Effects of a 60-day confinement on the blood pressure, hormonal responses and body fluids of a mixed crew

During the EXEMSI experiment, an international crew of 4 subjects (1 woman and 3 men) was confined for 60 days in a normobaric diving chamber (with 1060 mbar atmospheric pressure) to simulate life in a space station and to assess the effects of confinement on psychological and physiological factors. Blood pressure and blood volume regulating hormones (atrial natriuretic peptide, renin, aldosterone) and urine data (24-h urine outputs, ionogram) were measured before (BDC: baseline data collection), during (D: day) and after (R: recovery) confinement. We also measured energy expenditure and total body water, 14 days before, and after 27 days of confinement, by the double-labeled water method. We found a marked increase in 24-h urine output during most of the confinement in the men and the woman. Body weight (-1.8 +/- 0.9 kg) and energy expenditure (-1064 +/- 143 kcal/d, p<0.01) decreased in the 3 men. The total body water (TBW) decreased by 1.5 +/- 1.2 l in the men. Stress was not indicated by plasma and urine catecholamines but plasma growth hormone was elevated on D2 (p<0.01 vs. BDC) in the men. This study shows that confinement conditions can modify body fluid (increases in 24-h urine outputs and TBW changes) and energetic metabolisms.     

Effects of Chromium(III) Picolinate on Cortisol and DHEAs Secretion in H295R Human Adrenocortical Cells

Dietary chromium(III) picolinate (CrPic) effects on circulating steroid hormones have been reported in various experimental animals. However, direct effects of CrPic on adrenocortical steroidogenesis are uncertain. Therefore, the objective was to determine the effects of CrPic on cortisol and dehydroepiandrosterone sulfate (DHEAs) secretion from H295R cells. In experiment 1, a 24-h exposure to CrPic (0 to 200 μM) had both linear (p < 0.001) and quadratic (p < 0.001) effects on cortisol secretion from forskolin-stimulated cells with the highest cortisol secretion at 0.1 μM of CrPic and the lowest at 200 μM of CrPic. In experiment 2, a 48-h exposure to CrPic (200 μM) decreased cortisol (p < 0.07) release from forskolin-stimulated cells during a 24-h collection period. In experiment 3, a 48-h exposure to CrPic (100 μM) decreased cortisol (p < 0.05) and DHEAs (p < 0.01) from forskolin-stimulated cells during a 24-h sampling period. In experiment 4, a 24-h exposure to forskolin followed by a 24-h exposure to both forskolin and CrPic (100 and 200 μM) decreased both cortisol and DHEAs secretion (p < 0.01). This study suggests that at high concentrations, CrPic inhibits aspects of steroidogenesis in agonist-stimulated adrenocortical cells.     

Urinary Exosomal microRNA-451-5p Is a Potential Early Biomarker of Diabetic Nephropathy in Rats

Non-invasive renal signatures can help in serial monitoring of diabetic patients. We tested whether urinary exosomal (UE) microRNA (miR) analysis could non-invasively predict renal pathology in diabetic rats during the course of diabetes. Diabetes mellitus (DM) was induced in male Wistar rats by a single intraperitoneal injection of streptozotocin (STZ, 50 mg/kg body weight). Non-diabetic control (CTRL) rats were injected with vehicle. Insulin (INS) treatment (5U/d, s.c.) was provided to 50% of the DM rats. Urine samples were collected at weeks 3, 6, and 9 following injections and UE prepared. An increase in miR-451-5p and miR-16, observed by pilot small RNA sequencing of UE RNA, was confirmed by quantitative real-time polymerase chain reaction (qPCR) and selected for further study. Subsets of rats were euthanized after 3, 6, and 9 weeks of diabetes for renal pathology analysis, including determination of the tubulointerstitial fibrotic index (TFI) and glomerulosclerotic index (GI) scores. qPCR showed a substantial rise in miR-451-5p in UE from DM rats during the course of diabetes, with a significant rise (median fold change >1000) between 3 and 6 weeks. Moreover, UE miR-451-5p at 6 weeks predicted urine albumin at 9 weeks (r = 0.76). A delayed but significant rise was also observed for miR-16. In contrast, mean urine albumin only increased 21% between 3 and 6 weeks (non-significant rise), and renal TFI and GI were unchanged till 9 weeks. Renal expression of miR-451-5p and miR-16 (at 10 weeks) did not correlate with urine levels, and moreover, was negatively associated with indices of renal pathology (r≥-0.70, p = 0.005 for TFI and r≥-0.6, p≤0.02 for GI). Overall, a relative elevation in renal miR-451-5p and miR-16 in diabetes appeared protective against diabetes-induced kidney fibrosis; while UE miR-451-5p may hold prognostic value as an early and sensitive non-invasive indicator of renal disease.     

Bone resorption is induced on the second day of bed rest: results of a controlled crossover trial.

The aim of the study was to analyze the kinetics of short-term changes in bone turnover. We studied in a randomized crossover design the effects of 6 days of bed rest on eight healthy male subjects (mean body wt: 70.1 +/- 5.7 kg; mean age: 25.5 +/- 2.9 yr). The metabolic ward period was divided into three parts: 4 ambulatory days, 6 days of either bed rest or non-bed rest periods, and 1 recovery day. The diet was identical in both bed rest and non-bed rest phases. Continuous urine collection started on the first day in the metabolic ward to analyze excretion of bone resorption markers, namely C-telopeptide (CTX) and N-telopeptide (NTX), creatinine, urea, and 3-methylhistidine. On the second ambulatory day and on the fifth day of bed rest or during the non-bed rest phase, blood was drawn to analyze bone formation markers and amino acid concentrations. Urinary calcium excretion was increased as early as the first day of bed rest (P < 0.01). CTX and NTX excretion stayed unchanged during the first 24 h of bed rest compared with the non-bed rest period. However, already on the second day, both resorption markers had increased significantly. NTX excretion increased by 28.7 +/- 14.0% (P < 0.01), whereas CTX excretion rose by 17.8 +/- 8.3% (P < 0.001). Creatinine, urea, and 3-methylhistidine excretion did not change. We conclude that 24 h of bed rest are sufficient to induce a significant rise in osteoclast activity in healthy subjects.     

Comparison of the urinary excretion time courses of pyrene-1,6-dione, pyrene-1,8-dione and 1-hydroxypyrene in rats intravenously exposed to pyrene.

The urinary excretion time courses of pyrene-1,6-dione (P16D), pyrene-1,8-dione (P18D) and 1-hydroxypyrene (1-OHP) were compared in Sprague-Dawley and Wistar rats. Groups of five male rats, of about 200 g of body weight, were injected intravenously with 0.05, 0.5, 5 and 50 micromol pyrene kg-1 of body weight. Urine was collected at 2, 4, 6, 8, 10, 12, 18, 24, 30, 42 and 48 h post-dosing. Pyrene metabolites were measured by high-performance liquid chromatography (HPLC)/fluorescence after enzymatic hydrolysis of the glucurono- and sulfo-conjugates, extraction on Sep-Pak C18 cartridges and, for the analysis of dione metabolites, derivatization to stable diacetoxypyrene molecules. Over the 48-h sampling period, on average 17.4-25.6% of the injected pyrene was excreted overall as P16D, 6.4-8.8% as P18D and 0.6-0.8% as 1-OHP in the urine of Sprague-Dawley rats. By comparison, on average 10.3-14.7% of the intravenous pyrene dose was recovered as P16D, 4.8-6.4% as P18D and 0.3-0.4% as 1-OHP in the urine of Wistar rats. In both strains of rats there was no clear effect of the dose on the 0-48-h cumulative urinary excretion of P18D and 1-OHP over the entire dose range, while the percentage of dose recovered overall as P16D in urine at the highest dose (50 micromol kg-1) was statistically lower than at the other doses. The 0-48-h cumulative percentage of pyrene dose excreted as metabolites in the urine of Sprague-Dawley rats was also significantly higher than in Wistar rats (p<0.01) exposed under identical conditions. As for the urinary excretion-time courses of the different metabolites, for a given dose and strain of rats, excretion curves of P16D, P18D and 1-OHP generally evolved in parallel. There was also no clear effect of the dose on the excretion rate, thus half-life, of pyrene metabolites, except for P16D in Sprague-Dawley rats at the highest dose where elimination tended to be slower compared with the other doses (p<0.01). The average first-order elimination half-life of P16D, P18D and 1-OHP was 4.0, 5.7 and 4.1 h, respectively, in Sprague-Dawley rats, and 5.1, 6.1 and 5.1 h, respectively, in Wistar rats (all doses combined but excluding the highest dose for P16D). This study showed the relative importance of metabolic pathways leading to diones compared with 1-OHP. These dioxygenated metabolites appear to be interesting biomarkers of pyrene exposure at environmentally and occupationally relevant doses. Their adequacy as biomarkers of human exposure has yet to be confirmed.     

Urine volume dependency of specific dehydroepiandrosterone (DHEA) and cortisol metabolites in healthy children

Urine volume should be considered as a confounder when using urinary free cortisol (UFF) and cortisone (UFE) to assess glucocorticoid (GC) status. We aimed to examine whether adrenal androgen (AA) metabolites may be also affected by urine volume in healthy children. To compare the flow dependence of GC and AA metabolites, specific GC metabolites were examined. In 24-h urine samples of 120 (60 boys) healthy children (4-10 yr), steroid profiles were determined by GC-MS analysis, UFF and UFE by radioimmunoassay. To assess daily AA and GC secretion rates, 7 quantitatively most important AA (∑C19) and GC (∑C21) metabolites were summed. Sum of DHEA and its 16α-hydroxylated metabolites were denoted as DHEA&M. Association of urine volume with AA (∑C19, DHEA&M, DHEA, 16α-hydroxy-DHEA, 3β,16α,17β-androstenetriol) and GC (∑C21, UFF, UFE, 6β-hydroxycortisol, 20α-dihydrocortisol) were examined in linear regression models. Among the examined AA metabolites, 16α-hydroxy-DHEA (β=0.56, p<0.0001) and DHEA (β=0.43, p=0.05) showed relatively strong association with urine volume. A trend was seen for ∑C19 (β=0.23, p=0.08), but not for DHEA&M (p>0.1). Regarding GC metabolites, urine volume showed a stronger association with cortisol's direct metabolites, i.e., cortisone, 6β-hydroxycortisol and 20α-dihydrocortisol (β=0.4-0.6, p<0.01) than with cortisol itself (β=0.28, p<0.05). ∑C21 was not associated with urine volume. In conclusion, like UFF and UFE, renal excretion of DHEA, 16α-hydroxy-DHEA, 6β-hydroxycortisol, and 20α-dihydrocortisol may also depend on urine volume. The intrarenal production of the latter three and cortisone might explain their relative strong water-flow-dependency. Total AA or GC secretion marker appears not to be relevantly confounded by urine volume.     

Inactivation of Ascaris Eggs in Source-Separated Urine and Feces by Ammonia at Ambient Temperatures

Sustainable management of toilet waste must prevent disease transmission but allow reuse of plant nutrients. Inactivation of uterus-derived Ascaris suum eggs was studied in relation to ammonia in source-separated urine without additives and in human feces to which urea had been added, in order to evaluate ammonia-based sanitation for production of safe fertilizers from human excreta. Urine was used concentrated or diluted 1:1 and 1:3 with tap water at 4, 14, 24, and 34°C. Fecal material, with and without ash, was treated with 1% or 2% (wt/wt) urea at 24 and 34°C. At 34°C eggs were inactivated in less than 10 days in urine and in amended feces. At 24°C only feces with 2% (wt/wt) urea or 1% (wt/wt) urea at high pH (10) inactivated all eggs within 1 month, and no inactivation was observed after 75 days in urine diluted 1:3 (18 ± 11 mM NH₃). At temperatures of ≥24°C, NH₃ proved to be an efficient sanitizing agent in urine and feces at concentrations of ≥60 mM. Treating fecal material at 34°C can give a 6-log₁₀ egg inactivation within 1 month, whereas at 24°C 6 months of treatment is necessary for the same level of egg inactivation. At temperatures of 14°C and below, inactivation rates were low, with viable eggs after 6 months even in concentrated urine.     

Comparison of the urinary excretion time courses of pyrene-1,6-dione,pyrene-1,8-dione and 1-hydroxypyrene in rats intravenously exposed to pyrene

Abstract

The urinary excretion time courses of pyrene-1,6-dione (P16D), pyrene-1,8-dione (P18D) and 1-hydroxypyrene (1-OHP) were compared in Sprague–Dawley and Wistar rats. Groups of five male rats, of about 200 g of body weight, were injected intravenously with 0.05, 0.5, 5 and 50 µmol pyrene kg^?1 of body weight. Urine was collected at 2, 4, 6, 8, 10, 12, 18, 24, 30, 42 and 48 h post-dosing. Pyrene metabolites were measured by high-performance liquid chromatography (HPLC)/fluorescence after enzymatic hydrolysis of the glucurono- and sulfo-conjugates, extraction on Sep-Pak C18 cartridges and, for the analysis of dione metabolites, derivatization to stable diacetoxypyrene molecules. Over the 48-h sampling period, on average 17.4–25.6% of the injected pyrene was excreted overall as P16D, 6.4–8.8% as P18D and 0.6–0.8% as 1-OHP in the urine of Sprague–Dawley rats. By comparison, on average 10.3–14.7% of the intravenous pyrene dose was recovered as P16D, 4.8–6.4% as P18D and 0.3–0.4% as 1-OHP in the urine of Wistar rats. In both strains of rats there was no clear effect of the dose on the 0–48-h cumulative urinary excretion of P18D and 1-OHP over the entire dose range, while the percentage of dose recovered overall as P16D in urine at the highest dose (50 µmol kg^?1) was statistically lower than at the other doses. The 0–48-h cumulative percentage of pyrene dose excreted as metabolites in the urine of Sprague–Dawley rats was also significantly higher than in Wistar rats (p<0.01) exposed under identical conditions. As for the urinary excretion-time courses of the different metabolites, for a given dose and strain of rats, excretion curves of P16D, P18D and 1-OHP generally evolved in parallel. There was also no clear effect of the dose on the excretion rate, thus half-life, of pyrene metabolites, except for P16D in Sprague–Dawley rats at the highest dose where elimination tended to be slower compared with the other doses (p<0.01). The average first-order elimination half-life of P16D, P18D and 1-OHP was 4.0, 5.7 and 4.1 h, respectively, in Sprague–Dawley rats, and 5.1, 6.1 and 5.1 h, respectively, in Wistar rats (all doses combined but excluding the highest dose for P16D). This study showed the relative importance of metabolic pathways leading to diones compared with 1-OHP. These dioxygenated metabolites appear to be interesting biomarkers of pyrene exposure at environmentally and occupationally relevant doses. Their adequacy as biomarkers of human exposure has yet to be confirmed.     

Urinary zinc excretion and zinc status of patients with Β-thalassemia major

In this study, zinc status and urinary zinc excretion with and without desferrioxamine (DFO) infusion and the relationship between urinary zinc excretion and renal tubular dysfunction in thalassemia major (TM) patients were investigated. Forty TM patients were given four DFO infusions on alternate days over a 1-wk period prior to the transfusion. On each day that DFO was given, a 24-h urine collection initiated. DFO was omitted for 1-wk before the following transfusion and during the period four 24-h urine collections were performed. Twenty healthy children provided 24-h urine collection as controls. Blood samples were taken on each of two consecutive transfusion days of the patients and from the controls. Urinary zinc excretion was measured and plasma and red blood cell (RBC) zinc analysis were performed by inductively coupled plasma-atomic emission spectrophotometry. UrinaryN-acetyl-Β-D-glucosaminidase (NAG) activity and creatinine were determined in morning urine specimens. The mean plasma zinc concentration was significantly lower in the patients not given DFO compared to the values of the patients given DFO and the control group. The mean RBC zinc concentration (Μmol/g Hb) in the patients (with and without DFO) and the control group were similar. Urinary zinc excretion was significantly higher in the patients receiving DFO compared to the control group, whereas urinary zinc excretion in the patients not given DFO was not different from the controls. Urinary NAG indices (U/g Cr) were significantly higher in the patients compared to controls. Urinary zinc excretion was correlated with the urinary NAG indices.     

Standardization of factors that influence human urine metabolomics

In nutritional metabolomics a large inter- and intra-subject variability exists, and thus, it becomes important to limit the variance introduced by external factors. In a composite controlled study with full provision of all food for the standardized intervention, human urinary metabolite profiles were investigated for different factors, such as handling of urine collections, diet standardization, diet culture, cohabitation and gender. In study A, 8 healthy subjects (4 men; 4 women) collected 24-h urine, splitting each void into two specimens stored either at 4°C or at room temperature. In study B, 16 healthy subjects (7 men; 9 women) collected 24-h urine for three days while being on a standardized diet. Samples were analyzed by ¹H NMR and chemometrics. The NMR profiles indicated the presence of metabolites presumably originating from bacterial contamination in 3 out of 16 sample collections stored at room temperature. On the contrary, no changes in the NMR profiles due to contamination occurred in the 24-h urine samples stored at 4°C. The study also showed a trend towards a reduced inter- and intra-individual variation during 3 days of diet standardization. In study A, the urine metabolome showed a clear effect of diet culture and cohabitation, but these effects significantly attenuated after diet standardization (study B). Besides, gender-specific differences were found in both studies. Our results emphasize that best practice for any metabolomic study is a standardized, chilled sample collection procedure, and recommend that diet standardization is performed prior to dietary interventions in order to reduce intra- and inter-subject variability.     

Free fatty acid-induced peripheral insulin resistance augments splanchnic glucose uptake in healthy humans

To investigate the effect of elevated plasma free fatty acid (FFA) concentrations on splanchnic glucose uptake (SGU), we measured SGU in nine healthy subjects (age, 44 +/- 4 yr; body mass index, 27.4 +/- 1.2 kg/m(2); fasting plasma glucose, 5.2 +/- 0.1 mmol/l) during an Intralipid-heparin (LIP) infusion and during a saline (Sal) infusion. SGU was estimated by the oral glucose load (OGL)-insulin clamp method: subjects received a 7-h euglycemic insulin (100 mU x m(-2) x min(-1)) clamp, and a 75-g OGL was ingested 3 h after the insulin clamp was started. After glucose ingestion, the steady-state glucose infusion rate (GIR) during the insulin clamp was decreased to maintain euglycemia. SGU was calculated by subtracting the integrated decrease in GIR during the period after glucose ingestion from the ingested glucose load. [3-(3)H]glucose was infused during the initial 3 h of the insulin clamp to determine rates of endogenous glucose production (EGP) and glucose disappearance (R(d)). During the 3-h euglycemic insulin clamp before glucose ingestion, R(d) was decreased (8.8 +/- 0.5 vs. 7.6 +/- 0.5 mg x kg(-1) x min(-1), P < 0.01), and suppression of EGP was impaired (0.2 +/- 0.04 vs. 0.07 +/- 0.03 mg x kg(-1) x min(-1), P < 0.01). During the 4-h period after glucose ingestion, SGU was significantly increased during the LIP vs. Sal infusion study (30 +/- 2 vs. 20 +/- 2%, P < 0.005). In conclusion, an elevation in plasma FFA concentration impairs whole body glucose R(d) and insulin-mediated suppression of EGP in healthy subjects but augments SGU.     

Degradation of quercetin-3-glucoside in gnotobiotic rats associated with human intestinal bacteria

AIM: The two bacterial species, Eubacterium ramulus and Enterococcus casseliflavus, which had previously been isolated from human faeces using the flavonoid quercetin-3-glucoside as the growth substrate, were tested for their ability to utilize this compound in vivo. METHODS AND RESULTS: Germ-free rats were associated with Eu. ramulus and subsequently with Ent. casseliflavus and vice versa. Identification and enumeration of the bacterial cell counts in faeces and intestinal contents were performed by whole cell fluorescence in situ hybridization. Eubacterium ramulus and Ent. casseliflavus occurred in caecal and colonic contents at cell counts of up to 10(10) g(-1) dry weight. In the jejunum, only Ent. casseliflavus was found (10(9) g(-1) dry weight). Upon oral administration of 32 micromol quercetin-3-glucoside, quercetin was detected in the faeces and urine of germ-free rats (2.2 x 10(-1)-8.1 x 10(-1) micromol 24-h(-1) faeces collection and 1.0 x 10(-2)-2.8 x 10(-1) micromol 24-h(-1) urine collection, respectively) and of rats monoassociated with Ent. casseliflavus (7.9 x 10(-1)-2.7 micromol 24-h(-1) faeces and 1.0 x 10(-1)-5.9 x 10(-1) micromol 24-h(-1) urine, respectively). In contrast, the faeces and urine of rats associated with Eu. ramulus contained 3,4-dihydroxyphenylacetic acid (4.7 x 10(-2)-3.6 micromol 24-h(-1) faeces and 2.4 x 10(-2)-1.0 micromol 24-h(-1) urine, respectively) but only low, or undetectable, concentrations of faecal quercetin (up to 9.3 x 10(-2) micromol 24-h(-1) faeces; detection limit 2.5 x 10(-2) micromol). Urinary quercetin concentrations varied markedly from undetectable amounts up to 1.0 micromol 24-h(-1) urine (detection limit 1.0 x 10(-2) micromol). Isorhamnetin was found in the urine of all animals independent of their bacterial status. There were no significant differences between the groups (2.0 x 10(-2)-2.8 x 10(-1) micromol 24-h(-1) urine). In complete intestinal tissues of animals, associated with both species, quercetin-3-glucoside and its metabolites were detected by a more sensitive and selective method at concentrations that were two to three orders of magnitude lower than in faeces or urine. CONCLUSIONS: These results indicate that Eu. ramulus may be a key organism for the bacterial transformation of flavonoids in the gut.     

AT1a receptor signaling is required for basal and water deprivation-induced urine concentration in AT1a receptor-deficient mice

It is well recognized that ANG II interacts with arginine vasopressin (AVP) to regulate water reabsorption and urine concentration in the kidney. The present study used ANG II type 1a (AT(1a)) receptor-deficient (Agtr1a(-/-)) mice to test the hypothesis that AT(1a) receptor signaling is required for basal and water deprivation-induced urine concentration in the renal medulla. Eight groups of wild-type (WT) and Agtr1a(-/-) mice were treated with or without 24-h water deprivation and 1-desamino-8-d-AVP (DDAVP; 100 ng/h ip) for 2 wk or with losartan (10 mg/kg ip) during water deprivation. Under basal conditions, Agtr1a(-/-) mice had lower systolic blood pressure (P < 0.01), greater than threefold higher 24-h urine excretion (WT mice: 1.3 ± 0.1 ml vs. Agtr1a(-/-) mice: 5.9 ± 0.7 ml, P < 0.01), and markedly decreased urine osmolality (WT mice: 1,834 ± 86 mosM/kg vs. Agtr1a(-/-) mice: 843 ± 170 mosM/kg, P < 0.01), without significant changes in 24-h urinary Na(+) excretion. These responses in Agtr1a(-/-) mice were associated with lower basal plasma AVP (WT mice: 105 ± 8 pg/ml vs. Agtr1a(-/-) mice: 67 ± 6 pg/ml, P < 0.01) and decreases in total lysate and membrane aquaporin-2 (AQP2; 48.6 ± 7% of WT mice, P < 0.001) and adenylyl cyclase isoform III (55.6 ± 8% of WT mice, P < 0.01) proteins. Although 24-h water deprivation increased plasma AVP to the same levels in both strains, 24-h urine excretion was still higher, whereas urine osmolality remained lower, in Agtr1a(-/-) mice (P < 0.01). Water deprivation increased total lysate AQP2 proteins in the inner medulla but had no effect on adenylyl cyclase III, phosphorylated MAPK ERK1/2, and membrane AQP2 proteins in Agtr1a(-/-) mice. Furthermore, infusion of DDAVP for 2 wk was unable to correct the urine-concentrating defects in Agtr1a(-/-) mice. These results demonstrate that AT(1a) receptor-mediated ANG II signaling is required to maintain tonic AVP release and regulate V(2) receptor-mediated responses to water deprivation in the inner medulla.     

Hydration status affects urea transport across rat urothelia

Although mammalian urinary tract epithelium (urothelium) is generally considered impermeable to water and solutes, recent data suggest that urine constituents may be reabsorbed during urinary tract transit and storage. To study water and solute transport across the urothelium in an in vivo rat model, we instilled urine (obtained during various rat hydration conditions) into isolated in situ rat bladders and, after a 1-h dwell, retrieved the urine and measured the differences in urine volume and concentration and total quantity of urine urea nitrogen and creatinine between instilled and retrieved urine in rat groups differing by hydration status. Although urine volume did not change >1.9% in any group, concentration (and quantity) of urine urea nitrogen in retrieved urine fell significantly (indicating reabsorption of urea across bladder urothelia), by a mean of 18% (489 mg/dl, from an instilled 2,658 mg/dl) in rats receiving ad libitum water and by a mean of 39% (2,544 mg/dl, from an instilled 6,204 mg/dl) in water-deprived rats, but did not change (an increase of 15 mg/dl, P = not significant, from an instilled 300 mg/dl) in a water-loaded rat group. Two separate factors affected urea nitrogen reabsorption rates, a urinary factor related to hydration status, likely the concentration of urea nitrogen in the instilled urine, and a bladder factor(s), also dependent on the animal's state of hydration. Urine creatinine was also absorbed during the bladder dwell, and hydration group effects on the concentration and quantity of creatinine reabsorbed were qualitatively similar to the hydration group effect on urea transport. These findings support the notion(s) that urinary constituents may undergo transport across urinary tract epithelia, that such transport may be physiologically regulated, and that urine is modified during transit and storage through the urinary tract.     

Effects of water dilution, housing, and food on rat urine collected from the metabolism cage

The objective of the study reported here was to investigate three factors that may affect the amounts of water consumed and urine excreted by a rat in the metabolism cage: water dilution, housing, and food. Young F344/N rats (eight per group) were used for all experiments. Food was withheld from rats before each 16-h urine collection, then rats were transferred into a metabolism cage. For trial A (water dilution), urine was collected from rats supplied with dyed water (0.05%, vol/vol). This was repeated three times over a 2-week period. Dye in water or urine was quantified, using a spectrophotometer. For trial B (housing), rats were individually housed in wire cages for 3 weeks before the first urine collection. Then they were group housed in the solid-bottom cage (four per cage). After 2 weeks of acclimation, urine collection was repeated. For trial C (food), one group of rats was provided with food, the other was not, during urine collection. About 8% of urine samples of small volume (< or = 3 ml) from trial A were contaminated with drinking water up to 13% of volume. The average urine volume associated with individual housing was approximately twice as large as that associated with group housing. When food was provided during urine collection, rats consumed similar amounts of water but excreted significantly smaller amounts of urine than did rats without food. It was concluded that water dilution of a urine sample from a sipper bottle is relatively small; rats individually housed in wire caging before urine collection can consume and excrete a larger quantity of water, compared with rats group housed in solid-bottom cages; and highly variable urine volumes are, in part, associated with lack of access to food during urine collection.     

Manipulation of citrulline availability in humans

To determine whether circulating citrulline can be manipulated in vivo in humans, and, if so, whether citrulline availability affects the levels of related amino acids, nitric oxide, urinary citrulline, and urea nitrogen, 10 healthy volunteers were studied on 3 separate days: 1) under baseline conditions; 2) after a 24-h treatment with phenylbutyrate (0.36 g.kg(-1).day(-1)), a glutamine "trapping" agent; and 3) during oral L-citrulline supplementation (0.18 g.kg(-1).day(-1)), in randomized order. Plasma, erythrocyte (RBC), and urinary citrulline concentrations were determined by gas chromatography-mass spectrometry at 3-h intervals between 1100 and 2000 on each study day. Regardless of treatment, RBC citrulline was lower than plasma citrulline, with an RBC-to-plasma ratio of 0.60 +/- 0.04, and urinary citrulline excretion accounted for <1% of the citrulline load filtered by kidney. Phenylbutyrate induced an approximately 7% drop in plasma glutamine (P = 0.013), and 18 +/- 14% (P < 0.0001) and 19 +/- 17% (P < 0.01) declines in plasma and urine citrulline, respectively, with no alteration in RBC citrulline. Oral L-citrulline administration was associated with 1) a rise in plasma, urine, and RBC citrulline (39 +/- 4 vs. 225 +/- 44 micromol/l, 0.9 +/- 0.3 vs. 6.2 +/- 3.8 micromol/mmol creatinine, and 23 +/- 1 vs. 52 +/- 9 micromol/l, respectively); and 2) a doubling in plasma arginine level, without altering blood urea or urinary urea nitrogen excretion, and thus enhanced nitrogen balance. We conclude that 1) depletion of glutamine, the main precursor of citrulline, depletes plasma citrulline; 2) oral citrulline can be used to enhance systemic citrulline and arginine availability, because citrulline is bioavailable and very little citrulline is lost in urine; and 3) further studies are warranted to determine the mechanisms by which citrulline may enhance nitrogen balance in vivo in humans.     

Magnesium,Calcium, and Trace Elements Excretion in 24-h Urine

Urine is a clinical specimen often used in medical diagnostics for monitoring of elements concentrations and kidneys function. We determined the contents of magnesium (Mg), calcium (Ca), zinc (Zn), copper (Cu), iron (Fe), lead (Pb), and cadmium (Cd) in 74 samples of 24-h urine (from 46 women and 28 men). The measurements were realized by the atomic absorption spectrometry (AAS) with atomization in the flame (FAAS) and in the graphite furnace (GFAAS). The received results were the subject of statistical analysis including the sex and age of volunteers. Moreover, correlations between the elements and the relationships between age and amounts of excreted elements with urine were tested. We found the statistically significant higher content of Zn in men’s urine than in women^’s one. Moreover, both adult women and men (>18 years) excreted much more Ca in urine in comparison to young subjects. Only in case of Pb the significant positive correlation between its amount in 24-h urine of all donors and age was stated. The correlation analysis has shown the significant positive relationships between Ca–Mg, Ca–Fe, Mg–Fe, Cu–Fe, Cu–Cd, Fe–Cd, and Pb–Cd in total samples of urine. Basing on our results, we concluded that the gender and age of donors may impact on the elemental status of 24-h urine.     

Pathophysiological changes in urine and blood from calves experimentally infected with Sarcocystit cruzi.

Two 8-week experiments were conducted to determine the relationships between nutritional stress and pathophysiological changes in male Holstein calves infected with Sarcocystis cruzi. Calves were infected by oral inoculation with 200 000 S. cruzi sporocysts. In the first experiment weight gain reduction was greatest in inoculated calves during weeks 4 and 5 after inoculation. Feed intake was reduced during the 5th week. Erythrocyte count was reduced during week 5 and haemoglobin was reduced during week 6. The 24-h excretion of urinary and urea nitrogen from the inoculated calves was increased by treatment. In the 2nd experiments, both the feed-restricted and inoculated calves lost weight during weeks 4 and 5; feed intake was lower from week 5 to 8 inclusive. Urine volume from inoculated calves was lower during week 8. Lower urine excretions of sodium and potassium resulted from S. cruzi inoculation. There was a non-significant trend for higher urinary zinc excretion in the inoculated group during week 4. Urine nitrogen excretion from inoculated calves was higher during week 6. The urinary excretion of 3-methylhistidine from the inoculated calves was higher during week 4 and excretion of guanine was higher during weeks 4, 5 and 8. S. cruzi has several specific pathophysiological effects on calves beyond those induced by nutritional stress.     

Changes in body fluids of the cocooning fossorial frog Cyclorana australis in a seasonally dry environment

We investigated changes in the lymph (equivalent to plasma) and urine of the cocooning frog Cyclorana australis during the dry season in monsoonal northern Australia. Frogs in moist soil for two days were fully hydrated (lymph 220 mOsm kg(-1), urine 49 mOsm kg(-1)). From five weeks onwards the soil was dry (matric potential <-8000 kPa). Aestivating frogs at three and five months formed cocoons in shallow (<20 cm) burrows and retained bladder fluid (25-80% of standard mass). After three months, urine but not lymph osmolality was elevated. After five months, lymph (314 mOsm kg(-1)) and urine (294 mOsm kg(-1)) osmolality and urea concentrations were elevated. Urea was a major contributing osmolyte in urine and accumulated in lymph after five months. Lymph sodium concentration did not change with time, whereas potassium increased in urine after five months. Active animals had moderate lymph osmolality (252 mOsm kg(-1)), but urea concentrations remained low. Urine was highly variable in active frogs, suggesting that they tolerate variation in hydration state. Despite prolonged periods in dry soil, osmolality increase in C. australis was not severe. Aestivation in a cocoon facilitates survival in shallow burrows, but such a strategy may only be effective in environments with seasonally reliable rainfall.     

Effect of wilting and the additives,straw, molasses and urea on the fermentation pattern of maize silage

The process of ensiling was studied in fresh maize (15% dry matter (DM)), wilted maize (18 and 24% DM) and maize mixed with 5–20% of wheat straw (18, 25 and 29% DM). Silages with 24% DM were preserved better than those with lower dry matter content. There was a significant change, with time, in pH, titrable acidity, volatile fatty acids, lactic acid, number of lactic acid bacteria, volatile nitrogen and soluble sugars in all the treatments. There was a significant decline in volatile fatty acids (P<0.05) and ammonia (P<0.01) production, and a significant increase in soluble sugar (P<0.01) in silages made after wilting. A significant decline in titrable acidity (P<0.01), volatile fatty acid production (p<0.05) and ammonia nitrogen (P<0.01), and a significant increase in pH (P<0.01) were found in silages of maize mixed with wheat straw. The overall rate of fermentation decreased during the first few days of fermentation in wilted and wheat straw silages, but the final products had characteristics of a good silage. In the second experiment the effect of urea and molasses was studied on wheat straw plus maize (15:85) silage with an initial DM content of 31–34%. Three levels of molasses (0, 3 and 6% of fresh weight) and two levels of urea (0 and 0.5% of fresh weight) were studied. Urea treatment with 3% molasses was found to be the best on the basis of silage characteristics.