Formation of N tau-ribosylhistidine, a novel histidine derivative found in the urine in histidinemia, from histidine and NAD(P)+ catalyzed by an NAD(P)+ glycohydrolase system

The formation of N tau-ribosylhistidine (His-R), a novel histidine derivative found in the urine of histidinemic patients, was studied. A most possible synthetic pathway catalyzed by imidazole acetic acid (ImAA) phosphoribosyltransferase was not substantiated, because p.o. administration to humans and rats of aspirin, an inhibitor of the enzyme, did not change the urinary excretion of His-R, whereas aspirin decreased the excretion of ImAA-R with concomitant increase in that of ImAA. His-R was produced on incubation of a rat liver homogenate or its membrane fraction with histidine, NAD(P)+ and MgCl2, but not with only histidine or NAD(P)+. Nicotinamide inhibited the formation of His-R. Thus the enzymes responsible for the formation of His-R were suggested to be NAD(P)+ nucleosidase, nucleotide pyrophosphatase and 5'-nucleotidase.     

Increased urinary 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine excretion in thalassemia patients: markers for lipid peroxidation-induced DNA damage

Thalassemic diseases including homozygous beta-thalassemia and beta-thalassemia/Hb E (beta-Thal/Hb E) are prevalent in Southeast Asia. Iron overload is a common complication in beta-thalassemia patients which induces intracellular oxidative stress and lipid peroxidation (LPO). LPO end products generate miscoding etheno adducts in DNA which after their repair are excreted in urine. We investigated whether urinary levels of 1,N6-ethenodeoxyadenosine (epsilondA) and 3,N4-ethenodeoxycytidine (epsilondC) can serve as putative cancer risk markers in beta-Thal/Hb E patients. epsilondA and epsilondC levels were assayed in collected urine samples by immunoprecipitation-HPLC-fluorescence and 32P-postlabeling TLC, respectively. Mean epsilondA (fmol/micromol creatinine) levels in urine of beta-Thal/Hb E patients ranged from 4.8 to 120.4 (33.8+/-3.9; n=37) and were 8.7 times higher compared to asymptomatic controls (1.4-13.8; 3.9+/-0.8; n=20). The respective epsilondC levels ranged from 0.15 to 32.5 (5.2+/-1.3; n=37) and were increased some 13 times over controls (0.04-1.2; 0.4+/-0.7; n=20). epsilondC levels were correlated positively with NTBI (r=0.517; P=0.002), whereas epsilondA showed only a trend (r=0.257; P=0.124). We conclude that the strongly increased urinary excretion of etheno adducts indicates elevated LPO-induced DNA damage in internal organs such as the liver. These highly promutagenic lesions may contribute to the increased risk of thalassemia patients to develop hepatocellular carcinoma.     

A sensitive liquid chromatographic method for the spectrophotometric determination of urinary trans,trans-muconic acid

Benzene is a human carcinogen and its metabolite, urinary trans,trans-muconic acid (ttMA), is a biomarker for risk assessment. However, most of the existing methods were not sensitive enough for monitoring of low level exposure. This paper describes a HPLC-UV method for ttMA determination with enhanced selectivity and sensitivity. A 30 mg OasisMAX cartridge was used to clean-up 50 microl of urine sample and gradient elution was performed on a Zorbax SB-C(18) column (30 degrees C). ttMA was detected at wavelength 263 nm using a UV diode array detector (DAD). The two mobile phases used were (A) 150 mM ortho-phosphoric acid containing of 9% (v/v) methanol; and (B) 125 mM ortho-phosphoric acid containing 30% (v/v) acetonitrile. The method was validated with 61 urine samples collected from non-occupationally benzene exposed individuals and 14 quality control specimens from an international quality assessment scheme. The urinary ttMA concentrations (mean+/-S.D.microg/g creatinine) were 90+/-34 for smokers (n=26), 49+/-39 for non-smokers (n=21) and 23+/-18 for non-smoking hospital staff (n=14). A correlation coefficient, r=0.99 was found with 14 external quality specimens for ttMA ranged from 0.4 to 6.8 mg/l. The recovery and reproducibility were generally over 90% and the detection limit was 5 microg/l.     

Urinary dopamine, noradrenaline and adrenaline in type 2 diabetic patients with and without nephropathy

We measured the urinary excretions of dopamine, noradrenaline and adrenaline, their conjugated metabolites, urinary excretion of sodium and creatinine clearance simultaneously in 21 patients with Type 2 (non-insulin-dependent) diabetes and 6 normal subjects. The mean (+/- SEM) value for urinary excretion of dopamine (52.4 +/- 8.8 micrograms/day) in diabetic patients with nephropathy (Group C, n = 12) was significantly lower (P less than 0.01) than in the normal subjects (Group A, 179.7 +/- 15.5 micrograms/day) and in diabetic patients without nephropathy (Group B, n = 9, 131.5 +/- 16.5 micrograms/day). The mean values for the urinary excretions of noradrenaline and adrenaline were also significantly lower (P less than 0.01) in Group C than in Groups A and B. In addition, the mean urinary excretion of conjugated metabolite of dopamine in Group C was significantly lower (P less than 0.05) than in Group A. There was a trend toward the observation that the mean 24-h urinary excretion of sodium in Group C (121.6 less than 12.9 mEq) was lower as compared with that in Group A (140.8 +/- 8.9 mEq) or B (150.7 +/- 17.9 mEq). A multiple regression analysis revealed that the 24-h urinary excretion of dopamine correlated significantly with creatinine clearance, systolic (P less than 0.01) and diastolic (P less than 0.05) blood pressures. The results indicate that synthesis or secretion of renal dopamine might decrease with a progression of diabetic nephropathy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the protein-bound, lipophilic, uremic toxin p-cresol in healthy rats.

P-Cresol, a partially lipophilic and protein-bound compound is related to several biochemical alterations in uremia. Because p-cresol kinetics have never been studied, we investigated its kinetic behavior in rats. Results were compared with those obtained with creatinine, a water soluble, non-protein-bound uremic retention solute, which is currently used as a marker of uremic retention. Healthy rats were divided into 3 groups with comparable body weight: (1) a control group (n=6); (2) a group (n=7) which received an intravenous bolus of 3 mg p-cresol; and (3) a group (n=5) which received an intravenous bolus of 18 mg creatinine. Blood samples were collected at 0, 5, 30, 60, 120, 180 and 240 minutes after administration for the determination of p-cresol and creatinine. Urine was collected at 1-hour intervals. p-Cresol concentrations were assessed by HPLC. Pharmacokinetic parameters of p-cresol and creatinine were calculated from the serum concentration-time curves using non-compartmental analysis. Each compound showed a concentration at time point 5 min (p-cresol: 6.7 +/- 1.4 mg/L and creatinine: 141 +/- 12 mg/L) which was comparable with values observed in uremic patients; these concentrations decreased gradually towards min 240 (p-cresol: 0.6 +/- 0.3 mg/L and creatinine: 4 +/- 2 mg/L, p<0.05 vs. 5 min in both cases). No p-cresol was found in the serum of control rats and these rats showed no changes in serum concentration of creatinine. Urinary excretions were strikingly different (p-cresol: 23 +/- 10% and creatinine: 95 +/- 25% of the administered dose, p<0.05). The half-life of p-cresol was twice as long as that of creatinine (1.5 +/- 0.8 vs. 0.8 +/- 0.1 h, p<0.05). Total clearance (CLt) was much higher for p-cresol than for creatinine (23.2 +/- 4.5 vs. 8.1 +/- 0.4 mL/min/kg, p<0.01); renal clearance (CLr), however, was substantially lower for p-cresol (4.8 +/- 2.0 vs. 8.2 +/- 1.9 mL/min/kg, p<0.05). Whereas CLt and CLr were similar for creatinine, CLt of p-cresol largely exceeded its CLr (p<0.05). The volume of distribution (Vd) was also much larger for p-cresol than for creatinine (2.9 +/- 1.4 vs. 0.6 +/- 0.1 L/kg, p<0.01). After injection of p-cresol, an additional chromatographic peak appeared in serum and in urine samples. Although at min 240 serum concentration of p-cresol had decreased to 10% of the peak value, only 23% of the administered amount was excreted in the urine and the CLr was +/- 50% lower compared to that of creatinine. Non-renal clearance and Vd of p-cresol were, however, substantially larger. These data may be of value to explain the different behavior of p-cresol in renal failure and dialysis, compared to creatinine.     

Identification and quantification of N(epsilon)-(Hexanoyl)lysine in human urine by liquid chromatography/tandem mass spectrometry

The identification and quantification of N(epsilon)-(hexanoyl)lysine (N(epsilon)-HEL), which was found from the reactions between lipid hydroperoxide and lysine, from human urine was examined using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The N(epsilon)-HEL in the partially purified urine fraction was identified using LC/MS/MS by several approaches including precursor/product ion scans. The peak found by the multiple-reaction monitoring (MRM) of the collision-induced fragmentation of N(epsilon)-HEL was clearly observed in urine, and the elution position coincided with the synthetic standard N(epsilon)-HEL. The product, estimated N(epsilon)-HEL, was absorbed by a specific antibody to N(epsilon)-HEL. Moreover, N(alpha)-HEL, one of the plausible hexanoyl adducts from the reaction between the N(alpha) moiety of L-lysine and the peroxidized lipid, was hardly detected in urine samples, suggesting that the origin of the N(epsilon)-HEL is the peroxidized lipid-modified proteins but not artificial hexanoylated L-lysine. Using the MRM technique, the amount of urinary N(epsilon)-HEL from the control subjects (observed healthy) was estimated to be 1.58 +/- 0.23 mumol/mol of creatinine. A comparative study of the urinary N(epsilon)-HEL with an oxidative stress marker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, showed a high correlation (r = 0.844) between the two biomarkers. Furthermore, the quantification of N(epsilon)-HEL in the control and diabetic urines revealed that the urinary N(epsilon)-HEL from diabetic subjects (3.21 +/- 0.65 mumol/mol of creatinine) was significantly higher than that from the control subjects.     

Liquid chromatography-based determination of urinary free and total N(epsilon)-(carboxymethyl)lysine excretion in normal and diabetic subjects

We propose a specific, reproducible and sensitive HPLC method for the determination of N(epsilon)-(carboxymethyl)lysine (CML) excreted in urine. Total CML was measured in acid hydrolysates of urine samples, while free CML was measured in acetonitrile-deproteinised urine samples using a RP-HPLC method with ortho-phtaldialdehyde (OPA)-derivatisation and fluorescence detection suited for automation. We compared the CML excretion of 51 non-proteinuric patients with diabetes mellitus (DM) (age 57+/-14 years, HbA1c 8.0+/-1.8%) to 42 non-diabetic controls (C) (age 45+/-17 years). The urinary excretion of total CML in diabetic patients was increased by approximately 30% (DM: 0.58+/-0.21; C: 0.45+/-0.14 microM/mmol creatinine; P<0.001). While urinary excretion of free CML was not significantly different, excretion of bound CML was increased (DM: 0.36+/-0.17; C: 0.27+/-0.14; P<0.05) in diabetic patients. CML excretion was correlated with protein and albumin excretion, but did not correlate with HbA1c, duration of DM or diabetic complications such as neuropathy or retinopathy. Furthermore, no age-dependent change of total CML excretion was found, while free CML excretion was lower in younger subjects. The specific and sensitive determination of CML by RP-HPLC of its OPA-derivative is well suited for automation and better than that of less defined glycoxidation products (AGEs).     

Quantification of 8-iso-prostaglandin-F(2alpha) and 2,3-dinor-8-iso-prostaglandin-F(2alpha) in human urine using liquid chromatography-tandem mass spectrometry

Quantification of 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) has been suggested to be a reliable indicator of lipid peroxidation that may be related to in vivo free radical generation, oxidative damage, and antioxidant deficiency. We have developed a LC-MS/MS method to quantify 8-iso- PGF(2alpha) and its dinor metabolite, 2,3-dinor-8-iso-prostaglandin F(2alpha) (2,3-dinor-8-iso-PGF(2alpha)), in human urine samples. After an initial purification step using an automated C18 solid phase extraction procedure, the urine sample was injected directly into a liquid chromatography (LC) system and detected with tandem mass spectrometry. The detection limit of the assay was 9 pg for 8-iso-PGF(2alpha) and 3 pg for 2,3-dinor-8-iso-PGF(2alpha) with both inter- and intraday variations of less than 12%. The inaccuracies were less than 3% for both analytes at three different levels. The urinary excretion rate of 2,3-dinor-8-iso-PGF(2alpha) was higher than that of 8-iso-PGF(2alpha), and changed in proportion to the parent compound (R = 0.70, n = 60). Values obtained with this method showed good linear correlation to duplicate 8-iso-PGF(2alpha) measurements performed with GCMS (R = 0.97, n = 15). The mean excretion rates of 8-iso-PGF(2alpha) and 2,3-dinor-8-iso-PGF(2alpha) were significantly higher in smokers than in nonsmokers (0.53 +/- 0.37 vs. 0.25 +/- 0.15 microg/g creatinine, p = 0.002 for 8-iso-PGF(2alpha) and 8.9 +/- 3.8 vs. 4.6 +/- 2.6 microg/g creatinine, p = 0.003 for 2,3-dinor-8-iso-PGF(2alpha), respectively). The excellent accuracy, reproducibility, and high throughput of this method should permit it to be used in large clinical studies and standard clinical laboratories.     

Effects of age, sex and renal function on urinary insulin-like growth factor I (IGF-I) levels in adults.

Insulin-like growth factor I (IGF-I) levels in urine were measured in adults using specific RIA after extraction with acid-ammonium sulfate. Mean (+/- SD) total urine IGF-I values were 267.9 +/- 112.9 ng/day and 167.8 +/- 73.2 ng/g creatinine (Cr) in 17 normal young adults. There was a positive correlation (r = 0.785, P < 0.001) between IGF-I values in early morning urine and those of 24 h urine when they were corrected by urinary Cr. IGF-I values in early morning urine were ranged from 60 to 1,100 ng/gCr with a mean value of 309.6 ng/gCr in 178 normal adults aged 21-80 yr. There was a consistent trend towards higher urinary IGF-I values in males during aging and this trend did not reach statistical significance until the sixth and seventh decades. There was a positive correlation (r = 0.465, P < 0.005) between urinary IGF-I values and age in males but not in females. Although urinary IGF-I values were higher in females than in males of the second and third decades, no sex difference was found in older adults. Urinary IGF-I values were correlated reversely with 24 h Cr clearance (CCr) and positively with urinary beta 2-microglobulin (beta 2-MG) levels in patients with renal dysfunction. These findings indicate that urinary IGF-I levels are influenced by age, sex and renal function in adults.     

Determination of eumelanin in human urine

Normal and malignant melanocytes produce melanins and melanin-related metabolites, most of which are retained in the cells but some are secreted into the blood and then excreted in the urine. In this study, we developed a method to measure levels of eumelanin in urine samples and evaluated its clinical significance in comparison with the melanin-related metabolites 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) and 5-S-cysteinyldopa (5-S-CD), and with pheomelanin, measured after degradation as 4-amino-3-hydroxyphenylalanine (4-AHP). The method is based on the production of pyrrole-2,3,5-tricarboxylic acid (PTCA) on permanganate oxidation of eumelanin, followed by quantification by liquid chromatography. For 118 urine samples from 10 control subjects, mean urinary excretions of PTCA, 6H5MI2C, 5-S-CD and 4-AHP were 19, 67, 37 and 59 micromol/mol creatinine respectively. In melanoma patients (n = 45), the mean urinary excretions of PTCA, 6H5MI2C, 5-S-CD, and 4-AHP were 91, 926, 4070 and 3530 micromol/mol creatinine respectively. Median level of PTCA in melanoma patients was elevated 2.1-fold compared with control subjects. The degrees of elevation for 6H5MI2C, 5-S-CD, and 4-AHP were 1.8-, 22- and 6.2-fold respectively. Thus, although urinary PTCA is of little clinical value in following the progression of melanoma, urinary 4-AHP appears to be of considerable value in this respect.     

Urinary excretion of 8-hydroxy-2'-deoxyguanosine measured by high-performance liquid chromatography with electrochemical detection

There is good evidence that oxidative DNA damage permanently occurs in living cells. The oxidative DNA damage product 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the predominant forms of radical-induced lesions to DNA, and has therefore been widely used as a biomarker for oxidative stress, either in cellular DNA or as DNA repair product in urine. In this paper we describe the use of a high-performance liquid chromatographic procedure with electrochemical detection for the measurement of urinary 8-OHdG. Our study has addressed the questions (i) of baseline urinary levels of 8-OHdG in spot urine and 24-h urine, (ii) of inter- and intra-individual variation of this biomarker, and (iii) of confounding factors for the excretion of 8-OHdG. No significant difference between the mean group levels of 8-OHdG/creatinine in spot urine (2.03+/-1.21 micromol/mol, n=148) and in 24-h urine (1.86+/-1.09 micromol/mol, n=67) was observed. However, when only 24-h urine was used for analysis, 8-OHdG was found to be statistically significantly higher in smokers. By multiple linear regression analysis, urinary creatinine was identified as the only predictor of 8-OHdG/24 h (r(p)=0.33, P=0.007). High intra-individual coefficients of variation of 8-OHdG/24 h were observed in two healthy subjects over a period of 10 consecutive days (37 and 57%, respectively), indicating that the intra-individual fluctuation of urinary 8-OHdG has so far been underestimated. Therefore, we suggest that single values of 8-OHdG should be considered with caution, in particular in small study groups and when spot urine is used.     

Human epidermal growth factor concentrations in urine, but not in saliva and serum, depend on thyroid state

To evaluate the effects of thyroid hormones on the concentration of epidermal growth factor (EGF), we determined values for the immunoreactive EGF concentration in the urine (U-irEGF) of newborn infants with congenital hypothyroidism (N = 19), and in urine, saliva and serum of adult patients with hypothyroidism (N = 11) and hyperthyroidism (N = 8). The values were expressed as SD score (SDS), i.e. deviation in SD units from their mean value of healthy subjects of the same age and sex. The SDS of relative U-irEGF (ng/mg creatinine) was lower (P less than 0.01) in newborn infants with congenital hypothyroidism (-0.8 +/- 0.2; mean +/- SEM) than in healthy infants. Their relative U-irEGF correlated with their serum T4 concentrations (r = 0.59, P less than 0.01). The SDS of relative U-irEGF was lower (P less than 0.01) in adult hypothyroid patients (-1.2 +/- 0.5) and higher (P greater than 0.05) in adult hypothyroid patients (0.9 +/- 0.6) than in healthy adult subjects. When subsequently euthyroid, their SDS of relative U-irEGF increased to -0.5 +/- 0.3 (P less than 0.01), and decreased to -0.7 +/- 1.1 (P less than 0.05), respectively. The irEGF concentrations in saliva and serum were not significantly different between the hypothyroid and hyperthyroid patients. Our results indicate that urinary excretion of irEGF in man is dependent on thyroid hormone.     

Measurement of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine by high-performance liquid chromatography with direct ultraviolet detection

A new high-performance liquid chromatographic (HPLC) method is described for the determination of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), the final product of the conjugation reaction between a metabolic intermediate of N,N-dimethylformamide (DMF) and glutathione. Urine samples were purified by C(18) solid-phase extraction and then directly analysed by HPLC with an Aminex Ion Exclusion HPX-87H column maintained at 25 degrees C and a UV detector set at 196 nm. Under isocratic conditions (2.4 mM sulphuric acid, flow-rate=0.6 ml/min) AMCC eluted at 20.2 min. The reproducibility (C.V.%) was 1.3-2.7% (intra- and inter-assay, N = 5); the accuracy was 98.0+/-1.7% at 10 mg/l and 101.9+/-1.5% at 800 mg/l (mean+/-SD, N = 3). AMCC was measured in urine from 22 exposed subjects. A strong correlation was found between AMCC and environmental DMF [AMCC (mg/g creatinine)=3.40xDMF (mg/m(3)) + 3.07; r=0.95], while in the urine of 20 unexposed subjects the concentration of AMCC was constantly below the detection limit of the method (0.9 mg/l in urine). The method described appears to be useful for the biological monitoring of DMF exposure.     

Renal function and fractional clearances of American river otters (Lutra canadensis)

The finely lobulated kidneys of American river otters (Lutra canadensis) are not visualized on plain abdominal radiographs. Similar values for blood urea nitrogen (BUN), creatinine, and uric acid were obtained on different analytical systems used in 1984 and 1985. The mean +/- SD for measured plasma osmolalities (309.80 +/- 8.86 mOsmol/kg) of otters in 1985 was significantly (P less than 0.01) less than that of calculated serum osmolalities in the same 1985 specimens (321.61 +/- 5.64 mOsmol/kg) and in 1984 specimens (322.20 +/- 7.16 mOsmol/kg). Urine specific gravities and osmolalities were highly correlated (r = 0.92). On routine urinalysis, protein and bilirubin were frequent chemical findings, and urobilinogen was present in all urine samples. White and red blood cells and epithelial cells were frequent findings on urine microscopic examinations. Proteus mirabilis was cultured from four of four female otters with genitourinary infections. The mean +/- SD creatinine values for paired serum and urine samples (n = 13) were serum creatinine (Scr) 0.66 +/- 0.09 mg/dl and urine creatinine (Ucr) 186.9 +/- 55.6 mg/dl. Corresponding values for serum electrolytes (Se) and urine electrolytes (Ue) yielded mean +/- SD calculated renal fractional clearances (FC = Ue/Se x Scr/Ucr) of sodium 9.65 +/- 5.81 x 10(-4), potassium 4.15 +/- 2.01 x 10(-2), chloride 10.81 +/- 5.33 x 10(-4), calcium 4.52 +/- 4.46 x 10(-3), and phosphate 6.58 +/- 3.44 x 10(-3).     

Influence of metabolic genotype GSTM1 on levels of urinary mutagens in patients treated topically with coal tar

Fifteen hospitalized, non-smoking, dermatological patients were treated with ointment containing 2% coal tar (CT) in order to assess the influence of metabolic genotype GSTM1 on urinary mutagen levels. Urinary 1-pyrenol, the main metabolite of pyrene, was used to check the high exposure to PAH of this population. The mean levels of urinary 1-pyrenol found in the 24-h urine of our patients were 467. 8+/-211.0 nmoles-24 h (range 94.6-890.1 nmoles-24 h). Mutagenicity was assessed on urine samples collected over a period of 24 h, after three consecutive days of topical application, using the bacterial mutagenesis test on Salmonella typhimurium strains TA98 and YG1024 in the presence of microsomal enzymes. The latter strain turned out to be more sensitive than the former in revealing urinary mutagens in these patients (42 693+/-30 867 vs. 6877+/-6040 net revertants-24 h). The mutagenicity on YG1024 strain and 1-pyrenol levels of urine samples were correlated (Spearman's rank correlation coefficient=0. 6678, P<0.01, z=2.795). The influence of genotype GSTM1 on urinary mutagen levels was assessed on strain YG1024. The values of urinary mutagenicity of subjects with genotype GSTM1-null (n=6) were on average higher than those of GSTM1-positive subjects (n=9) (55 498+/-45 957 vs. 34 156+/-11 933 net rev.-24 h), a non-significant statistical difference. The mean total excretion of mutagens corrected for PAH exposure (net rev./nmoles of urinary 1-pyrenol) in GSTM1-null patients was double that of GSTM1-positive ones (136. 8+/-34.7 vs. 70.8+/-23.3 net rev./nmoles of urinary 1-pyrenol; one-tailed Mann-Whitney U-test, U=11.5, P<0.05). These results indicate a greater body burden of promutagens, resulting from skin application of CT, in GSTM1-null subjects.     

Isolation and identification of urinary beta-aspartyl dipeptides and their concentrations in human urine.

beta-Aspartyl-methionine, -aspartic acid and -glutamic acid and gamma-glutamyl-threonine and -glycine were isolated and identified in human urine by ion-exchange chromatography, high-voltage paper electrophoresis, acid hydrolysis and determination of N-terminal amino acids of the isolated compounds, and comparison of their behaviors in paper electrophoresis and chromatography with those of the authentic compounds. The concentrations of acidic beta-aspartyl dipeptides in human urine were determined using an amino acid analyzer. Their concentrations were as follows: beta-aspartyl-glycine, male, 44.4 +/- 8.5, female, 61.4 +/- 18.9, child, 83.7 +/- 27.1; -alanine, male, 11.0 +/- 4.9, female, 20.7 +/- 12.0, child, 25.3 +/- 9.1; -glutamic acid, male, 10.0 +/- 3.7, female, 23.0 +/- 8.5, child, 20.4 +/- 7.5; -serine, male, 9.9 +/- 2.8, female, 13.6 +/- 3.8, child, 14.9 +/- 4.7; -aspartic acid, male, 4.3 +/- 1.0, female, 9.1 +/- 2.2, child, 18.4 +/- 6.5; -threonine, male 3.9 +/- 0.9, female, 5.8 +/- 1.1, child, 13.2 +/- 4.9 mumol/g creatinine (mean +/- S.D.). The order of the sum of their concentrations tended to be child greater than female greater than male. Patients receiving intravenous hyperalimentation also excreted acidic beta-aspartyl dipeptides into urine in amounts similar to those in females and in a pattern similar to that observed in healthy persons. This finding indicates that urinary beta-aspartyl dipeptides were probably of endogenous origin because oral nutrition was stringently excluded in these patients.     

F(2)-isoprostane and prostaglandin F(2 alpha)metabolite excretion rate and day to day variation in healthy humans.

Isoprostanes are mainly formed in vivo by a non-enzymatic free radical catalysed oxidation of arachidonic acid. Studies have indicated that a major isoprostane, 8-iso-PGF(2 alpha)in plasma and urine is a reliable biomarker of oxidative stress. Prostaglandins are formed by enzymatic oxidation of arachidonic acid catalysed by cyclooxygenase (COX). 15-Keto-dihydro-PGF(2 alpha), a major metabolite of prostaglandin F(2 alpha)in plasma, and also found in urine, is considered to be a useful biomarker of inflammation. To investigate the excretion pattern and day to day variation of 8-iso-PGF(2 alpha)and 15-keto-dihydro-PGF(2 alpha)in healthy individuals, morning urine samples were collected from 13 volunteers on 10 successive days. The samples were analysed for free 8-iso-PGF(2 alpha)and 15-keto-dihydro-PGF(2 alpha)by radioimmunoassay. The mean excretion rate of 8-iso-PGF(2 alpha)was 0.27+/-0.11 nmol/mmol creatinine (mean+/-SD, n=13) and the coefficient of variation was 42% during the 10 days. The mean excretion rate of 15-keto-dihydro-PGF(2 alpha)was 0.46+/-0.19 nmol/mmol creatinine, giving a coefficient of variation of 41%. The mean values of 8-iso-PGF(2 alpha)were significantly correlated with the mean values of 15-keto-dihydro-PGF(2 alpha)(r=0.68, P=0.01). In conclusion, day to day biological variation in urinary excretion rate of 8-iso-PGF(2 alpha)and 15-keto-dihydro-PGF(2 alpha)should be taken into account in evaluating a clinical study unless a large increase or decrease of these parameters has been obtained.     

Zea mays L. extracts modify glomerular function and potassium urinary excretion in conscious rats

Diuretic and uricosuric properties have traditionally been attributed to corn silk, stigma/style of Zea mays L. Although the diuretic effect was confirmed, studies of the plant's effects on renal function or solute excretion were lacking. Thus, we studied the effects of corn silk aqueous extract on the urinary excretion of water, Na+, K+, and uric acid. Glomerular and proximal tubular function and Na+ tubular handling were also studied. Conscious, unrestrained adult male rats were housed in individual metabolic cages (IMC) with continuous urine collection for 5 and 3 h, following two protocols. The effects of 25, 50, 200, 350, and 500 mg/kg body wt. corn silk extract on urine volume plus Na+ and K+ excretions were studied in water-loaded conscious rats (2.5 ml/100 g body wt.) in the IMC for 5 h (Protocol 1). Kaliuresis was observed with doses of 350 (100.42 +/- 22.32-120.28 +/- 19.70 microEq/5 h/100 g body wt.; n = 13) and 500 mg/kg body wt. (94.97+/- 29.30-134.32 +/- 39.98 microEq/5h/100 g body wt.; n = 12; p<0.01), and the latter dose resulted in diuresis as well (1.98 +/- 0.44-2.41 +/- 0.41 ml/5 h/100 g body wt.; n = 12; p<0.05). The effects of a 500 mg/kg body wt. dose of corn silk extract on urine volume, Na+, K+ and uric acid excretions, and glomerular and proximal tubular function, were measured respectively by creatinine (Cler) and Li+ (ClLi) clearances and Na+ tubular handling, in water-loaded rats (5 ml/100 g body wt.) in the IMC for 3 h (Protocol 2). Clcr (294.6 +/- 73.2, n = 12, to 241.7 +/- 48.0 microl/ min/100 g body wt.; n = 13; p<0.05) and the Na+ filtered load (41.9 +/- 10.3, n = 12, to 34.3 +/- .8, n = 13, p<0.05) decreased and ClLi and Na+ excretion were unchanged, while K+ excretion (0.1044 +/- 0.0458, n=12, to 0.2289 +/- 0.0583 microEq/min/100 body wt.; n = 13; p<0.001) increased. For Na+ tubular handling, the fractional proximal tubular reabsorption (91.5 +/- 3.5, n = 12, to 87.5 +/- 3.4%; n = 13; p<0.01) decreased, and both fractional distal reabsorptions--I and II--increased (96.5 +/- 1.5, n = 12, to 97.8 +/- 0.9%; n = 13; p<0.01; and 8.2 +/- 3.5, n = 12, to 12.2 +/- 3.4%, n = 13, p<0.01, respectively). To summarize, in water-loaded conscious rats (2.5 ml/100 body wt.), corn silk aqueous extract is diuretic at a dose of 500 mg/kg body wt. and kaliuretic at doses of 350 and 500 mg/kg body wt. In water-loaded conscious rats (5.0 ml/100 g body wt.), corn silk aqueous extract is kaliuretic at a dose of 500 mg/kg body wt., but glomerular filtration and filtered load decrease without affecting proximal tubular function, Na+, or uric acid excretion.     

Determination of estradiol-17-sulfate in human urine by a direct radioimmunoassay: urinary levels throughout the menstrual cycle

The measurement of urinary estradiol-17-sulfate concentration by direct radioimmunoassay was established. The urinary estradiol-17-sulfate levels measured by the radioimmunoassay correlated well with those determined by high-performance liquid chromatography equipped with electrochemical detector. Estradiol-17-sulfate concentrations in early morning urine of six healthy adult men was 8.2 +/- 2.0 ng/mL, or 5.7 +/- 1.8 ng/mg creatinine. The urinary levels in women throughout the menstrual cycle showed a characteristic three-peak excretion pattern: the first and the second peaks appeared just after and three days before the urinary LH peak, and the third peak appeared a few days before menstruation.     

Systems for collection of urine in the captive common marmoset, Callithrix jacchus

Two systems are described for the collection of 24 h urine samples from the common marmoset (Callithrix jacchus). Using 84 adult animals, 1210 24-h samples were collected. Mean urinary excretion was 14.4 +/- 7.5 ml/24 h (n = 1210, mean +/- SD). No differences were observed between sexes (for 52 females, 24 h volume = 15.1 +/- 8.0 ml; for 32 males, 24 h volume = 12.5 +/- 6.0 ml). No significant differences were observed between pregnant and non-pregnant females with respect to 24 h urine volume, and bilateral gonadectomy did not influence subsequent urinary excretion in either sex. For 161 pairs of observations, the intake of drinking water (11.7 +/- 10.2 ml/24 h) and the volume of urine excreted (12.6 +/- 7.1 ml/24 h) showed a positive correlation (r = 0.406 d.f. 159, P less than 0.001: y = 0.558x + 4.247).