Urinary Constituents Research Articles

Huge Primary Vaginal Stone in a Recumbent Woman

Summary Objective Vaginal stones are rare and primary vaginal stones are extremely rare. A primary vaginal stone originating from urinary stasis in the vagina may be due to anatomic abnormalities, vaginal outlet obstruction, infection, or vesicovaginal fistulae, while secondary vaginal stones result from crystallization of urinary constituents around a foreign body in the vagina. Case Report This 43-year-old female had cerebral palsy due to tuberculosis meningitis when she was 3 years old. She had been bedridden with urinary and fecal incontinence since then. She was admitted to our medical ward due to acute abdominal distension with poor appetite and weight loss. On arrival, leukocytosis with dehydration was noted and medical treatment was given initially. Imaging studies showed stool impaction with abdominal ileus and a huge calcified density measuring 10 cm in diameter in the pelvis. Exploratory laparotomy showed that the mass was in the vagina. A laminated and pear-shaped mass was removed through an incision in the anterior vaginal cuff. Pathologic examination showed lithiasis composed of calcification mixed with fibrinous exudates and fragments of reactive squamous epithelium. The postoperative course was smooth with uneventful convalescence and she was discharged and doing well at the time of writing. Conclusion Although vaginal stones are very rare, they may be formed in recumbent women due to urinary stasis, and the calculi may cause obstruction of the bowel or urinary tract.

Determination of methylmalonic acid in urine by HPLC with intramolecular excimer-forming fluorescence derivatization

We developed and validated an HPLC method with intramolecular excimer-forming fluorescence derivatization to determine methylmalonic acid, a unique biochemical marker for methylmalonic aciduria. Methylmalonic acid in urine and an internal standard were derivatized with pyrenebutyric hydrazide and separated on a C8 column. The derivatives were detected by monitoring the fluorescence at 475 nm (excitation wavelength 345 nm). At a signal-to-noise ratio of 3, the detection limit was 0.33 pmol on the column and the calibration curve was linear up to 1 mmol[sol ]L in urine. In a retrospective study on a relatively large number of known methylmalonic aciduria cases (n = 48), the method enabled us to differentiate methylmalonic aciduria cases from healthy controls (n = 52), regardless of age of patients at sampling or years of specimen storage. No interference was observed from isomeric or other dicarboxylic acids, or other urine constituents. As described, the method can be used retrospectively or prospectively for the diagnosis of methylmalonic aciduria and can be easily adopted by laboratories with no access to gas chromatography-mass spectrometry.

Adhesion at calcium oxalate crystal surfaces and the effect of urinary constituents

Kidney stones, aggregates of microcrystals, most commonly contain calcium oxalate monohydrate (COM) as the primary constituent. The aggregation of COM microcrystals and their attachment to epithelial cells are thought to involve adhesion at COM crystal surfaces, mediated by anionic molecules or urinary macromolecules. Identification of the most important functional group-crystal face adhesive combinations is crucial to understanding the stability of COM aggregates and the strength of their attachments to epithelial cell surfaces under flow in the renal tubules of the kidney. Here, we describe direct measurements of adhesion forces, by atomic force microscopy, between various functional groups and select faces of COM crystals immersed in aqueous media. Tip-immobilized carboxylate and amidinium groups displayed the largest adhesion forces, and the adhesive strength of the COM crystal faces decreased in the order (100) > (121) > (010), demonstrating that adhesion is sensitive to the structure and composition of crystal faces. The influence of citrate and certain urinary proteins on adhesion was examined, and it was curious that osteopontin, a suspected regulator of stone formation, increased the adhesion force between a carboxylate tip and the (100) crystal face. This behavior was unique among the various combinations of additives and COM crystal faces examined here. Collectively, the force measurements demonstrate that adhesion of functional groups and binding of soluble additives, including urinary macromolecules, to COM crystal surfaces are highly specific in nature, suggesting a path toward a better understanding of kidney stone disease and the eventual design of therapeutic agents.

ELUCIDATION OF FACTORS DETERMINING FORMATION OF CALCIUM PHOSPHATE STONES

In this study we compared the effect of the urinary concentration of calcium with that of oxalate on the urinary relative saturation ratio (RSR) of calcium oxalate and brushite in patients with pure or mixed calcium phosphate stones. A retrospective data analysis was performed of urinary stone risk analysis in 133 patients with pure or mixed calcium phosphate stones (30% or greater). Correlation was sought between stone events and urinary stone risk factors, and between brushite and urinary constituent RSRs before and after adjustment for variables. The formation rates of total events and stone surgeries weakly but significantly correlated with RSR urinary brushite (total events r = 0.22, p = 0.01) but not with RSR calcium oxalate. Urinary RSR brushite significantly correlated positively with urinary calcium (r = 0.67, p <0.0001), phosphorus and pH. After adjustment RSR brushite inversely correlated with citrate (r = -0.3, p = 0.0006). However, it did not correlate with oxalate. In stone formers with calcium phosphate or mixed calcium oxalate-calcium phosphate stones the stone formation rate depends on the urinary saturation of brushite but not of calcium oxalate. RSR brushite is determined by the urinary concentration of calcium, phosphate, citrate and pH but not by oxalate. Hypercalciuria should be controlled in patients with idiopathic calcareous renal calculi who form stones of pure or mixed calcium phosphate.

Occurrence and production of chloramines in the chlorination of creatinine in aqueous solution

Occurrence and production of stable chloramines in the chlorination of creatinine, a constituent of perspiration and urine, in aqueous media were studied. Creatinine (5 x 10(-5)M) was treated with free chlorine in aqueous solutions at molar ratios of 0.5-8 (chlorine/creatinine) at pH 7.0 at room temperature for several days. At lower ratios of chlorine, two stable N-chlorocreatinine derivatives, which were determined as dichloramine fractions by the DPD method, were isolated by HPLC and identified by EI-MS and (1)H-NMR. One was 2-chloroamino-1-methylimidazolin-4-one (creatinine chloramine) and the other was 2-chloroamino-5-hydroxy-1-methylimidazolin-4-one (hydroxycreatinine chloramine). In addition, the formation of methylamine was identified by GC-MS analyses of its imine derivative formed with pentafluorobenzaldehyde. Methylamine forms stable chloramines, which might be determined as mono- and/or di-chloramine fractions together with free chlorine by the DPD method in the reaction mixtures at higher molar ratios of chlorine. In practice, small amounts of methylamine (ca. 19 microg/L) were detected in water samples collected from several swimming pools. Hence, methylamine may be an origin of elusive organic chloramine formed in the chlorination of swimming pools. A probable mechanism of the occurrence and processing of chlorination products of creatinine is suggested.

Influence of a variable differential function on the stone-growth-related urinary depletion effect.

Recently, we showed that urine flowing past growing stone material on its way through the urinary tract becomes systematically depleted of its lithogenic substances (1). This effect can be large enough to be clinically relevant (2). However, only the altered urine can be assessed by urine collection, which leads to a urinalysis that does not sufficiently reflect the patient’s true metabolic status. The excessively low concentrations and excretion values of the lithogenic urinary constituents may lead to “optimistic” misinterpretation of the patient’s actual health status and overestimation of treatment success. Apart from geometric and physiologic simplifications, the previously used model assumes that the differential volume function of the kidneys amounts to 50%. Consequently, both the stone-free and the stone-forming kidney contribute equally to the total urine volume. However, even in healthy individuals, the differential function varies by ±6%. Parenchymal defects, such as tumors, renal arteriopathy, and posttraumatic failures, or morphologic abnormalities, such as pyeloureteral junction stenosis and vesico-ureteral-renal reflux, can lead to a dramatic decrease in renal function, causing a reduction in the glomerular filtration rate. Patients suffering from these diseases often present with calcium nephrolithiasis and proteinuria, occasionally with progression to nephrocalcinosis, urinary concentrating defects, and renal insufficiency caused by tubular atrophy, intestinal fibrosis, and glomerulosclerosis. The process of kidney stone formation follows fundamental physical principles such as mass conservation. In a recently introduced model (1), we evaluated the influence of in vivo-growing uroliths on urinary composition. The material from which the stones are formed originates completely from the urine streaming through the kidneys; therefore, the urinary concentrations of the lithogenic components, such as calcium and oxalate, in the excreted urine must be depleted by the mass deposited in the stone(s). As long as no method exists to measure those concentrations in vivo, mathematical models describing the depletion process …

Separation and Quantification of Enkephalin and Vasopressin Related Peptides in Reversed-Phase Capillary Liquid Chromatography

A method for determination of some biologically active penta- and nona-peptides under isocratic conditions in capillary liquid chromatography was developed. Separation system consisting of XTerra C18 stationary phase and mobile phase composed of a mixture of acetonitrile with 0.1% trifluoroacetic acid (TFA) and water with 0.1% TFA in the ratios 75/25 (v/v) and 85/15 (v/v) was suitable not only for a good resolution of enkephalin and vasopressin related peptides, respectively, but it also enabled separation of the respective biopeptides from other constituents of human urine. Calibration curves for the studied peptides were linear in the measured concentration range from 1.00 to 1.57×10−2 mg mL−1. The limit of detection and limit of quantification were in the range of units of μg mL−1 and tens of μg mL−1, respectively; slightly higher values were obtained for nonapeptides. Determination of certain biologically active peptides in urine can serve in future as a tool for diagnosis of various diseases, e.g. autism.

The value of urine citrate/calcium ratio in the estimation of risk of urolithiasis.

The urine saturation is considered as the better parameter for the estimation of risk of urolithiasis than any single urinary constituent. However, the determination of urine saturation is unsuitable for routine clinical practice. To evaluate a simpler and cheaper test than urine saturation for distinguishing stone formers from healthy individuals, urinary citrate/calcium ratio was determined in 30 children with urolithiasis, 36 children with isolated hematuria, and 15 healthy control children. The ratio was significantly lower in urolithiasis group comparing to controls, and significantly higher in hematuria than in urolithiasis group. The cut-off points between normal children and children with urolithiasis, accuracy, specificity and sensitivity were determined and compared with those of the urine saturation calculated with the computer program EQUIL 2. The data mining Weka software was used for the determination of the cut-off points. Children with urolithiasis had citrate/calcium ratio below 1.38 and urine saturation above 5.285. The citrate/calcium ratio showed in comparison to urine saturation similar high accuracy (91.11 vs. 88.89%), somewhat lesser specificity (73.33% vs. 93.33%) and much better sensitivity (100% vs. 86.89%) in discrimination of stone formers from normal children. The advantage in comparison to urine saturation is that it can be easily performed in clinical practice.

Schedule of taking calcium supplement and the risk of nephrolithiasis

Variation in the timing of calcium supplement may affect gastrointestinal absorption of both calcium and oxalate differently and may associate with variable risk of calcium oxalate nephrolithiasis. There are few human studies addressing specifically the appropriate time for taking calcium supplement. Therefore, this study was performed to compare calcium bioavailability and the risk of calcium oxalate stone formation for calcium supplement taken with meal vs. taken at bedtime. Thirty-two healthy male navy privates, 22.7 +/- 1.9 years old (mean +/- SD), who had normal renal function (serum creatinine less than 150 umol/L) and no history of renal stone, participated in the study. The subjects were randomly allocated into two groups of 16 each. Group A took 1 g of calcium carbonate with meal, 3 times/day; and group B took 3 g/day of calcium carbonate at bedtime. After taking the regimens for 1 week, followed by 1 month of washout period, crossover between both groups was done. The diet was controlled throughout the study. Twenty-four-hour urine collections for the determination of urinary constituents were obtained at baseline and after taking both regimens of calcium supplement. Activity product for calcium oxalate was determined to assess the risk of calcium oxalate stone formation. Urinary excretions of calcium were significantly elevated above the baseline values when taking calcium supplement both with meal (3.48 +/- 2.13 mmol/day vs. 5.17 +/- 2.61 mmol/day, P < 0.05) and at bedtime (3.09 +/- 1.70 mmol/day vs. 5.08 +/- 2.34 mmol/day, P < 0.05). There was no difference between the two regimens in the urinary calcium excretions. The urinary oxalate was decreased significantly when the subjects took calcium supplement with meal compared with the corresponding baseline value (0.13 +/- 0.05 vs. 0.17 +/- 0.07 mmol/d, P= 0.01). In contrast, there was no alteration in urinary oxalate when calcium supplement was taken at bedtime compared to the baseline values (0.15 +/- 0.05 mmol/day vs. 0.15 +/- 0.06 mmol/day, P= 0.9). Compared with the corresponding baseline values, there was no significant increase in the activity product for calcium oxalate when taking calcium with meal (0.54 +/- 0.25 vs. 0.57 +/- 0.22, P= 0.54), but it was increased significantly when calcium supplement was taken at bedtime (0.47 +/- 0.21 vs. 0.72 +/- 0.27, P < 0.01). Calcium supplement should be taken with meal in order to avoid increasing the risk of calcium oxalate nephrolithiasis.

Expression, localization, and regulation of urea transporter B in rat urothelia.

Although mammalian urothelia are generally considered impermeable to urinary constituents, in vivo studies in several species suggest urothelial transport of water, urea, and solutes under certain conditions. This study investigates the expression, localization, and regulation of urea transporter-B (UT-B) in rat renal pelvis, ureter, and bladder tissues. Immunoblots of homogenates of tissues identified characteristic approximately 40- to 55- and approximately 32-kDa bands in the ureter, bladder, and renal inner medulla, but not renal cortex. UT-B was localized by immunocytochemistry and was strongly expressed in all cell membranes (and to a limited extent in intracellular vesicles in the cytoplasm) of epithelial cells lining the rat bladder, ureter, and renal pelvis lumens except the apical membrane of the umbrella cells. It was also present in single-layer papillary surface epithelial cells. There was no difference in immunoblot expression of UT-B in the bladder or ureteral homogenates between groups of rats fed high- or low-protein or high- or low-sodium diets. Water restriction resulted in an increase in UT-B expression in ureters (49%, P = 0.001) but not in bladders (14%, P = not significant). The functional role of UT-B in the genitourinary tract epithelia is unknown. UT-B may participate in the regulation of epithelial cell volume and osmolality, in the dissipation of urea gradients, and in possible net urea transport across uroepithelia.

The molecular basis of kidney stones.

To emphasize an exploration of mechanisms of kidney stone disease based on a molecular understanding of excess urinary excretions of calcium, oxalate, cystine, and uric acid. Hypercalciuria is discussed relative to mutations in the renal chloride genes CLCN5 and CLCNKB, WNK kinases, ATPB61, and NPT2. Hyperoxaluria is discussed relative to mutations in AGXT and GRHPR. Cystinuria is discussed relative to mutations in SLC3A1 and SLC7A9. Hyperuricosuria is discussed with novel gene findings, and hyperxanthinuria with new findings in XDH. An enhanced understanding of the diagnosis, course, and prognosis for genetic causes of kidney stone diseases has been made available to the clinician caring for patients with kidney stones and to the scientist interested in their cause, as a result of molecular breakthroughs in the kidney handling of normal urinary constituents. We look forward to a new era of the therapeutics of kidney stones based on such advances.

An improved method for the extraction of mutagens from human urine and cooked meat using blue rayon

A reproducible method has been developed and validated that allows the detection of mutagenic material in human urine following the intake of a meal containing pan-fried beef patties. The mutagens are extracted from the urine with blue rayon and eluted with methanol/ammonia (100:1). Using 14C-2-amino-3-methylimidazo[4,5-f]quinoline (14C-IQ) as a tracer, the extraction efficiency of heterocyclic amines was consistently found to exceed 90%. It was observed that the critical factor in attaining high extraction efficiencies was the concentration of blue rayon, which depended on the nature of the matrix. Constituents of human urine were demonstrated to compete with IQ for the binding sites on the blue rayon. For the determination of the mutagenic activity in the extracted urine, the O-acetylase over-expressing Salmonella typhimurium strain YG1024 was utilised, and the Ames test procedure was optimised with respect to S9 concentration and incorporation of a pre-incubation step. Using the developed procedure, a marked rise in mutagenic activity, determined both in the absence and presence of an activation system, was evident in human urine following the intake of a beef patty meal. Normal mutagenicity levels were restored within 24h. Finally, a method is described, once again employing blue rayon, for the extraction of heterocyclic amines from cooked beef patties with a 92% efficiency, as exemplified by IQ.

Therapeutic efficacy of lipoic acid in combination with dimercaptosuccinic acid against lead-induced renal tubular defects and on isolated brush-border enzyme activities

The combined therapeutic potentials of lipoic acid and dimercaptosuccinic acid were compared against their sole administrations in restoring the altered lead sensitive indices in urine and isolated renal brush-border preparations. Toxicity was induced in male albino rats (Wistar strain) by administering lead acetate (0.2%) in drinking water for 5 weeks, followed by therapy comprising lipoic acid (25 mg/kg body weight) and dimercaptosuccinic acid (20 mg/kg body weight) solely as well as combined during the 6th week. Changes in kidney weights encountered upon lead administration improved after therapy with lipoic acid and dimercaptosuccinic acid. Renal integrity was assessed by measuring the activities of alkaline phosphatase, acid phosphatase, lactate dehydrogenase, leucine aminopeptidase, N-acetyl-β- d-glucosaminidase, γ-glutamyl transferase and β-glucuronidase in urine along with some urinary constituents (urea, uric acid, creatinine, protein and phosphorous). The effects of lead were also studied on isolated brush-border enzymes (alkaline phosphatase, acid phosphatase, γ-glutamyl transferase and β-glucuronidase) that showed a decline upon its administration. Increased activities of urinary enzymes were accompanied by increase in the urinary constituents. Increase in renal lead content was paralleled by a drastic fall in the renal δ-aminolevulinic acid dehydratase and a rise in urinary lead levels. Relative to the administration of lead, the combined therapy showed betterment on the renal integrity with respect to the functional parameters assessed, thereby indicating its efficacy over the monotherapies.

Molecular modulation of calcium oxalate crystallization by osteopontin and citrate.

Calcium oxalate monohydrate (COM), which plays a functional role in plant physiology, is a source of chronic human disease, forming the major inorganic component of kidney stones. Understanding molecular mechanisms of biological control over COM crystallization is central to development of effective stone disease therapies and can help define general strategies for synthesizing biologically inspired materials. To date, research on COM modification by proteins and small molecules has not resolved the molecular-scale control mechanisms. Moreover, because proteins directing COM inhibition have been identified and sequenced, they provide a basis for general physiochemical investigations of biomineralization. Here, we report molecular-scale views of COM modulation by two urinary constituents, the protein osteopontin and citrate, a common therapeutic agent. Combining force microscopy with molecular modeling, we show that each controls growth habit and kinetics by pinning step motion on different faces through specific interactions in which both size and structure determine the effectiveness. Moreover, the results suggest potential for additive effects of simultaneous action by both modifiers to inhibit the overall growth of the crystal and demonstrate the utility of combining molecular imaging and modeling tools for understanding events underlying aberrant crystallization in disease.

Approach to the Patient With Recurrent Nephrolithiasis

Although recurring nephrolithiasis may not be as common as single-episode disease, it poses greater problems to the patient's daily lifestyle and general future health status. The general need for an adequate history, examination, and radiological assessment is acknowledged, but a more intensive investigative and management approach is required. Investigations for secondary causes are paramount and may lead to identification of unusual metabolic abnormalities and specific therapies. Such medical approaches entail consideration of dietary adjustments to lower the urinary excretion of components of the renal stones, pharmacological therapies to modulate urinary constituents and limit renal stone risk, and monitoring efficacy of the therapy over time. This approach will prevent significant deterioration of renal function and other serious metabolic consequences.

5-hydroxytryptophol as a marker for recent alcohol intake.

To review the mechanism behind the alcohol-induced shift in serotonin metabolism, and the use of urinary 5-hydroxytryptophol (5-HTOL) as a biochemical marker of acute alcohol consumption. The serotonin metabolite 5-HTOL is a normal, minor constituent of urine and is excreted mainly in conjugated form with glucuronic acid. The formation of 5-HTOL increases dramatically after alcohol intake, due to a metabolic interaction, and the elevated urinary excretion remains for some time (>5-15 hours depending on dose) after ethanol has been eliminated. This biochemical effect can be used for detection of recent alcohol intake. 5-HTOL is determined by the gas chromatography-mass spectrometry (GC-MS) or liquid chromatography and mass spectrometry (LC-MS) techniques. A new ELISA method for 5-HTOL glucuronide provides a promising clinical assay. The most robust way to use the marker is by measuring the ratio of 5-HTOL to 5-hydroxyindoleacetic acid, because this compensates for urine dilution and dietary intake of serotonin. 5-HTOL is a very sensitive and specific indicator of recent alcohol consumption and, as such, a valuable complement to self-report. In clinical use, 5-HTOL is effective for monitoring lapses into drinking during out-patient treatment and for objective evaluation of treatment efforts. Other applications include detection of high-risk patients in elective surgery, monitoring of disulfiram treatment and a method to rule out artefactual ethanol formation in forensic toxicology. 5-HTOL can also be used as a sensitive reference method for validation of self-report data in clinical alcohol research. An elevated urinary 5-HTOL level can serve as a sensitive and reliable marker for recent alcohol intake with a number of clinical and forensic applications.

Biosensors for the Rapid Repetitive Detection of Adrenaline Using Stabilized Bilayer Lipid Membranes (BLMs) with Incorporated Calix[4]resorcinarene Receptor

AbstractThis work uses lipid film based biosensors with incorporated calix[4]resorcinarene receptor (lipophilic macrocyclic host molecule) for the rapid electrochemical detection of adrenaline. Freely‐suspended and metal supported BLMs (composed of egg phosphatidylcholine (PC) and 35% (w/w) dipalmitoyl phosphatidic acid) modified with the resorcin[4]arene receptor were used as one shot sensors to rapidly detect this catecholamine. The interactions of this compound with freely‐suspended BLMs were found to be electrochemically transduced in the form of a transient current signal with duration of seconds, which reproducibly appeared about 14 s after exposure of the membranes to adrenaline. The response time for these BLMs without incorporated receptor for adrenaline was about 1.5 min. The magnitude of the transient current signal was related to the concentration of adrenaline in bulk solution in the micromolar range. Differential scanning calorimetric (DSC) experiments were performed to explore the mechanism of interactions of BLMs with incorporated receptor with adrenaline. The interactions of adrenaline with surface‐stabilized bilayer lipid membranes (sBLMs) with incorporated receptor produced electrochemical ion current increases, which reproducibly appeared within a few seconds after exposure of the membranes to the stimulant. The use of the receptor in sBLMs increased the sensitivity of the method 6‐fold. The current signal increases were related to the concentration of adrenaline in bulk solution in the micromolar range. Stabilized lipid membranes formed by polymerization on glass fiber microfilters were used as practical chemical biosensors for the rapid detection of adrenaline. The interactions of polymerized lipid films with adrenaline were also found to provide transient current signals similar to those of freely‐suspended BLMs. The magnitude of the transient current signal was also related to the concentration of the stimulating agent in bulk solution in the micromolar range and these stabilized lipid films can be used again after storage in air. No interferences from ascorbic acid were noticed because of the negatively charged lipids in membranes. The effect of other compounds such as proteins and other compounds closely related to adrenaline was also investigated. Results of recovery experiments using human urine have shown recoveries ranged between 94 to 105%, which shows no interferences from matrix effects due to the presence of urine constituents. The present sensor based on stabilized lipid films can be used for the rapid repetitive detection of this pharmaceutical substance and keep prospects for the selective determination of catecholamines in biofluids.

Low molecular weight constituents of male mouse urine mediate the pregnancy block effect and convey information about the identity of the mating male.

Mouse urine contains a complex mixture of chemosignals including a variety of small volatile molecules that are bound to major urinary proteins. In addition to signalling maleness, male urine also conveys information about individuality, which allows recently mated female mice to distinguish the urinary chemosignals of the mating male from those of an unfamiliar male. The highly polymorphic nature of the major urinary proteins makes them a likely candidate for conveying individuality information in the context of the pregnancy block effect. This was investigated by comparing the pregnancy-blocking effectiveness of a high molecular weight urinary fraction, containing major urinary proteins, with that of a low molecular weight fraction containing volatile ligands. Not only was the high molecular weight fraction ineffective in blocking pregnancy, but it also appeared to be less important in signalling individuality than the low molecular fraction. The high molecular weight fraction was ineffective in inducing expression of the immediate early gene product egr-1 in the accessory olfactory bulb. In contrast, the low molecular weight fraction induced egr-1 expression in the mitral/tufted neurons in the anterior subregion of the accessory olfactory bulb, suggesting that they activate the V1R class of vomeronasal receptor neuron.

The Biology and Enzymology of Protein Tyrosine O-Sulfation

The Biology and Enzymology of Protein Tyrosine O-Sulfation

Inhibitory effects of urinary metabolites on platelet aggregation after orally administering Shimotsu-To, a traditional Chinese medicine, to rats.

Shimotsu-To, which consists of four herbal extracts, has been used clinically for improving abnormal blood coagulation, fibrinolysis, atherosclerosis and chronic inflammation in Japan and China. We have investigated the pharmacological relationship between the effects and chemical components of Shimotsu-To after oral administration to rats. The urinary constituents were separated and identified by three dimensional (3D-) HPLC equipped with a photodiode array detector as a new tool and the chemical structures were determined by spectroscopic methods to be trans-ferulic acid-3-O-sulfate (1), vanillic acid (2), m-hydroxyphenylpropionic acid (3), trans-ferulic acid (4) and cis-ferulic acid (5). Of these compounds, 2-5 strongly inhibited platelet aggregation induced by ADP and arachidonic acid. Compound 1, the sulfate conjugate of 4, did not show any inhibitory effect, which suggested that the inhibitory effect on platelet aggregation was inactivated by sulfate conjugation. These results indicated that compounds 2-5 partly contributed to the anti-Oketsu effect of Shimotsu-To through the inhibition of platelet aggregation.