Metabolite profiles evaluated, according to sex, do not predict resting energy expenditure and lean body mass in healthy non-obese subjects.

PURPOSE: Differences in resting energy expenditure (REE) between men and women mainly result from sex-related differences in lean body mass (LBM). So far, a little is known about whether REE and LBM are reflected by a distinct human metabolite profile. Therefore, we aimed to identify plasma and urine metabolite patterns that are associated with REE and LBM of healthy subjects. METHODS: We investigated 301 healthy male and female subjects (18-80 years) under standardized conditions in the cross-sectional KarMeN (Karlsruhe Metabolomics and Nutrition) study. REE was determined by indirect calorimetry and LBM by dual X-ray absorptiometry. Fasting blood and 24 h urine samples were analyzed by targeted and non-targeted metabolomics methods using GC × GC-MS, GC-MS, LC-MS, and NMR. Data were evaluated by predictive modeling of combined data using different machine learning algorithms, namely SVM, glmnet, and PLS. RESULTS: When evaluating data of men and women combined, we were able to predict REE and LBM with high accuracy (> 90%). This, however, was a clear effect of sex, which is supported by the high degree of overlap in identified important metabolites for LBM, REE, and sex, respectively. The applied machine learning algorithms did not reveal a metabolite pattern predictive of REE or LBM, when analyzing data for men and women, separately. CONCLUSIONS: We could not identify a sex independent predictive metabolite pattern for REE or LBM. REE and LBM have no impact on plasma and urine metabolite profiles in the KarMeN Study participants. Studies applying metabolomics in healthy humans need to consider sex specific data evaluation.

Plasma beta-carotene is not a suitable biomarker of fruit and vegetable intake in german subjects with a long-term high consumption of fruits and vegetables.

BACKGROUND/OBJECTIVE: beta-Carotene is often used as a marker for the amount of fruit and vegetables consumed, but little is known about plasma beta-carotene concentrations in subjects whose habitual (long-term) diets are characterized by different amounts of foods of plant origin. We compared dietary beta-carotene intake and plasma concentrations in women on habitual diets differing in the consumed amounts of foods of plant origin. METHODS: A comparison of dietary beta-carotene intakes and plasma beta-carotene concentrations in women adhering to an average Western diet (n = 172), wholesome nutrition (following preventive recommendations) (n = 238) or a raw food diet (n = 104). RESULTS: Dietary beta-carotene intake was 5.5, 9.3, 14.7 mg/day for women adhering to an average Western diet, wholesome nutrition and raw food diet, respectively (p < 0.001). Corresponding multivariate adjusted plasma beta-carotene concentrations were 1.07, 1.65, and 1.16 micromol/l, respectively (p < 0.001). Comparable dietary beta-carotene intake resulted in lower multivariate adjusted plasma beta-carotene in women adhering to a raw food diet and average Western diet compared to those on wholesome nutrition (p < 0.001 for all intake groups up to 20 mg/day). The amount of fruit and vegetable intake did not predict plasma beta-carotene levels in women consuming a raw food diet. CONCLUSIONS: Plasma beta-carotene concentrations differed among the diet groups, with highest plasma levels in women adhering to wholesome nutrition. Plasma beta-carotene concentrations may not reflect beta-carotene intake and the amount of fruit and vegetables consumed.

Effects of combined supplementation with B vitamins and antioxidants on plasma levels of asymmetric dimethylarginine (ADMA) in subjects with elevated risk for cardiovascular disease.

Elevated plasma asymmetric dimethylarginine (ADMA) concentrations have been suggested as a potential risk factor for cardiovascular disease (CVD). Studies indicate a linkage between hyperhomocysteinemia, oxidative stress and ADMA metabolism. We tested the hypothesis that combined supplementation of B vitamins and antioxidants reduces ADMA concentrations in subjects with at least two CVD risk factors. A total of 123 men and women (58+/-8.1 years) were randomly assigned to take either a preparation including B vitamins and antioxidants (verum) or placebo for 6 months in a double-blind design. Blood concentrations of ADMA, symmetric dimethylarginine (SDMA), L-arginine, B vitamins, total homocysteine (tHcy), alpha-tocopherol, antioxidant capacity (TEAC), and oxLDL were measured pre- and post-intervention. Treatment with verum significantly decreased tHcy (-2.14 micromol/L; P<0.001) and significantly increased TEAC values (+39.3 microM; P<0.022), but no effect on ADMA was observed. OxLDL was significantly reduced in verum (-7.3 U/L; P=0.001) and placebo (-9.2U/L; P<0.001). At baseline, significant correlations were found only between ADMA and SDMA (r=0.281; P=0.002), L-arginine/ADMA and SDMA (r=-0.294; P<0.001), L-arginine/ADMA and oxLDL (r=-0.281; P=0.016), and L-arginine/ADMA and age (r=-0.231; P=0.010). Our results indicate that combined supplementation of B vitamins and antioxidants is not an adequate strategy to reduce ADMA plasma levels in subjects with elevated CVD risk.

Cloudy apple juice decreases DNA damage, hyperproliferation and aberrant crypt foci development in the distal colon of DMH-initiated rats.

Clear (CleA) and cloudy (CloA) apple juices containing different amounts of analyzed procyanidins and pectin were investigated for preventive effects of colon cancer and underlying molecular mechanisms in F344 rats given intraperitoneal injections of 1,2-dimethylhydrazine (DMH; 20 mg/kg body wt) once a week for 4 weeks. Rats received either water (Cont), CleA or CloA (ad libitum) for 7 weeks starting 1 week before the first DMH injection. CloA inhibited DMH induced genotoxic damage in mucosa cells of the distal colon compared with Cont as investigated by single-cell microgel electrophoresis assay. The mean tail intensity in mucosa cells of DMH-treated controls (Cont/DMH: 6.1+/-0.9%) was significantly reduced by CloA (2.4+/-0.8%; P<0.01) but not by CleA intervention (4.1+/-1.2%; P>0.05). The crypt cell proliferation index induced by DMH (Cont/NaCl: 10.0+/-0.7%; Cont/DMH: 19.9+/-1.0%; P<0.001) was significantly decreased by CleA (15.7+/-0.7%; P<0.001) and CloA intervention (11.9+/-0.4%; P<0.001). CloA but not CleA significantly reduced the number of large aberrant crypt foci (ACF) consisting of more than four aberrant crypts (AC) (Cont/DMH: 37.4+/-5.4; CleA/DMH: 32.8+/-4.4, P>0.05; CloA/DMH: 18.8+/-2.5 ACF; P<0.05) and the overall mean ACF size in the distal colon (Cont/DMH: 2.31+/-0.09; CleA/DMH: 2.27+/-0.05; CloA/DMH: 2.04+/-0.03 AC/ACF; P<0.05). After treatment with DMH and/or apple juices there were no changes in transcript levels of colonic cyclooxygenase isoforms (COX-1, COX-2) or glutathione-associated enzymes (GST-M2, gamma-GCS, GST-P), the splenocyte natural killer cell activity and plasma antioxidant status. However, CloA but not CleA prevented the DMH-induced reduction of splenocyte CD4/CD8 (T-helper cells to cytotoxic lymphocytes) ratio. Since both formulations contained comparable concentrations and types of monomeric polyphenols, complex polyphenols or non-polyphenolic compounds, such as pectin might be responsible for the stronger cancer-preventive effect by CloA.

Daily moderate amounts of red wine or alcohol have no effect on the immune system of healthy men.

OBJECTIVE: To investigate whether the daily intake of red wine (RW) at a dose which inversely correlates with cardiovascular disease (CVD) risk modulates immune functions in healthy men. DESIGN: Randomized single-blind trial with four intervention periods. SETTING: The Institute of Nutritional Physiology, Federal Research Centre for Nutrition, Karlsruhe, Germany. SUBJECTS: A total of 24 healthy males with moderate alcohol consumption patterns were recruited and all completed the study. INTERVENTION: Participants consumed 500 ml of RW (12% ethanol (ETOH)) or 500 ml of a 12% ETOH dilution per day for a period of 2 weeks. To control the potential effects of RW polyphenols, accordingly 500 ml/day of dealcoholized red wine (DRW) and of red grape juice (RGJ) were given. The following immune parameters were measured before beverage consumption and at 1 and 2 weeks following beverage consumption: phagocytic activity of neutrophils and monocytes, production of tumor necrosis factor-alpha (TNFalpha), interleukin-2 and -4, transforming growth factor-beta, TNFalpha mRNA, lymphocyte proliferation, lytic activity of natural killer cells, and percentage of apoptotic lymphocytes. RESULTS: Consumption of a moderate volume of alcohol with RW and with a 12% ETOH dilution had no effect on immune functions in healthy males. Consumption of polyphenol-rich beverages (DRW and RGJ) did not affect immunity-related parameters. CONCLUSIONS: Daily moderate consumption of alcohol and of RW for 2 weeks at doses which inversely correlate with CVD risk has no adverse effects on human immune cell functions. Polyphenol-rich beverages such as RGJ and DRW further do not suppress immune responses in healthy men.

Malvidin-3-glucoside bioavailability in humans after ingestion of red wine, dealcoholized red wine and red grape juice.

BACKGROUND & AIMS: Dietary polyphenols, including anthocyanins, are suggested to be involved in the protective effects of red wine against cardiovascular diseases. Very little data are available concerning the bioavailability of anthocyanins, major sources of red pigmentation in red wine. The aim of this study was to compare changes in plasma malvidin-3-glucoside (M-3-G), a red wine anthocyanin, and its urinary excretion after ingestion of red wine, dealcoholized red wine and red grape juice. DESIGN: Six healthy male subjects were studied in a randomized cross over setting in a human nutrition research unit under controlled conditions. All subject consumed 500 mL of each beverage on separate days providing the following M-3-G quantities: red wine 68 mg, dealcoholized red wine 58 mg, and red grape juice 117 mg. M-3-G was measured by HPLC and photodiode detection. RESULTS: M-3-G was found in plasma and urine after ingestion of all the beverages studied. The aglycon, sulfate or glucuronate conjugates of M-3-G were not detected in plasma and urine. Increases in plasma M-3-G concentrations were not significantly different after the consumption of either red wine or dealcoholized red wine and were about two times less than those measured after consumption of red grape juice. This difference may be caused by the about two times higher M-3-G concentration determined in red grape juice. Area under the plasma concentration curves were as follows: 288 +/- 127 nmol x h/L (red wine), 214 +/- 124nmol x h/L (dealcoholized red wine) and 662 +/- 210 nmol x h/L (red grape juice) and showed a linear relationship with the amount of anthocyanin consumed (mean +/- SD). CONCLUSIONS: M-3-G is poorly absorbed after a single ingestion of red wine, dealcoholized red wine, or red grape juice and seems to be differentially metabolized as compared to other red grape polyphenols. Our results suggest that not anthocyanins such as M-3-G themselves but rather not yet identified anthocyanin metabolites and/or other polyphenols in red wine might be responsible for the observed antioxidant and health effects in vivo in subjects consuming red wine.

Moderate intervention with carotenoid-rich vegetable products reduces lipid peroxidation in men.

Because of their antioxidant properties, carotenoids may have beneficial effects in preventing cancer and cardiovascular disease. However, in humans consuming carotenoid-rich vegetables, data concerning the antioxidant effects of carotenoids are rather scarce. A human intervention trial was conducted, therefore, to determine whether a moderately increased consumption of carotenoid-rich vegetables would influence the antioxidant status in 23 healthy men. This short-term feeding study lasted 8 wk during which the men consumed a low carotenoid diet. A 2-wk low carotenoid period was followed by daily consumption of 330 mL tomato juice, then by 330 mL carrot juice and then by 10 g of spinach powder, each for 2 wk. Antioxidant status [water-soluble antioxidants in serum, ferric reducing ability of plasma (FRAP) and antioxidant enzyme activities] and lipid peroxidation (plasma malondialdehyde and ex vivo oxidation of LDL) were determined. In a subgroup of 10 men, lipoprotein carotenoids were measured. The consumption of carotenoid-rich vegetables significantly increased selected carotenoids in lipoproteins but had only minor effects on their relative distribution pattern. Tomato juice consumption reduced plasma thiobarbituric acid reactive substances (TBARS) by 12% (P: < 0.05) and lipoprotein oxidizability in terms of an increased lag time (18%, P: < 0.05). Carrot juice and spinach powder had no effect on lipid peroxidation. Water-soluble antioxidants, FRAP, glutathione peroxidase and reductase activities did not change during any study period. In evaluating the low carotenoid diet, we conclude that the additional consumption of carotenoid-rich vegetable products enhanced lipoprotein carotenoid concentrations, but only tomato juice reduced LDL oxidation in healthy men.

Prolonged tomato juice consumption has no effect on cell-mediated immunity of well-nourished elderly men and women.

The immunomodulatory potential of carotenoids has been investigated thoroughly only for beta-carotene. Data on the immunomodulatory activity of other carotenoids such as lycopene are scarce. The objective of this study was to investigate the effects of prolonged tomato juice consumption on cell-mediated immunity of well-nourished healthy elderly persons. In an intervention study, 33 female and 20 male subjects (aged 63-86 y) consumed 330 mL/d tomato juice (47.1 mg/d lycopene) or mineral water for 8 wk. Immune status was assessed by measuring number and lytic activity of natural killer (NK) cells, secretion of cytokines [interleukin (IL)-2, IL-4, tumor necrosis factor-alpha (TNF-alpha)] by activated peripheral blood mononuclear cells (PBMC), lymphocyte proliferation, and delayed-type hypersensitivity (DTH) skin responses. Tomato juice consumption resulted in significantly increased plasma lycopene and beta-carotene concentrations over time. In both treatment groups, TNF-alpha and IL-4 secretion were increased at the end of the intervention period, whereas IL-2 secretion was decreased. Tomato juice consumption had no effect on lymphocyte proliferation, DTH or the number of NK cells. Lytic activity of NK cells was increased in both groups at the end of the intervention period. In conclusion, these results show that prolonged tomato juice consumption increased plasma lycopene concentrations without significantly affecting cell-mediated immunity in well-nourished elderly subjects.

Plasma concentrations of carotenoids in healthy volunteers after intervention with carotenoid-rich foods.

AIM OF THE STUDY: The present study was conducted to investigate changes in the plasma concentration of carotenoids and carotenoid oxidation products, vitamin A, alpha- and gamma-tocopherol, and ubiquinone-10 during a dietary intervention trial with 23 male healthy volunteers. METHOD: A two week carotenoid depletion period was followed by a daily consumption of 330 mL tomato juice (40 mg lycopene), then by 330 mL carrot juice (15.7 mg alpha-carotene and 22.3 mg beta-carotene), and then by a 10 g spinach powder preparation (11.3 mg lutein and 3.1 mg beta-carotene) served with main meals for two weeks, respectively. Blood samples were collected in the morning after an overnight fasting and carotenoids, vitamin A, tocopherols, and ubichinone were analyzed by reversed-phase HPLC. RESULTS: During the tomato juice intervention, plasma concentrations of trans- and cis-lycopene increased 3-fold compared to the depletion period. Lycopene oxidation products could be demonstrated in plasma and were significantly elevated compared to control (p < 0.001). After two weeks of carrot juice consumption, alpha-carotene and beta-carotene concentrations increased 8.6- and 3.2-fold, respectively. Finally, during the spinach consumption period the lutein concentration increased 2-fold, while the beta-carotene concentrations were still elevated 2-fold. CONCLUSIONS: The moderate change in dietary habits, e.g., the consumption of 330 mL of carotenoid-rich vegetable juices caused significant changes in the plasma carotenoid concentrations, indicating a high bioavailability of carotenoids from these processed vegetable products. The changes in plasma carotenoid concentrations reflected the carotenoid composition of the consumed foods. However, particularly during the tomato juice intervention period the occurrence of lycopene oxidation products and cis-lycopene isomers in plasma was eminent. The formation may be due to antioxidant reactions of lycopene in the organism.

Anthocyanins are potent antioxidants in model systems but do not reduce endogenous oxidative DNA damage in human colon cells.

Anthocyanins are common colored plant flavonoids, occurring as glycosides of the respective anthocyanidin-chromophores. Like other flavonoids, anthocyanidins are also expected to have antioxidative and anti-mutagenic properties in vivo, although only few data are available. To gain more knowledge on possible protective mechanisms in mammalian cells, we have compared their extracellular and intracellular antioxidative potential in vitro and in human colon tumor cells. We used Aronia melanocarpa Elliot anthocyanin (AA) concentrates, fractions thereof, concentrates from Elderberry, Macqui, and Tintorera fruits, as well as pure compounds. In vitro, antioxidative properties of the samples were studied with the ferric reducing ability assay (FRA assay). As a measure of intracellular oxidative/antioxidative effects, H2O2-induced strand breaks as well as oxidized DNA bases were determined in human tumor HT29 clone 19A cells using a microgelelectrophoresis assay (comet test). Major results were that isolated compounds (aglycons and glycosides) and complex plant samples are powerful antioxidants in vitro. In fact their activities by far exceeded those of Trolox and vitamin C in the FRA assay. Also, H2O2-induced DNA strand breaks were reduced in cells treated with the complex plant extracts. In contrast, endogenous generation of oxidized DNA bases was not prevented. In summary, the intracellular steady state of oxidized DNA bases is not altered by anthocyanins or anthocyanidins. This finding raises questions with respect to the cancer preventive potential of anthocyanidins within specific tissues, such as the colon. Extracellularly, however, the compounds are potent antioxidants. This points to their potential for providing systemic protection in vivo, e.g., by scavenging oxidants in the blood stream and in the colon. Notably, both aglycons and glycosides have equally strong antioxidant activity.

Modulation of human T-lymphocyte functions by the consumption of carotenoid-rich vegetables.

A human intervention study was conducted to determine the effect of the consumption of carotenoid-rich vegetables on the immune system. Subjects, (twenty-three men), who were non-smokers, were not restricted in their daily diet, except that they had to abstain from fruit and vegetables high in carotenoids throughout the whole study period. The study was divided into four periods, each lasting 2 weeks: weeks 1-2: low-carotenoid period; throughout weeks 3-8: daily consumption of 330 ml tomato juice (40 mg lycopene/d, 1.5 mg beta-carotene/d) (weeks 3-4), 330 ml carrot juice (21.6 mg beta-carotene/d, 15.7 mg alpha-carotene/d, 0.5 mg lutein/d) (weeks 5-6), 10 g dried spinach powder (11.3 mg lutein/d, 3.1 mg beta-carotene/d) (weeks 7-8). Blood was collected weekly from subjects after a 12 h fast. T-lymphocyte functions were assessed by measuring proliferation and secretion of immunoreactive cytokines. The consumption of a low-carotenoid diet resulted in a significantly reduced proliferation of peripheral blood mononuclear cells (PBMC) cultured with concanavalin A. After 2 weeks of tomato juice consumption and until the end of the intervention period lymphocyte proliferation was not significantly changed compared with proliferation at the end of the depletion period. Secretion of cytokines by T-helper-1-like lymphocytes (interleukin (IL)-2) and by T-helper-2-like lymphocytes (IL-4) was influenced by the dietary intervention. IL-2 and IL-4 secretion values were significantly suppressed after the low-carotenoid diet (P < 0.001 and P < 0.05 respectively compared with baseline). Tomato juice consumption significantly enhanced IL-2 (P < 0.001) and IL-4 secretion (P < 0.05) compared with the end of depletion period. After carrot juice and spinach powder consumption the cytokine secretion capacity of PBMC was not significantly different from that at the end of the depletion period. In conclusion, the results of the present study indicate that a low-carotenoid diet reduces T-lymphocyte functions and addition of tomato juice restores these functions. This modulation could not be explained by changes in the plasma carotenoid concentrations. The active constituents in tomato juice as well as the biological significance of this immunomodulation remain to be determined.

Mechanisms by which vegetable consumption reduces genetic damage in humans.

A previous intervention study had shown that consumption of carotenoid-containing vegetable juices reduces oxidative DNA damage in lymphocytes of 23 male subjects. It was the aim of this study to elucidate the potential mechanisms involved. Specifically, we studied the modulation of protein expression and determined susceptibility factors. Cryopreserved lymphocytes from the study were analyzed for genetic polymorphisms of glutathione S-transferase (GSTM1, GSTP1, and GSTT1) using multiplex PCR, GSTP1-protein with an ELISA, total protein by a colorimetric enzyme reaction, and DNA-repair enzymes with the Comet Assay. Analyses of the genotoxicity data revealed a more steady state of protection for GSTM1*+ than for GSTM1*0 (15 and 8 of 23, respectively) genotypes. Increased expression of cytosolic protein was observed in 11 of 23 subjects, increased expression of GSTP1 in 6 of 23 subjects, and capacity of repair of oxidized DNA bases in 9 of 21 subjects. GSTP1 induction was independent of the GSTP1 genotype (GSTP1a or GSTP1b/c alleles). Kinetics of induction of cytosolic protein and of GSTP1 were compared in one GSTM1*+ and one GSTM1*0 subject and showed an efficacy of tomato and carrots, but not of spinach. Reduced genetic DNA damage in lymphocytes may be due to the enhancement of cytosolic GSTP1, and DNA-repair proteins by tomato and carrot juices. Enhancement of cytosolic proteins may be indicative of increased gene expression by vegetable juices, some of which may be associated with protective activities.

Is urodilatin the missing link in exercise-dependent renal sodium retention?

The purpose of the present study was to investigate the behavior of plasma atrial natriuretic peptide [ANP-(99-126)] concentration ([ANP]) and renal urodilatin [Uro; ANP-(95-126)] excretion during and after exercise and their possible effects on renal Na+ retention. Ten male subjects performed a cycle ergometer test for 60 min at 60% of maximum workload. Blood and urine samples were collected before, during, and up to 24 h after exercise. During exercise, plasma [ANP] and renal Uro excretion were oppositely affected: whereas [ANP] increased from 46.5 +/- 5.1 to 124.1 +/- 10.6 pg/ml, urinary Uro excretion decreased from 120.8 +/- 16.0 to 49.5 +/- 9.8 fmol/min and remained at a lower level until 1 h after exercise. Glomerular filtration rate showed lowest values during exercise (from 164.9 +/- 15.3 to 75.8 +/- 10.1 ml/min), and urine flow and the fractional excretion rate of Na+ (FENa+) and Cl- (FECl-) had their nadir during the first hour after exercise. Positive relationships were observed between Uro excretion and FENa+ (P < 0.05) and FECl-, whereas a tendency toward a negative correlation was obtained between [ANP] and FENa+. It seems possible that Uro may be, among other factors, involved in the exercise-related regulation of renal Na+ retention. The specific roles Uro and ANP play during exercise, however, remain to be investigated.

Consumption of vegetables reduces genetic damage in humans: first results of a human intervention trial with carotenoid-rich foods.

A human intervention study with vegetable products has been performed in twenty three healthy, non smoking males aged 27-40. It was the aim of the study to assess whether consumption of vegetables containing different carotenoids could protect against DNA damage and oxidative DNA damage. The subjects consumed their normal diets, but abstained from vegetables high in carotenoids throughout the study period. After a 2 week depletion period, they received daily 330 ml tomato juice with 40 mg lycopene (weeks 3 and 4), 330 ml carrot juice with 22.3 mg beta-carotene and 15.7 mg alpha-carotene (weeks 5 and 6), and 10 g dried spinach powder (in water or milk) with 11.3 mg lutein (weeks 7 and 8). Blood was collected weekly and DNA damage was detected in peripheral blood lymphocytes with the 'COMET' assay. Oxidised DNA bases were detected by including an incubation step with endonuclease III. The supplementation of the diet with tomato, carrot or spinach products resulted in a significant decrease in endogenous levels of strand breaks in lymphocyte DNA. Oxidative base damage was significantly reduced during the carrot juice intervention. These findings support the hypothesis that carotenoid containing plant products exert a cancer-protective effect via a decrease in oxidative and other damage to DNA in humans.

Gene expression of aphrodisin in female hamster genital tract segments.

Aphrodisin is a glycoprotein originally isolated from hamster vaginal discharge which was demonstrated to be involved in pheromonal effects on male hamsters. In the present study, we investigated the localization of aphrodisin-synthesizing and -storing cells in the entire genital tract of the female golden hamster using immunohistochemical and molecular biological methods. By use of immunohistochemical methods, significant aphrodisin immunoreactivity was detected within the cervical glandular tissue. Western blot analysis revealed high concentration of aphrodisin in vaginal discharge and in tissue extracts from the vagina and the cervix uteri. According to intracellular localization of aphrodisin, this protein is confined to cytoplasm of the immunoreactive cells. Immunoreactivity was also detected extracellularly on the surface of the anterior vaginal pluristratified epithelium. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed an extremely high level of aphrodisin gene expression in the vagina and in the lower part of the uterus comprising the cervix. However, aphrodisin gene expression was also demonstrated in the middle part of the uterus and at a low level even in the ovaries. No aphrodisin gene expression was detectable in the upper part of the uterus and the uterine horns. In situ hybridization confirmed that the maximum expression of the aphrodisin gene is encountered in glandular cells of the cervix uteri. These results indicate that within the female hamster genital tract aphrodisin is predominantly synthesized throughout the vagina and cervical uterus. The protein is then secreted into the vaginal lumen. It is under discussion whether the accumulation of aphrodisin in the vaginal discharge facilitates the transfer of pheromone of low molecular weight to the male hamster's vomeronasal organ during investigatory behavior.

Volume changes in the forearm and lower limbs during 2 h of acute hypobaric hypoxia in nonacclimatized subjects.

To investigate the role of fluid shifts during the short-term adjustment to acute hypobaric hypoxia (AHH), the changes in lower limb (LV) and forearm volumes (FV) were measured using a strain-gauge plethysmograph technique in ten healthy volunteers exposed to different altitudes (450 m, 2500 m, 3500 m, 4500 m) in a hypobaric chamber. Arterial blood pressure, heart rate, arterial oxygen saturation (SaO2), endtidal gases, minute ventilation and urine flow were also determined. A control experiment was performed with an analogous protocol under normobaric normoxic conditions. The results showed mean decreases both in LV and FV of 0.52 (SD 0.39) ml x 100 ml(-1) and -0.65 (SD 0.32) ml x 100 ml(-1), respectively, in the hypoxia experiments [controls: LV 0.28 (SD 0.37), FV 0.41 (SD 0.47) ml x 100 ml(-1)]. Descent to normoxia resulted in further small but not significant decreases in mean LV [-0.02 (SD 0.11) ml x 100 ml(-1)], whereas mean FV tended to increase slightly [ + 0.02 (SD 0.14) ml x 100 ml(-1)]; in the control experiments mean LV and FV decreased continuously during the corresponding times [-0.19 (SD 0.31), -0.18 (SD 0.10) ml x 100 ml(-1) , respectively]. During the whole AHH, mean urine flow increased significantly from 0.84 (SD 0.41) ml x min(-1) to 3.29 (SD 1.43) ml x min(-1) in contrast to the control conditions. We concluded that peripheral fluid volume shifts form a part of the hypoxia-induced acute cardiovascular changes at high altitude. In contrast to the often reported formation of peripheral oedema after prolonged exposure to hypobaric hypoxia, the results provided no evidence for the development of peripheral oedema during acute induction to high altitude. However, the marked increase in interindividual variance in SaO2 and urine flow points to the appearance of the first differences in the short-term adjustment even after 2 h of acute hypobaric hypoxia.

Secretion of a urodilatin-like immunoreactive (URO-like-IR) substance from a human kidney cell line (HEK-293).

The aim of this study was to explore whether sodium chloride is involved in the release of urodilatin from epithelial human embryonic kidney cells (HEK-293). Using a highly specific urodilatin radioimmunoassay combined with HPLC, gel chromatography, and a cyclic GMP generating bioassay, we demonstrate that HEK-293 cells release a biologically active 3.5 kD, urodilatin-like immunoreactive substance. To show the effect of sodium on urodilatin release, HEK-293 cells were incubated with cell culture buffer containing 120, 130, 140, and 150 mmol/l sodium, respectively. Urodilatin secretion from HEK-293 cells is increased to 176% when extracellular sodium is raised to 150 mmol/l (control: 120 mmol/l = 100%). There was no significant difference when exposing the cells to 140 mmol/l or 150 mmol/l sodium. It is suggested, that beside the known extrarenal factors influencing urodilatin secretion, high cephalic sodium and cardiac volume load, urodilatin secretion might also be regulated by an intrarenal sodium sensitive mechanism.

Urodilatin secretion in salt-loaded Wistar rats.

The aim of our study was to investigate whether urodilation (URO, INN: ularitide) is present in rat urine and if URO excretion in the rat is influenced by dietary sodium intake. Therefore, three groups of Wistar rats were placed in metabolic cages where they received different sodium diets for 9 days (0.05%, 0.4%, and 8.0% NaCl, respectively). Food and water intake were determined by weight. At days -4, 2, 5, and 8 blood pressure was measured non invasively using the tail cuff method. After nine days rats were anesthetized and blood was drawn for serum electrolyte, plasma A-type natriuretic peptide (CDD/ANP-99-126), and plasma aldosterone concentration measurements. Using a highly specific antibody against URO combined with high performance liquid chromatography and gel chromatography, we were able to show that a URO-like substance of approx. 3.5 kD that is distinct from CDD/ANP-99-126, brain natriuretic peptide, and C-type natriuretic peptide, is present in rat urine. Sodium chloride loaded rats showed significantly increased urinary excretion rates of URO (p < 0.001), chloride (p < 0.001), sodium (p < 0.001), and the fractional excretion of sodium (p < 0.001). In the plasma, sodium (p < 0.01) and chloride (p < 0.001) increased, while potassium, hematocrit, osmolality, plasma CDD/ANP-99-126, as well as glomerular filtration rate (GFR), and systolic blood pressure did not change. Since CDD/ANP-99-126 is believed to be a natriuretic peptide, it is suggested that CDD/ANP-99-126 might participate in the natriuresis due to high dietary sodium intake. In sodium-loaded rats, however, plasma CDD/ANP-99-126 remains unchanged, while URO excretion increases with sodium excretion, independent of GFR and blood pressure. We conclude that URO secretion is stimulated by dietary salt loading and might be involved in the regulation of water and electrolyte metabolism in the rat.

Urodilatin inhibits sodium reabsorption in the isolated perfused rat kidney.

The effect of human urodilatin (hURO) on kidney function has been examined in stable recirculating isolated perfused rat kidney. The concentration-dependent effects of urodilatin (URO) on isolated perfused rat kidney were analyzed at different perfusion pressures. The experiments show that at perfusion pressures of 100 or 130 mm Hg, 100 nmol/l hURO cause a significant increase of urine flow and a significant decrease of the fractional sodium reabsorption from 95.2 (SD 0.9, n = 5) to 85.6% (SD 0.9, n = 5) and 86.8 (SD 1.6, n = 5) to 77.6% (SD 3.2, n = 5), respectively. In contrast, at a perfusion pressure of 80 mm Hg sodium excretion was not increased by URO. The glomerular filtration rate was unchanged. Furthermore, the renal effects of hURO were compared with rat urodilatin (rURO) and the circulating form of rat cardiodilatin/atrial natriuretic peptide (rCDD/ANP 99-126). hURO, rURO, and rCDD/ANP produced a concentration-dependent increase in sodium excretion. However, differences in natriuretic efficiency are observed. rCDD/ANP causes a decrease in fractional sodium reabsorption from 93.6 (SD 2.6, n = 5) to 86.8% (SD 2.4, n = 5), similar to hURO. rURO shows a significantly higher natriuretic effect decreasing fractional sodium reabsorption from 92.0 (SD 0.7, n = 5) to 79.1% (SD 1.2, n = 5). From these results we conclude that the response of the isolated kidney to URO is critically dependent on the perfusion pressure and that URO exhibits tubular action on renal sodium excretion. The observed differences among the tested peptides on renal function may be due to species differences in the peptide sequence.

Development and application of a urodilatin (CDD/ANP-95-126)-specific radioimmunoassay.

Urodilatin, a renal natriuretic peptide that is an analogue to circulating atrial natriuretic peptide [alpha-ANP(99-126)], is measurable with a highly specific and sensitive radioimmunoassay. While most ANP antibodies cannot distinguish between urodilatin and other ANP analogues, the polyclonal urodilatin antibody specifically measures human urodilatin without any cross-reactivity to other ANP analogues. Urodilatin is not detected in blood from healthy volunteers nor from cardiac patients. Urinary urodilatin accounts for only a part of total urinary ANP immunoreactivity. Urodilatin excretion closely parallels sodium excretion in response to an acute volume load while changes in urinary immunoreactive ANP excretion do not reflect this renal response. We conclude that specific urodilatin assays are required to explore further the physiological role of the renal natriuretic peptide.