Urea-selective concentrating defect in transgenic mice lacking urea transporter UT-B.

Urea transporter UT-B has been proposed to be the major urea transporter in erythrocytes and kidney-descending vasa recta. The mouse UT-B cDNA was isolated and encodes a 384-amino acid urea-transporting glycoprotein expressed in kidney, spleen, brain, ureter, and urinary bladder. The mouse UT-B gene was analyzed, and UT-B knockout mice were generated by targeted gene deletion of exons 3-6. The survival and growth of UT-B knockout mice were not different from wild-type littermates. Urea permeability was 45-fold lower in erythrocytes from knockout mice than from those in wild-type mice. Daily urine output was 1.5-fold greater in UT-B- deficient mice (p < 0.01), and urine osmolality (U(osm)) was lower (1532 +/- 71 versus 2056 +/- 83 mosM/kg H(2)O, mean +/- S.E., p < 0.001). After 24 h of water deprivation, U(osm) (in mosM/kg H(2)O) was 2403 +/- 38 in UT-B null mice and 3438 +/- 98 in wild-type mice (p < 0.001). Plasma urea concentration (P(urea)) was 30% higher, and urine urea concentration (U(urea)) was 35% lower in knockout mice than in wild-type mice, resulting in a much lower U(urea)/P(urea) ratio (61 +/- 5 versus 124 +/- 9, p < 0.001). Thus, the capacity to concentrate urea in the urine is more severely impaired than the capacity to concentrate other solutes. Together with data showing a disproportionate reduction in the concentration of urea compared with salt in homogenized renal inner medullas of UT-B null mice, these data define a novel "urea-selective" urinary concentrating defect in UT-B null mice. The UT-B null mice generated for these studies should also be useful in establishing the role of facilitated urea transport in extrarenal organs expressing UT-B.     

The flehmen response of bulls and cows

The response of dairy bulls to the urine of cows in various stages of the reproductive cycle was quantified by presenting 200 ml of urine in a stainless steel bowl to the stanchioned bulls for 10 min. Estrous mucus was also presented in the same manner. Sniffs, nose licks and flehmen responses were recorded. Of 15 bulls tested only 7, or 47%, met the criteria of two flehmens in response to estrous urine. Among these bulls, the rate of flehmen was higher to estrous urine 6.1 +/- 1 flehmen/10 min) than to nonestrous urine (3.5 +/- 0.6 flehmen/10 in, paired t = 3.1, P < 0.03). Flehmen duration was also longer in response to estrous urine (6.4 +/- 0.4 sec) than to nonestrous urine (5.7 +/- 0.4 sec, t = 2.65, P < 0.03). There were no significant differences between the sniff frequencies and durations or in the number of licks to estrous and diestrous urine. There were significantly more flehmen responses to estrous urine (7 +/- 1.4 10 min ) than to mucus (2.25 +/- 0.9 10 min , t = 4.75, P < 0.01). The response to water (0.6 +/- 0.3 10 min ) was not different from that to mucus (t = 2.37, P < 0.10). The spontaneous flehmen rate of dairy bulls in their home stalls was 3.2 +/- 0.7 24 h . Although estrous cows did not exhibit flehmen frequently, they did sniff bull urine more frequently (3.6 +/- 0.6 10 min ) than nonestrous cows (1.8 +/- 0.3 10 min , t = 2.4, P < 0.03).     

Effects of atrial natriuretic factor on urinary concentration of catecholamines and renin secretion in dogs

This study evaluated the effects of synthetic atrial natriuretic factor (ANF) on renal hemodynamics, urinary excretion of electrolytes, norepinephrine (NE), and dopamine (DA); and renal production of renin in anesthetized dogs. Following a bolus (1 micrograms/kg body weight) and infusion (0.1 microgram/kg/min) for 30 min, there was significant increase in urine flow (220 +/- 41%), glomerular filtration rate (72 +/- 14%), and urinary sodium excretion (170 +/- 34%). There was a decrease in renin secretory rate and the concentration ratio of urine NE to DA following ANF was decreased (p less than 0.05). These data suggest that ANF decreases renal production of NE and renin.     

Aglomerular kidney function when challenged with exogenous MgSO4 loading or environmental MgSO4 depletion

The goal of this study was to investigate the role of MgSO4 in aglomerular kidney function, independent of changes in NaCl. The renal handling of MgSO4 was manipulated by intravenous infusion of an isoosmotic solution containing 80 mmol/L MgSO4 or through exposure to an environment that was reduced in MgSO4 concentration by 90%. Intravenous infusion resulted in a transient increase in circulating Mg2+ and SO4 (2-) levels; however, the concentration of both divalent ions in the urine remained elevated throughout the entire infusion period. Infusion also resulted in a transient increase in urine flow rate and apparent glomerular filtration rate, measured using the glomerular filtration rate marker, [3H] PEG 4000. Exposure to MgSO4-depleted conditions resulted in a significant decrease in plasma and urine concentrations of Mg2+ and in the urine concentrations of SO4 (2-); correspondingly, urine flow rate was significantly depressed. The urinary excretion of both Mg2+ and SO4 (2-) demonstrated nonlinear saturation kinetics. The urinary excretion of Mg2+ was significantly correlated with plasma Mg2+ concentration (r=0.75, P=0.04) and yielded a Michealis constant (Km) of 1.67+/-1.43 mmol/L; P=0.26 and a maximal velocity (Vmax) of 117.4+/-47.0 micromol/kg/hr; P=0.046. The urinary excretion of SO4 (2-) was significantly correlated with plasma SO4 (2-) concentration (r=0.94, P<0.02) with a Km of 0.76+/-0.54; P=0.26 and a Vmax of 59.3+/-13.1; P=0.02.     

Changes in the plasma and urine alpha human atrial natriuretic peptide (alpha hANP) concentration in patients with thyroid disorders

In order to assess the possible involvement of thyroid hormone in alpha human atrial natriuretic peptide (alpha hANP), we investigated the plasma and urine ANP concentration in patients with primary hyperthyroidism and hypothyroidism. Plasma and urine were extracted through Sep-Pak C18 cartridges and the urine ANP concentration was corrected by urine creatinine (cre. mg/dl) and expressed as fmol/mg.cre.. The plasma ANP concentration in patients with untreated hyperthyroidism (32.3 +/- 7.0 fmol/ml; n = 22) was higher than in normal subjects (p less than 0.01 vs control; 6.2 +/- 0.7 fmol/ml). After restoration to euthyroidism, the plasma ANP concentration (patients with treated hyperthyroidism) fell to normal (8.9 +/- 1.9 fmol/ml). The plasma ANP concentration in patients with untreated hypothyroidism (14.1 +/- 3.0 fmol/ml; n = 7) was higher than normal, but in two of them there was mild renal dysfunction and an incomplete right blundle branch block in the electrocardiogram. It was possible that these factors contributed to the observed increase in plasma ANP. However, a significant positive correlation was found between plasma ANP and free thyroxine (n = 40, r = 0.449; p less than 0.01) and free triiodothyronine (n = 40, r = 0.546; p less than 0.01). The urine ANP concentration in patients with untreated hyperthyroidism was markedly higher than in normal subjects (p less than 0.01), but in untreated hypothyroidism not significantly different from normal.     

Quantification of apolipoprotein D in human urine by zone immunoelectrophoresis assay: a methodological and clinical study.

A zone immunoelectrophoresis assay (ZIA) has been developed for the quantification of apolipoprotein D (apo D) in unconcentrated native human urine. A standard curve, linear between 1 and 8 mg apo D/l was obtained with ZIA. The relative coefficients of variation for this method were 5-9% (n = 15 x 6) with a mean +/- SD of 7 +/- 1.4% and below 11% (n = 6 x 15) for within-run and between-run reproducibility, respectively. Equal amounts of apo D in unconcentrated and diluted urines, in serum and of the purified protein produced the same zone migration distances indicating parallelism between the immunologic reactions of apo D in different sample matrixes. Storage experiments with normal urines demonstrated good stability of apo D in both acidic and alkalinized urine over at least 2 days at +5 degrees C and during several days at -20 degrees C to -40 degrees C. Using ZIA, urine samples from 50 normal healthy men aged 23-65 years were analyzed for apo D. Mean and SD were: 2.8 +/- 2.1 mg/l, 2.6 +/- 1.8 micrograms/min and 0.24 +/- 0.13 mg/mmol for concentration, rate of excretion and mass/creatinine concentration, respectively.     

Reduction in urine C-peptide clearance rate after metabolic control in NIDDM patients

One hour urine C-peptide and creatinine clearance rates were determined simultaneously in 25 hospitalized patients with non-insulin-dependent diabetes mellitus (NIDDM) undergoing sulfonylurea and/or diet treatment. The studies had been performed after an overnight fast on the second day of admission and on a day soon before discharge, with intervals of 18.9 +/- 7.0 days. Fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) values decreased significantly at the second examination as compared to the initial values (FPG: 101 +/- 20 mg/dl vs. 161 +/- 47 mg/dl, p less than 0.005; HbA1c: 7.3 +/- 1.5% vs. 8.4 +/- 1.7%, p less than 0.005). The urine C-peptide clearance rate also decreased significantly after metabolic control (0.75 +/- 0.36 l/hr vs. 1.06 +/- 0.54 l/hr, p less than 0.005). Meanwhile, the urine creatinine clearance rate tended to decrease, but the difference was not significant (3.69 +/- 2.04 l/hr vs. 4.87 +/- 2.98 l/hr) at the second examination. The data suggest that the urine C-peptide clearance rate is susceptible to the effects of the fluctuation of metabolic states in NIDDM patients. In order to use urinary C-peptide for a follow up study of pancreatic B-cell secretion, the changes in C-peptide clearance under various metabolic conditions must be taken into account.     

Isolation of cyclo(His-Pro)-like immunoreactivity from human urine and demonstration of its immunologic, pharmacologic, and physico-chemical identity with the synthetic peptide

Measurements of cyclo(His-Pro) levels in human urine were carried out by specific radioimmunoassay. Cyclo(His-Pro)-like immunoreactivity in Human urine was found to be immunologically, pharmacologically, and physico-chemically identical to that of synthetic cyclo(His-Pro). The concentration of urinary cyclo(His-Pro) in 24-h collection was 1133.8 +/- 122.5 nmol/L, with a range of 606 to 1865 nmol/L. The daily excretion rate of cyclo(His-Pro) was 1812 +/- 248 nmol cyclo(His-Pro)/g creatinine, or 1814 +/- 199 nmol cyclo(His-Pro/day.     

Tamm-Horsfall protein excretion during chronic alterations in urinary concentration and protein intake in the rat.

Tamm-Horsfall protein (THP), a normal constituent of mammalian urine, has been determined in rat urine under various conditions in an attempt to elucidate the physiological role of this glycoprotein. Experiments were designed to assess whether THP production is related to the process of urine concentration or to the transport activity of the thick ascending limb of the loop of Henle (TAL), the nephron segment where it is produced. For this purpose, THP excretion was measured, by radioimmunoassay, in adult male rats under 4 different conditions induced by the following chronic treatments: (1) furosemide (12 mg/day in osmotic minipumps); (2) increased water intake; (3) antidiuretic hormone (ADH) infusion (50 ng DDAVP/day in osmotic minipumps) in rats of the Brattleboro strain with hereditary hypothalamic diabetes insipidus; (4) high-protein (32% casein) versus low-protein diet (10% casein). Each experiment included 6 experimental and 6 control rats. After treatment for 1-3 weeks, 24-h urines were collected for determination of urine flow rate, osmolality, and creatinine and THP concentrations. No significant changes in THP excretion were observed in experiments (1) and (2) despite 5- to 7-fold-differences in urine flow rate. Antidiuretic hormone treatment in (3) slightly lowered THP excretion (287 +/- 53 vs. 367 +/- 41 micrograms/day per 100 g body weight; p less than 0.005), whereas high-protein diet, in experiment (4), led to a 50% increase in THP excretion (446 +/- 57 vs. 304 +/- 79 micrograms/day per 100 g body weight; p less than 0.001). Expressing THP excretion relative to that of creatine did not change these findings. These results show (1) that chronically established changes in the level of diuresis, chronic furosemide-induced blockade of the Na,K,Cl-cotransporter or the absence of ADH in Brattleboro rats have little or no impact on the level of THP production, and (2) that THP production is independent of the intensity of transport in the TAL, since two conditions which both are known to increase the transport rate of solutes in the TAL (ADH infusion and high-protein diet), resulted in opposite changes in THP excretion. It is concluded that the rate of THP synthesis is neither linked to the process of urine concentration nor to the ion transport activity of the TAL.     

Oral loading of homogentisic acid in controls and in obligate heterozygotes for hereditary tyrosinemia type I.

Homogentisic acid (HGA) (50 mg/kg) was given orally to 22 obligate heterozygotes for hereditary tyrosinemia type 1 (HT) and to 11 controls. After 1 h the mean +/- standard error (SE) plasma level of HGA was 30.42 +/- 1.41 micrograms/ml in carriers and 19.29 +/- 1.62 in controls. Mean +/- SE fasting delta-amino-levulinate dehydratase (delta-ALD) was 40.05 +/- 1.79 m microM/min/g Hb in carriers, much lower than the 60.81 +/- 5.11 found in controls. After 3 h this difference in levels of delta-ALD remained, with mean +/- SE values of 25.70 +/- 2.89 m microM/min/g Hb in carriers, compared with 48.83 +/- 5.37 in controls. Three-hour mean +/- SE excretion of fumarylacetone "equivalent" [FAc] in urine in carriers, 51.597 +/- 5.580 micrograms/mg/creatinine, was significantly higher than the 27.941 +/- 5.916 in controls. Three-hour excretion of succinylacetone "equivalent" [SAc] was also significantly higher in the urine of carriers. FAc in 3-h urine was identified by thin-layer chromatography and confirmed by gas chromatography/mass spectrometry. Multivariate stepwise discriminant analysis showed that the inclusion order of significant variables was as follows: HGA levels at 1 hr, fasting level of delta-ALD, residual level of HGA at 3 h, and 3-h excretion of [FAc]. Non-significant variables were HGA tolerance, levels of delta-ALD at 3 h, sex, and 3-h excretion of [SAc].(ABSTRACT TRUNCATED AT 250 WORDS)     

Head-down tilt and restraint on renal function and glomerular dynamics in the rat

A model utilizing 25 degree head-down tilt (HDT) and incorporated with chronic catheterization and renal micropuncture techniques in rats was employed to study alterations in renal function induced by HDT. Renal function and extracellular volume measurements were performed after 24 h, 4 days, and 7 days of HDT in conscious rats and compared with their own control measurements and to nontilted but similarly restrained rats. After 24 h HDT, glomerular filtration rate (GFR) increased 19 +/- 8% and renal plasma flow (RPF) increased 18 +/- 8% with increases in urine flow rate, Na+, and K+ excretion in conscious rats. These increases after 24 h were associated with an increase in extracellular volume of 16 +/- 3% (P less than 0.01). In the nontilted controls, there was a decrease in extracellular volume after 24 h of suspension. After 7 days of HDT, GFR was decreased by 7 +/- 1% (P less than 0.01), but RPF and extracellular fluid volume were not different from control values. However, RPF and GFR increased in the nontilted rats after 7 days. After 7 days of HDT renal micropuncture studies demonstrated that single-nephron filtration rate was also decreased from 43 +/- 2 to 31 +/- 3 nl/min (P less than 0.05) due solely to reductions in the glomerular ultrafiltration coefficient (0.11 +/- 0.01 to 0.07 +/- 0.01 nl.s-1 X mmHg-1, P less than 0.05). There was a dissociation between GFR and water and Na+ excretion at days 4 and 7 of HDT not observed in the nontilt restraint controls.     

Elementary steps in the formation of horseradish peroxidase compound I: direct observation of compound 0, a new intermediate with a hyperporphyrin spectrum

The reaction of horseradish peroxidase (HRP) with H2O2 has been studied in 50% v/v methanol/water over the 25.0 to -36.0 degrees C temperature range by using the low-temperature stopped-flow technique. All reactions were carried out under pseudo-first-order conditions with [H2O2] much greater than [HRP]. Arrhenius plots for the pseudo-first-order rate constant kobs were linear over the 17.6 to -36.0 degrees C temperature range studied with an activation energy of 4.8 +/- 0.5 kcal/mol. Above 0 degrees C, kobs varies linearly with peroxide concentration. However, saturation kinetics are observed below -16.0 degrees C, indicating that there is at least one reversible elementary step in this reaction. Double-reciprocal plots at -26.0 degrees C at pH* 7.3 for the reaction give kappa max(obs) = 163 s-1 and KM = 0.190 mM. Rapid-scan optical studies carried out at -35.0 degrees C with [H2O2] much greater than KM reveal the presence of a transient intermediate referred to as compound 0 whose conversion to compound I is rate limiting. The Soret region of the optical spectrum of compound 0 resembles that of a "hyperporphyrin" with prominent bands near 330 and 410 nm. The temperature dependencies of kappa max(obs) and KM have been measured over the -16.0 to -26.0 degrees C range and give an activation energy for kappa max(obs) of 1.6 +/- 0.7 kcal/mol and an enthalpy of formation for compound 0 of 4.0 +/- 0.7 kcal/mol.     

Atrial natriuretic factor in liver cirrhosis--the influence of volume expansion

Plasma levels of atrial natriuretic factor (ANP) were examined in 12 patients with liver cirrhosis (6 with ascites) and 6 controls before and after the administration of the infusion of 2000 ml of saline solution per 70 kg of body weight during 2 hours. Basal concentration of ANF tended to be slightly, but nonsignificantly higher in patients with ascitic liver cirrhosis (5.5 +/- 1.3 fmol/ml) than in controls (3.0 +/- 1.0 fmol/ml) and in patients with non-ascitic liver cirrhosis (4.6 +/- 1.3 fmol/ml). Saline administration led to the comparable increase of plasma ANF in ascitic (14.2 +/- 4.0 fmol/ml) and non-ascitic cirrhotics (15.7 +/- 3.7 fmol/ml) and in controls (12.4 +/- 4.3 fmol/ml). The increase of plasma ANF was accompanied by the suppression of plasma renin activity (PRA) and plasma aldosterone (PA) in all groups; in ascitic patients, however, PRA and PA remained above the normal range. While in controls and non-ascitic cirrhotics saline administration led to the increase of urine flow rate /from 0.74 +/- 0.13 to 2.04 +/- 0.44 ml/min, P less than 0.01, in controls; from 0.83 +/- 0.05 to 1.28 +/- 0.07 ml/min, P less than 0.01, in non-ascitic cirrhotics) and urinary sodium excretion (from 110.7 +/- 21.3 to 364.8 +/- 74.4 umol/min, P less than 0.01, in controls; from 125.0 +/- 16.7 to 218.7 +/- 24.3 umol/min, P less than 0.01 in non-ascitic cirrhotics), in patients with ascitic liver cirrhosis neither urine flow rate (from 0.66 +/- 0.1 to 0.72 +/- 0.15 ml/min, n.s.), nor urinary sodium excretion (from 16.7 +/- 9.9 to 54.2 +/- 40.3 umol/min, n.s.) changed significantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in body fluids of the frog Leptodactylus fuscus during estivation (Anura, Leptodactylidae)

The frog, L. fuscus, becomes dormant during the dry season in southeastern Brazil. Plasma and urine were obtained and analyzed for K+, Na+, and osmotic concentrations in active and estivating frogs. Soil water potential from the estivation sites was compared with the osmotic concentrations of the frog. Plasma and urine osmotic concentrations (286.2 +/- 13.8 and 242.3 +/- 17.2 mOsm1(-1), respectively) were higher in the estivating than in active frogs (240.3 +/- 12.8 and 112.7 +/- 15.6 mOsm1(-1); plasma and urine), and the same holds true for plasma K+ content. The Na+ concentration was the same for active and estivating frogs. Soil water potential corresponded to osmotic pressure of 110 mOsm1(-1), showing that L. fuscus may uptake water from the soil during the estivation.     

Toxic effect of DDT and endosulfan in white shrimp postlarvae Litopenaeus vannamei (Decapoda: Penaeidae) from Chiapas, Mexico

We analized acute toxicity in white shrimp (Litopenaeus vannamei) postlarvae exposed to two chlorinated pesticides, DDT and endosulfan, under laboratory conditions during 168 hours, with controlled temperature (29 +/- 1 degrees C), salinity (3 +/- 1%o) and pH (8 +/- 1). Median lethal concentrations (LC50), "incipient" LC50, median lethal time (LT50) the "maximum acceptable concentration of the toxic compound" (MACT) and "the safety level" (SL) were determined. The concentration of the compounds at which organism growth was reduced by 5 and 50% (EC5 and EC50), as well as changes in oxygen consumption patterns were determined in the surviving postlarvae. They were very sensitive to both compounds and DDT was thrice as toxic as endosulfan. Growth rate decreased 50% and 80% with endosulfan and DDT, respectively, at the experimental pestice concentration. The low resistance of postlarvae to DDT and endosulfan suggests that additional inflow of these pesticides into the aquatic system could affect the rate of shrimp production in the area.     

Natural antimicrobial agent (reuterin) produced by lactobacillus reuteri for sanitization of biological tissues inoculated with pseudomonas aeruginosa

The study was done to evaluate the efficacy of using reuterin produced by Lactobacillus reuteri to sanitize biological tissues. The microorganism tested in the study was Pseudomonas aeruginosa, a common cause of nosocomial biomaterial-related infections. The inhibitory effect of reuterin on P. aeruginosa for an inoculated tissue was investigated at different conditions of concentration, temperature, and pH. Additionally, the cellular compatibility of the reuterin-sanitized tissue was evaluated. Glutaraldehyde was employed as a control. It was noted that the minimum inhibitory concentration (MIC, 33.0 +/- 2.9 ppm) and minimum bactericidal concentration (MBC, 50.0 +/- 0.0 ppm) values of reuterin for P. aeruginosa were significantly lower than their glutaraldehyde counterparts (MIC, 130.0 +/- 8.2 ppm and MBC, 180.0 +/- 18.3 ppm). This indicated that reuterin was more efficient than glutaraldehyde as an antimicrobial agent. The addition of reuterin on the inoculated tissue led to a reduced viability of P. aeruginosa. The reduction in the P. aeruginosa culture was more pronounced with increasing the concentration of reuterin (0-100 ppm). At increasing temperature (25-45 degrees C), there was an increasing effect of reuterin on its sanitization activity. However, it should be pointed out that the growth of P. aeruginosa in the nutrient broth was also significantly affected by temperature. The sanitization activity of reuterin was more evident with increasing the pH level (pH 6.5-8.5). The cytotoxicity of reuterin was significantly lower than that of glutaraldehyde. Additionally, the cellular compatibility of the reuterin-sanitized tissue was superior to its glutaraldehyde-sanitized counterpart.     

A stop-flow study of intrarenal effects of atrial natriuretic factor

We performed paired series of stop-flow studies on six mongrel dogs to determine a possible nephron site of action of synthetic atrial natriuretic factor (ANF). The initial free-flow response to intrarenal infusion of 5 micrograms/min of synthetic ANF into mannitol-expanded dogs resulted in an increased urine flow rate (6.81 +/- 0.88 to 9.00 +/- 1.17 ml/min, P less than 0.05) and a 40% increase in sodium excretion (496 +/- 110 to 694 +/- 166 meq/min, P less than 0.025) when compared to paired control periods. Renal blood flow did not change, but the glomerular filtration rate increased 4% (47 +/- 5 to 49 +/- 6 ml/min, P less than 0.05). The filtered load of sodium increased 4% (P less than 0.05), and the fractional sodium excretion increased by 35% (P less than 0.01). Stop-flow experiments showed no difference in tubular sodium concentration or in the fractional sodium-to-inulin ratio at the nadir of sodium concentration, suggesting that no differences existed in distal tubular sodium handling. Further, no apparent differences were detected in collections representing the more proximal portions of the nephron. While we were able to demonstrate marked natriuresis in response to synthetic ANF, no tubular effect was discernible, and the natriuresis obtained appears to be predominantly a function of hemodynamic effects.     

Extracellular volume estimation from ratios of bromide to chloride in urine or saliva.

Use of either urine or saliva samples to estimate extracellular water volume was investigated in 10 men using nonradioactive bromide (Br) and in seven newborn piglets using radioactive Br (82Br) and chloride (36Cl). The relation to Br to Cl concentrations in urine enabled an estimation of Br dilution volume from human urine (267 +/- 42 ml/kg, mean +/- SD) that was not significantly different (P = 1.0) from the Br dilution volume calculated from plasma Br concentration (268 +/- 20 ml/kg). Although the Br dilution volume estimated from saliva was not different from that of plasma, the error in the estimates of Br dilution volume from saliva was too large (mean difference, -36 +/- 64 ml/kg) to make its use practical. The data from piglets showed good agreement between 82Br and 36Cl dilution volumes calculated from 4-hr plasma samples (356 +/- 14 ml/kg and 347 +2- 12 ml/kg; P greater than 0.1) and between 82Br dilution volumes calculated from urine 82Br:36Cl and plasma 82Br (360 +/- 31 ml/kg and 356 +/- 14 ml/kg; P greater than 0.1). Extracellular water volume can be estimated in both adult and young animals using the Br dilution volume calculated from urine samples. It requires (i) two urine collections: one before and one 4 to 8 hr after administration of Br; (ii) a measurement or estimate of plasma Cl concentration; and (iii) a correction factor that describes the relationship of the ratio of Br to Cl in urine to that ratio in plasma.     

Low-volume,high-sensitivity assay for cadmium in blood and urine using conventional atomic absorption spectrophotometry

An assay for cadmium in whole blood and urine using deuterium background-correction electrothermal atomic absorption spectroscopy (D(2)-ETAAS) was developed. Cadmium (in a 1- to 2-ml sample) was bound to 15 mg anion-exchange resin, interfering ions were removed in a 2-ml Bio-Spin column, and cadmium was extracted into 100 microl 1M nitric acid for analysis. Cadmium in the sample extract was concentrated 7-fold for blood and 10-fold for urine over the starting material. These steps produced cadmium atomic absorption traces with high signal to background ratios and allowed analysis against aqueous standards. At approximately 0.1 ng Cd/ml, mean intra- and interassay coefficients of variation were 11-12%. Cadmium recovery for 0.1 to 0.6 ng added cadmium was 107+/-4% for blood and 94+/-4% for urine (mean+/-SE, n=3). The mean detection limit (mean + 3 x SD of blank) was 0.008 ng/ml for blood and 0.003 ng/ml for urine. Samples from "unexposed" animals including humans ranged from 0.051+/-0.000 to 0.229+/-0.035 ng/ml. Values were approximately 10-fold lower than those obtained by the method of Stoeppler and Brandt using Zeeman background-correction ETAAS. This new high-sensitivity, low-volume assay will be useful for epidemiological studies, even those involving children, and will provide a means to help determine the contribution of cadmium to disease incidence in the general population.     

Stability studies of selected doping agents in urine: caffeine

The stability of caffeine in urine samples has been studied. A high-performance liquid chromatography (HPLC) method for the quantification of caffeine in urine samples was validated for that purpose. The method consists of a liquid-liquid extraction at alkaline pH with chloroform-2-propanol (9:1, v/v) with a salting out effect. 7-Ethyltheophylline was used as internal standard (ISTD). Analyses were performed with an Ultrasphere ODS C18 column using water/acetonitrile (90:10, v/v) as a mobile phase at a flow rate of 1 ml/min. Ultraviolet absorption at 280 nm was monitored. Extraction recoveries for caffeine and 7-ethyltheophylline were 81.4+/-6.0 and 87.3+/-5.7%, respectively. The calibration curves were demonstrated to be linear in the working range of 6-30 microg/ml (r2>0.990). The limit of detection and the limit of quantitation were estimated as 0.7 and 2.0 microg/ml, respectively. Precisions in the range of 1.5-9.2 and 4.1-5.8% were obtained in intra- and inter-assay studies, respectively, using control samples containing 10, 14 and 26 microg/ml of caffeine. Accuracies ranging from 2.9 to 7.4% for intra-assay experiments, and from 3.9 to 5.4% in inter-assay studies were obtained. Stability of caffeine in urine samples was evaluated after long- and short-term storage at different temperature conditions. The batches of spiked urine were submitted to sterilization by filtration. No adsorption of the analyte on filters was observed. Before starting stability studies, batches of reference materials were tested for homogeneity. For long-term stability testing, caffeine concentration in freeze-dried urine stored at 4 degrees C and in liquid urine samples stored at 4, -20, -40 and -80 degrees C was determined at several time intervals for 18 months. For short-term stability testing, caffeine concentration was evaluated in liquid urine stored at 37 degrees C for 7 days. The effect of repeated freezing (at -20 degrees C) and thawing was also studied for up to three cycles. The stability of caffeine was also evaluated in non-sterile samples stored at -20 degrees C for 18 months. No significant loss of the compound was observed at any of the investigated conditions.