal injury models such as mice limits analyte detection. Here we develop an ultrasensitive (sub-pg/ml) assay utilizing surface enhanced Raman scattering (SERS) to measure KIM-1. Capture and detection antibodies of a commercially available KIM-1 ELISA kit (R&D Systems) were utilized. [email protected] nanocuboids ([email protected]) with 16 nm shell thickness, assembled on silicon wafers, were derivatized with cysteamine followed by glutaraldehyde to covalently attach the capture antibody. Separately, [email protected] were derivatized with the Raman reporter 4-aminothiophenol (pATP), glutaraldehyde followed by the detection antibody. After analyte capture on the silicon wafer, washing, incubating with the derivatized detection antibody and washing, the silicon wafers were subject to SERS. Among several prominent Raman bands, the intensity band at 1080 cm-1 of pATP was proportional to the concentration of human recombinant KIM-1 spiked into artificial urine. The urine of control individuals and patients with kidney disease was diluted prior to assay and the results compared to that using the conventional ELISA. The Raman intensity band at 1080 cm-1 of pATP was proportional to the KIM-1 concentration from to 400 pg/ml. As little as 1fg/ml above background could be reliably detected. The KIM-1 concentration, determined in patient urine using both the conventional ELISA and the SERS assay, was found to be synonymous with a correlation coefficient of 0.93 ( p<0.002). A similar strategy was used to develop a sub-pg/ml assay for the pro-inflammatory/myokine molecule IL-6. Using the power of SERS, capture and detection antibodies of a conventional ELISA kit were modified to measure sub-pg/ml amounts of KIM-1. This sensitivity allows dilution of samples to eliminate the influence of pH and/or solute concentration that would otherwise affect conventional ELISA results. Also the sensitivity of the assay allows the use of small samples of urine or plasma from infants, small children or from small animals such as mice with experimental renal diseases. It would be possible to detect preclinical changes in KIM-1 prior to disease manifestation. This assay strategy can be adapted to multiplexing to measure two or more analytes simultaneously in one sample by utilizing different Raman reporter molecules on different detection antibodies, an appropriate mix of capture antibodies on the silicon wafer, and monitoring non-overlapping Raman bands specific for each reporter.

An emergency bioassay method for 210Po in urine

A rapid method was developed to efficiently measure 210Po in urine samples in an emergency situation. Polonium-210 in small urine samples (10mL) was spontaneously deposited on a stainless steel disc in 1M HCl at room temperature for 4h in a polyethylene bottle. The metallic disc was then counted for 4h by alpha spectrometry. The developed method allowed the preparation of large sample batch in a short time. The method meets the requirements for an emergency bioassay procedure.

Emerging biomarkers and metabolomics for assessing toxic nephropathy and acute kidney injury (AKI) in neonatology.

Identification of novel drug-induced toxic nephropathy and acute kidney injury (AKI) biomarkers has been designated as a top priority by the American Society of Nephrology. Increasing knowledge in the science of biology and medicine is leading to the discovery of still more new biomarkers and of their roles in molecular pathways triggered by physiological and pathological conditions. Concomitantly, the development of the so-called “omics” allows the progressive clinical utilization of a multitude of information, from those related to the human genome (genomics) and proteome (proteomics), including the emerging epigenomics, to those related to metabolites (metabolomics). In preterm newborns, one of the most important factors causing the pathogenesis and the progression of AKI is the interaction between the individual genetic code, the environment, the gestational age, and the disease. By analyzing a small urine sample, metabolomics allows to identify instantly any change in phenotype, including changes due to genetic modifications. The role of liquid chromatography-mass spectrometry (LC-MS), proton nuclear magnetic resonance (1H NMR), and other emerging technologies is strategic, contributing basically to the sudden development of new biochemical and molecular tests. Urine neutrophil gelatinase-associated lipocalin (uNGAL) and kidney injury molecule-1 (KIM-1) are closely correlated with the severity of kidney injury, representing noninvasive sensitive surrogate biomarkers for diagnosing, monitoring, and quantifying kidney damage. To become routine tests, uNGAL and KIM-1 should be carefully tested in multicenter clinical trials and should be measured in biological fluids by robust, standardized analytical methods.

High throughput liquid and gas chromatography-tandem mass spectrometry assays for tobacco-specific nitrosamine and polycyclic aromatic hydrocarbon metabolites associated with lung cancer in smokers.

We developed and applied high throughput liquid and gas chromatography-tandem mass spectrometry (LC-MS/MS and GC-MS/MS) methods for the cigarette smoking-associated biomarkers 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT), which are urinary metabolites of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the polycyclic aromatic hydrocarbon phenanthrene. NNAL and PheT levels have been linked to lung cancer in previous studies of smokers. Confirmation of these relationships will require further molecular epidemiology studies, necessitating improved methodology applicable to large numbers of small urine samples. Furthermore, NNAL is excreted in urine either unconjugated or as an N- or O-glucuronide, but little data are available on the amounts of each in urine. For the high throughput analysis of NNAL, 3 aliquots were processed from each urine sample, one for the analysis of free NNAL, one for free NNAL plus NNAL-N-Gluc, and one for total NNAL (the sum of free NNAL, NNAL-N-Gluc, and NNAL-O-Gluc). Ninety-six well plate technology was used for sample enrichment by supported liquid extraction plates, mixed mode reverse-phase/cation exchange solid-phase extraction, and LC-MS/MS analysis. For the analysis of PheT, the urine samples were cleaned up by solid-phase extraction on styrene-divinylbenzene sorbent, silylated, and analyzed by GC-MS/MS, both in 96-well format. The methods were validated analytically with respect to accuracy and precision, and applied in an ongoing molecular epidemiology study of smokers. The amount of total NNAL in smokers' urine was (mean ± SD) 1.65 ± 2.13 pmol/mL (N = 2641). Free NNAL, NNAL-N-Gluc, and NNAL-O-Gluc represented (mean ± SD) 31 ± 11%, 22 ± 14%, and 48 ± 15% of total NNAL, respectively. The amount of PheT in smokers' urine was (mean ± SD) 1.43 ± 2.16 pmol/mL (N = 2613). The methodology described here should be widely applicable in future studies of tobacco use and cancer.

High throughput liquid chromatography-tandem mass spectrometry assay for mercapturic acids of acrolein and crotonaldehyde in cigarette smokers' urine.

3-Hydroxypropylmercapturic acid (3-HPMA) and 3-hydroxy-1-methylpropylmercapturic acid (HMPMA) are urinary metabolites of the toxicants acrolein and crotonaldehyde, respectively. Virtually all human urine samples contain these metabolites, resulting from the action of glutathione-S-transferases on acrolein and crotonaldehyde, which are lipid peroxidation products, environmental and dietary contaminants, and constituents of cigarette smoke. We have developed a high throughput liquid chromatography-tandem mass spectrometry method for quantitative analysis of 3-HPMA and HMPMA in large numbers of small urine samples, as would be required in molecular epidemiology and clinical studies relating levels of these metabolites to cancer risk. Solid-phase extraction on mixed mode reverse phase-anion exchange 96-well plates provided sufficient purification for LC-MS/MS analysis, which was performed by auto-injection using a 96-well format, and resulted in clean, readily interpretable chromatograms, with detection limits of 4.5pmol/mL urine for 3-HPMA and 3.5pmol/mL urine for HMPMA. Accuracy was 92% for 3-HPMA and 97% for HMPMA while inter-day precision was 9.1% (coefficient of variation) for 3-HPMA and 11.0% for HMPMA. The method was applied to more than 2600 urine samples from smokers; mean levels of 3-HPMA and HMPMA were 4800±5358 (S.D.)pmol/mL and 3302±3341pmol/mL, respectively.

Measurement of urinary catecholamines in small samples for mice

IntroductionAnalysis of catecholamines in small samples of urine is difficult and sensitive to stress. Current techniques require pooling of samples or expensive separation by double mass spectrometry. A method for extraction of unconjugated catecholamines in 20μL urine samples has been developed using alumina extraction prior to separation by high performance liquid chromatography (HPLC) and electrochemical detection (ECD). MethodsThree murine experiments tested the application of the procedure. In the first, collection occurred in the morning and evening prior to handling, and in the morning after three days of handling. In the second, passively obtained urine was compared to stressfully obtained urine in the same mice. Finally, basal collections were compared to urinary catecholamine levels 15 and 30min into novel cage stress. Urine was extracted alongside 2,3-dihydroxybenzoic acid (DHBA) internal standard via alumina and brought to pH 8.5 with tris buffer. The mixture underwent two wash steps for depuration and eluted with perchloric acid for analysis on HPLC with ECD. ResultsThis novel extraction method using low amounts of urine yielded 48% recovery in the samples and 60% recovery in the standard extraction on average. With a signal to noise ratio of 3:1, the limit of detection (LOD) of a standard is 1.2pg/mL, which allows for the detection of 3.6pg/mL in urine or 72fg in a 20μL sample. Thus resting catecholamine levels are 216 times higher than the LOD. Unconjugated norepinephrine and epinephrine levels were significantly increased 15min after novel cage stress and epinephrine remained elevated after 30min, but did not show significant differences when comparing collection time, handling exposure, or specific collection technique. DiscussionThe technique is an effective measure for sympathetic activity in micro samples, with a limit of detection in the attomole range for 20μL samples.

Comparison of the analysis of β-blockers by different techniques

In this work we have compared three analytical techniques (ELISA, GC–MS, and LC–MS) for the analysis of 16 β-blockers: acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, carteolol, labetalol, metipranolol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, sotalol, timolol, and bupranolol. Several sample-preparation methods were optimized for each technique and enabled compounds of interest to be extracted from small urine samples (1–2.5 mL). The results enabled us to assess the possibilities and the sensitivity of each technique for application to doping tests. ELISA, whose selectivity is very poor and sensitivity the lowest one, is, nevertheless, useful as a rapid screening method. GC/MS and LC/MS provide confirmation procedures with the identification and quantification of the β-blockers with good sensitivity, accuracy, precision. The LC–MS analytical procedure allows the determination of the target analytes in the lower ng/mL range (0.53–2.23 ng/mL). The methodology was applied to the analysis of β-blockers in different urines.

Ibuprofen metabolite profiling using a combination of SPE/column-trapping and HPLC–micro-coil NMR

Solid-phase extraction and column-trapping preconcentration are combined to enhance HPLC–nuclear magnetic resonance (HPLC–NMR) and applied to metabolite profiling in biological samples. Combining the two signal enhancement techniques improved the NMR signal substantially such that we were able to identify 2-hydroxyibuprofen, carboxyibuprofen, and unmetabolized ibuprofen molecules from a small urine sample after a therapeutic dose of ibuprofen. The hyphenated SPE/column-trapping method resulted in an excellent overall signal enhancement of up to 90-fold.

An isotope dilution gas chromatographic–mass spectrometric method for the simultaneous assay of estrogens and phytoestrogens in urine

The metabolism of endogenous estrogens is complicated and certain metabolic patterns may reflect an individual risk of estrogen-dependent diseases such as breast cancer. Since the 1960s we have been constantly involved in developing estrogen profiling methods, in the beginning using gas chromatography and later gas chromatography–mass spectrometry (GC–MS) in the selected ion monitoring mode (SIM) and finally utilizing isotope dilution (ID–GC–MS–SIM). The addition of the dietary phytoestrogens to the profile rendered the method even more complicated. The present work presents the final estrogen profile method for 15 endogenous estrogens, four lignans, seven isoflavonoids and coumestrol in one small urine sample (1/150th of a 24 h human urine sample, minimum 2.5–5 ml) with complete validation including investigations as to the precision, sensitivity, accuracy and specificity. The method does not include the minimal amounts of unconjugated estrogens in urine. It may also be used for animal (e.g. rat and mouse) urine using a minimum of 2 ml of usually pooled sample. Despite its complexity it was found to fulfill the reliability criteria, resulting in highly specific and accurate results.

Comparison of the analysis of corticosteroids using different techniques

In this work we have optimized the analysis of 18 human corticosteroids, some endogenous (tetrahydrocortisol, tetrahydrocortisone, cortisol, and cortisone) and others synthetic (betamethasone, budesonide, cortisone acetate, desonide, dexamethasone, dexamethasone acetate, flunisolide, fluocinolone acetonide, halcinonide, methylprednisolone, prednisolone, prednisone, triamcinolone, and triamcinolone acetonide). Three analytical techniques were developed: ELISA, gas chromatography coupled with mass spectrometry (GC-MS), and liquid chromatography coupled with mass spectrometry (LC-MS). Several sample-preparation methods were optimized for each technique and enabled compounds of interest to be extracted from small urine samples (several mL). The results enabled us to assess the possibilities and the sensitivity of each technique for application to doping tests.

Determination of methylnaltrexone in clinical samples by solid-phase extraction and high-performance liquid chromatography for a pharmacokinetics study

A high-performance liquid chromatographic (HPLC) method with electrochemical detection and solid-phase extraction (SPE) using cartridges of weak cation-exchange capacity as the primary retention mechanism is described for the separation and determination of methylnaltrexone (MNTX) in small clinical samples of plasma or urine. The procedure was performed using a Phenomenex Prodigy ODS-2, 5 μm, 150×3.2 mm analytical column and 50 m M potassium acetate buffer, with 11% methanol as organic modifier at pH* 4.5 at a flow-rate of 0.5 ml/min. The detection potential was 700 mV. The six-point standard calibration curves were linear over three consecutive days in the range from 2 to 100 ng/ml. The average goodness of fit ( r) was 0.9993. The lower limit of detection (LOD) and limit of quantification (LOQ) were found to be 2.0 and 5.0 ng/ml, respectively. At the LOQ, the coefficient of variation for the entire method was 8.0% and the accuracy was 10.0% ( n=10). Recovery of the drug from plasma was in the region of 94%. The method was applied to a pharmacokinetics study of methylnaltrexone after subcutaneous administration and in numerous assays of analytes in blood plasma and urine. The pharmacokinetics parameters for a single dose of 0.1 or 0.3 mg/kg in plasma were C max=110 (±55) and 287 (±101) ng/ml and t max=16.7 (±10.8) and 20.0 (±9.5) min, respectively. The method is simple, yet sensitive for the detection and determination of methylnaltrexone in biological samples at the level of the physiological response.

Improved determination of urinary cortisol and cortisone, or corticosterone and 11-dehydrocorticosterone by high-performance liquid chromatography with ultraviolet absorbance detection

A sensitive assay was developed for the determination of low levels of free (unconjugated) glucocorticoids in human, swine (cortisol and cortisone) and rat urine (corticosterone and 11-dehydrocorticosterone), using solid-phase extraction and HPLC with UV absorbance detection (254 nm). Precise quantitation is allowed by the use of internal standards (dexamethasone for swine urine and Reichstein's substance S for rat urine). This simple method allows the use of small urine samples (less than 2 ml), and is suitable for a wide range of applications in human and animal clinical and physiological studies.

Determination of amino acids in biological fluids by capillary gas chromatography with nitrogen-phosphorus selective detection

A selective and sensitive method for the determination of protein and non-protein amino acids in biological fluids by capillary gas chromatography (GC) has been developed. The amino acids in the samples were directly converted into their N( O, S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with nitrogen-phosphorus selective detection (NPD) using a DB-17ht capillary column. Using this method, the derivatives of the 21 protein amino acids and the 25 non-protein amino acids provided excellent NPD responses and were quantitatively and reproducibly resolved within 28 min. The lower detection limits of these amino acids, at a signal-to-noise ratio of 3, were ca. 6–150 pg injected. The calibration curves for each amino acid in the range of 0.02–2 μg were linear and sufficiently reproducible for quantitative analysis. This method was successfully applied to small urine and serum samples without prior clean-up; there was no evidence of interference from coexisting substances. Overall recoveries of amino acids added to urine and serum samples were 83–112%. The intra-assay and inter-assay R.S.D. of amino acids in these samples were 0.3–8.9% ( n = 3) and 1.9–15.8% ( n = 3) respectively.

Determination of urea in urine using a conductivity cell with surface acoustic wave resonator-based measurement circuit

A conductivity cell employing a 61 MHz surface acoustic wave resonator-based measurement circuit was applied to the detection of the urea/urease reaction. The kinetic enzymatic parameters of the urease were estimated from the frequency shifts. The effects of pH, temperature and inhibitor on the response of the enzyme conductivity measurement system were investigated. The system was applied to rapid determination of urea in small urine samples. The lowest detection limit of urea was 30 ng/ml.

Kinetic spectrophotometric method for the determination of urinary cobalt based on its catalytic effect on the oxidation of l-adrenaline hydrochloride by hydrogen peroxide

A catalytic for determination of nanomolar concentrations of Co(II), i.e., oxidation of l-adrenaline hydrochloride with H 2O 2 in alkaline medium, is proposed. The reaction gives a low limit of detection of 2.5 × 10 −9 M Co(II) in the reaction mixture, good reproducibility with a relative standard deviation (R.S.D.) of 4−5% in the Co(II) concentration range 8.0 × 10 −9−8.0 × 10 −8M and good selectivity. On the basis of this indicator reaction, a catalytic-spectrophotometric method for the determination of cobalt in small urine samples (5.00 ml) was elaborated. The analysis of 17 urine samples, taken from healthy persons of different ages, gave cobalt concentrations in the range 0.20–1.50 μmol 1 −1. The R.S.D. for ten replicate analyses of a urine sample with an average cobalt content of 0.63 μmol 1 −1 was 5.6%. The reliability of the method was verified by a comparative photometric method ( r = 0.9755) and by a determination based on known additions of cobalt ( r = 0.9894).

Determination of sulphur amino acids by gas chromatography with flame photometric detection.

A selective and sensitive method has been developed for the determination of sulphur amino acids by gas chromatography (GC). Sulphur amino acids were converted into their N(S)-isopropoxycarbonyl methyl ester derivatives and measured by GC with flame photometric detection using a DB-17 capillary column. The derivatives were sufficiently volatile and stable to give single symmetrical peaks. The detection limits of sulphur amino acids were ca. 0.5-1 pmol per injection, and the calibration curves were linear in the range 0.5-10 nmol for each sulphur amino acid. This method was successfully applied to small urine samples without prior clean-up, and sulphur amino acids in these samples could be analysed without any influence from coexisting substances. Overall recoveries of sulphur amino acids added to urine samples were 85-113%. The analytical results of free sulphur amino acid contents in urine samples of normal subjects are presented.

Determination of orotic acid in urine

Orotic acid was separated from other urinary constituents by ion-pair formation with tetrabutylammonium, and isocratic elution from a reversed-phase column. Absorbance at 280 nm was recorded for quantitation. Owing to the better column characteristics the separations are somewhat faster, and the sensitivity of the method is higher than those of analogous methods using anion-exchange columns. The method was used for the determination of orotic acid in human urine, in urine of rats with portacaval shunts and in small (30 μl) urine samples from sparse fur mice. Shunted rats excreted ca. 100% more orotic acid per 24 h than sham-operated controls, in spite of their considerably lower body weight. Excessive orotic acid in urine indicates a conditional deficiency of ornithine. Sparse fur mice are congenitally hyperammonemic because of a defective hepatic ornithine carbamoyltransferase. Determination of orotic acid in the urine is a suitable method to identify those animals among litter mates which have the hereditary enzyme defect.

Selective determination of secondary amines as their N-diethylthiophosphoryl derivatives by gas chromatography with flame photometric detection.

A selective and sensitive method has been developed for the determination of secondary amines by gas chromatography (GC). After removal of primary amines by the reaction with o-phthaldialdehyde, secondary amines were converted into their N-diethylthiophosphoryl derivatives and then measured by GC with flame photometric detection using a DB-1701 capillary column. The derivatives were sufficiently volatile and stable to give single symmetrical peaks. The detection limits of secondary amines were ca. 0.05-0.2 pmol per injection. N-Methylcyclohexylamine was used as an internal standard. The calibration curves for secondary amines in the range 1-20 nmol were linear and sufficiently reproducible for quantitative determination. This method was successfully applied to small urine samples without prior clean-up. Overall recoveries of secondary amines added to urine samples were 91-105%. By using this method, secondary amines in urine samples could be analysed without any influence from primary amines and other coexisting substances. The analytical results of secondary amine content in urine samples of normal subjects are presented.