Hypoxia causes increased monocyte nitric oxide synthesis which is mediated by changes in dimethylarginine dimethylaminohydrolase 2 expression in animal and human models of normobaric hypoxia.

BACKGROUND: Tissue hypoxia is a cardinal feature of inflammatory diseases and modulates monocyte function. Nitric oxide is a crucial component of the immune cell response. This study explored the metabolism of the endogenous inhibitor of nitric oxide production asymmetric dimethylarginine(ADMA) by monocyte dimethylarginine dimethylaminohydrolase 2(DDAH2), and the role of this pathway in the regulation of the cellular response and the local environment during hypoxia. METHODS: Peritoneal macrophages were isolated from a macrophage-specific DDAH2 knockout mouse that we developed and compared with appropriate controls. Cells were exposed to 3% oxygen followed by reoxygenation at 21%. Healthy volunteers underwent an 8 h exposure to normobaric hypoxia with an inspired oxygen percentage of 12%. Peripheral blood mononuclear cells were isolated from blood samples taken before and at the end of this exposure. RESULTS: Intracellular nitrate plus nitrite(NOx) concentration was higher in wild-type murine monocytes after hypoxia and reoxygenation than in normoxia-treated cells (mean(SD) 13·2(2·4) vs 8·1(1·7) pmols/mg protein, p = 0·009). DDAH2 protein was 4·5-fold (SD 1·3) higher than in control cells (p = 0·03). This increase led to a 24% reduction in ADMA concentration, 0·33(0.04) pmols/mg to 0·24(0·03), p = 0·002). DDAH2-deficient murine monocytes demonstrated no increase in nitric oxide production after hypoxic challenge. These findings were recapitulated in a human observational study. Mean plasma NOx concentration was elevated after hypoxic exposure (3·6(1.8)µM vs 6·4(3·2), p = 0·01), which was associated with a reduction in intracellular ADMA in paired samples from 3·6(0.27) pmols/mg protein to 3·15(0·3) (p < 0·01). This finding was associated with a 1·9-fold(0·6) increase in DDAH2 expression over baseline(p = 0·03). DISCUSSION: This study shows that in both human and murine models of acute hypoxia, increased DDAH2 expression mediates a reduction in intracellular ADMA concentration which in turn leads to elevated nitric oxide concentrations both within the cell and in the local environment. Cells deficient in DDAH2 were unable to mount this response.

Executive summary and conclusions from the European Hydration Institute Expert Conference on human hydration, health, and performance.

On April 7-8, 2014, the European Hydration Institute hosted a small group of experts at Castle Combe Manor House, United Kingdom, to discuss a range of issues related to human hydration, health, and performance. The meeting included 18 recognized experts who brought a wealth of experience and knowledge to the topics under review. Eight selected topics were addressed, with the key issues being briefly presented before an in-depth discussion. Presented here is the executive summary and conclusions from this meeting.

Symmetric dimethylarginine (SDMA) is raised in women with polycystic ovary syndrome: a pilot study.

Polycystic ovary syndrome (PCOS) is associated with endothelial dysfunction, which may be caused by elevated levels of asymmetric dimethylarginine (ADMA). ADMA reduces nitric oxide production in diabetes mellitus, hypertension and renal failure. Symmetric dimethylarginine (SDMA) is a stereoisomer produced alongside ADMA, and has recently been described as a risk factor for cardiovascular events. In this cross-sectional study based in a teaching hospital, 16 women with PCOS were recruited alongside 15 healthy controls, and fasting venous blood samples were obtained. Renal function was measured, and ADMA and SDMA were analysed using a high-performance liquid chromatography method. After controlling for BMI, mean ADMA and SDMA levels in women with PCOS were higher than in controls (p = 0.036 and p = 0.030, respectively). Renal function was not different between the two groups (p = 0.152). Women with PCOS have raised levels of SDMA, a molecule implicated in endothelial dysfunction and long-term cardiovascular risk.

The biology and therapeutic potential of the DDAH/ADMA pathway.

Asymmetric dimethylarginine (ADMA) is an endogenously produced molecule that inhibits nitric oxide synthase and consequently may have adverse effects on physiology, in particular in the cardiovascular system. This review highlights the mechanisms involved in the synthesis and metabolism of ADMA and their role in the control of nitric oxide (NO) synthesis. We describe how the effects of both cellular and circulating ADMA can alter physiological function involving both NO dependent and independent pathways and go on to describe how the metabolism of ADMA by dimethylarginine dimethylaminohydrolase (DDAH) is the major endogenous mechanism by which ADMA levels are regulated. Furthermore, we discuss the association of ADMA concentrations with cardiovascular disease and how ADMA levels can be modulated therapeutically by altering its production and/or metabolism. Finally we discuss the effects of some of the current pharmaceutical therapies used to treat cardiovascular disease and their involvement in the modulation of the ADMA/DDAH pathway.

Hormonal responses to a single session of wholebody vibration exercise in older individuals.

OBJECTIVE: Whole-body vibration (WBV) has been recently suggested as an alternative form of exercise. In this study, the acute effects of a single session of WBV exercise on anabolic hormones in aged individuals were analysed. DESIGN: A randomised cross-over trial design was used. SETTINGS: Geriatrics Department, Woodend Hospital. PARTICIPANTS: 20 individuals (9 men and 11 women; median age 70 years (range 66 to 85 years) volunteered in the experiment. Interventions Isometric squat on a platform with vibration or no vibration (control) conditions. MAIN OUTCOME MEASUREMENTS: Plasma cortisol, testosterone, growth hormone (GH) and insulin-like growth factor 1 (IGF-1) were measured before, immediately after, and 1 and 2 h after the interventions. REPORTS: A significant difference between treatments (p<0.001) and a time x treatment interaction (p<0.05) was found in IGF-1 levels. Cortisol levels were shown not to be significantly different between treatments (p = 0.43), but a difference over time (p<0.001) and a time6 treatment interaction (p<0.05) were identified. No significant differences were identified in GH and testosterone levels. CONCLUSIONS: As shown by the results of the study, 5 min of WBV exercise characterised by static squat with a frequency of 30 Hz can be performed by older individuals without apparent signs of stress and/or fatigue. Furthermore, WBV produced an acute increase in the circulating levels IGF-1 and cortisol greater than that observed following the same exercise protocol conducted without vibration.

Whole-body vibration can reduce calciuria induced by high protein intakes and may counteract bone resorption: A preliminary study.

Excess protein intake can adversely affect the bone via an increase in calcium excretion, while suitable mechanical loading promotes osteogenesis. We therefore investigated whether vibration exposure could alleviate the bone mineral losses associated with a metabolic acidosis. Ten healthy individuals aged 22 - 29 years (median = 25) underwent three 5-day study periods while monitoring their dietary intake. The study consisted of recording the participants' usual dietary intake for 5 consecutive days. Participants were then randomly divided into two groups, one of which received a protein supplement (2 g x kg(-1) body mass x day(-1); n = 5) and the other whole-body low-magnitude (3.5 g), low-frequency (30 Hz) mechanical vibration (WBV) delivered through a specially designed vibrating plate for 10 min each day (n = 5). Finally, for the third treatment period, all participants consumed the protein supplement added to their normal diet and were exposed to WBV exercise for 10 min per day. Daily urine samples were collected throughout the experimental periods to determine the excretion of calcium, phosphate, titratable acid, urea, and C-telopeptide. As expected, when the participants underwent the high protein intake, there was an increase in urinary excretion rates of calcium (P < 0.001), phosphate (P < 0.003), urea (P < 0.001), titratable acid (P < 0.001), and C-telopeptide (P < 0.05) compared with baseline values. However, high protein intake coupled with vibration stimulation resulted in a significant reduction in urinary calcium (P = 0.006), phosphate excretion (P = 0.021), and C-telopeptide (P < 0.05) compared with protein intake alone, but did not affect titratable acid and urea output. The participants showed no effect of WBV exercise alone on urinary excretion of calcium, phosphate, urea, titratable acid, or C-telopeptide. The results indicate that vibration stimulation can moderate the increase in bone resorption and reduction in bone formation caused by a metabolic acidosis.

A comparison of water turnover in older people in community and institutional settings.

BACKGROUND: Maintaining water balance is essential for health, but environmental factors, pathology and the ageing process can adversely affect water homeostasis. OBJECTIVE: This study examined the relationship between physical dependency and daily water turnover rate in an older population. DESIGN: Daily water turnover (DWT) was estimated, using deuterium oxide ((2)H(2)O) as a tracer for water, over two separate 7-day periods in summer and winter in two older populations. The independent group (N = 22) lived in their own homes and were self-caring. The dependent group (N = 15) lived in institutional care, and were more physically dependent. None of the subjects had significant mental impairment. Total body water (TBW) and DWT were estimated from the equilibration concentration of ingested (2)H(2)O and its subsequent elimination rate. RESULTS: The independent group had a median (range) age of 75(69-88) y, a mean Barthel Index (BI) of 19.8, and a mean Abbreviated Mental Test (AMT) score of 9.8. The dependent group were older (83(72-93) y), with a mean BI of 13 and a mean AMT of 9.3. Average median (range) DWT in the independent group was similar in summer (2.2(1.3-3.6) l.d(-1)) and winter (2.1(1.4-3.6) l.d(-1)), but faster than in the dependent group (1.5(0.9-2.9) and 1.6(1.0-2.8) l.d(-1), respectively) during the same two periods. Median urine output in the independent group was similar in summer (1.7(0.8-3.3) l.d(-1)) and winter (1.7(0.9-3.2) l.d(-1)), but greater than in the dependent group (1.1(0.6-2.7) and 0.9(0.5-1.6) l.d(-1), respectively). CONCLUSION: These results show that the water turnover rate of many older people is low, and that intake may be affected especially in those with physical disability.

Paroxetine administration failed [corrected] to influence human exercise capacity, perceived effort or hormone responses during prolonged exercise in a warm environment.

The purpose of the experiment was to examine whether selective serotonin (5-HT) re-uptake transporter blockade by paroxetine has any effect on perceived effort (RPE) during exercise or the time to reach volitional fatigue and on the prolactin and cortisol responses during prolonged exercise performed in a warm environment. Eight healthy males performed two cycle rides to exhaustion in a warm (32 degrees C) environment at 60% of maximum oxygen uptake. Paroxetine (20 mg) or placebo was administered 5 h before exercise trials in a randomised double blind fashion. Time to exhaustion was not significantly influenced by administration of paroxetine: median (range) time to exhaustion was 93.3 (76.2-175.0) min on the placebo trial and 92.5 (66.0-151.0) min on the paroxetine trial. Rectal temperature was higher at rest and throughout exercise on the paroxetine trial. The serum concentrations of prolactin and cortisol were determined throughout exercise as peripheral markers of central 5-HT activity. RPE increased over time but was not influenced by paroxetine administration. Prolactin and cortisol levels increased over time but paroxetine administration did not influence the hormone responses during exercise. In conclusion, acute administration of paroxetine failed to alter RPE, exercise capacity or the response of the determined peripheral hormone markers of central 5-HT activity during prolonged exercise in a warm environment.

Effects on health of fluid restriction during fasting in Ramadan.

During the 9th month (Ramadan) of the Islamic calendar (Hijra) many millions of adult Muslims all over the world fast during the daylight hours. Since Hijra is a lunar calendar, Ramadan occurs at different times in the seasonal year over a 33-year cycle. Fasting during Ramadan is partial because the abstention from food, fluid, tobacco and caffeine is from sunrise to sunset. Several categories of people are exempt or can postpone the Ramadan fast. The effect on health and well being of the month-long intermittent fast and fluid restriction has been studied in various potentially vulnerable groups in addition to normal healthy individuals in many countries. The majority of the studies have found significant metabolic changes, but few health problems arising from the fast. A reduction in drug compliance was an inherent negative aspect of the fast. Common findings of the studies reviewed were increased irritability and incidences of headaches with sleep deprivation and lassitude prevalent. A small body mass loss is a frequent, but not universal, outcome of Ramadan. During the daylight hours of Ramadan fasting, practising Muslims are undoubtedly dehydrating, but it is not clear whether they are chronically hypohydrated during the month of Ramadan. No detrimental effects on health have as yet been directly attributed to negative water balance at the levels that may be produced during Ramadan.

Common genetic variation in a basal promoter element alters DDAH2 expression in endothelial cells.

Synthesis of the vasodilator nitric oxide (NO) can be inhibited by the endogenous methylarginines L-NMMA and ADMA. ADMA is elevated in a number of cardiovascular disorders in which NO availability is reduced. Elimination of ADMA from the body occurs primarily by enzymatic breakdown through the action of DDAH, of which two isoforms exist, DDAH1 and DDAH2. In this study we have identified a core promoter region of the DDAH2 gene, and transcription factor sites that play an important role in the regulation of DDAH2 expression. Using PCR-SSCP analysis we also identified six common polymorphisms. One of these polymorphisms (an insertion/deletion at position -871) within the core promoter element influenced basal transcription. The discovery of a functional polymorphism within the DDAH2 promoter suggests that there may be common, individual differences in the ability to metabolise ADMA in vivo, that in turn, might underlie susceptibility to cardiovascular disease.

Gastric emptying of a carbohydrate-electrolyte drink during a soccer match.

PURPOSE: To investigate the effect on gastric emptying of intermittent exercise at varying intensities such as occurs during a soccer match. METHODS: We compared the emptying rate of a carbohydrate-electrolyte (CE) drink during two 15-min periods of a competitive 5-a-side indoor soccer match separated by 10 min and during two 15-min periods of low-intensity walking exercise. Seven healthy male subjects, all of whom regularly played soccer, completed the study. Exercise intensity on each trial was calculated from heart rate recordings. Subjects ingested 500 mL of the CE drink immediately before starting each exercise period. Before and after completing each 15-min exercise period, the gastric contents were aspirated and the volume recorded. Ambient room temperature ranged from 16-22 degrees C and humidity ranged from 57-72% on both trials. RESULTS: Mean +/- SD heart rate was higher during the Soccer trial than during the Walking trial at all time points. Exercise intensity was calculated to be 54 +/- 23% and 63 +/- 20% VO2max, respectively, for the first and second periods of exercise on the Soccer trial and 13 +/- 9% and 12 +/- 11% VO2max, respectively, for the first and second periods of exercise on the Walking trial. During the first 15-min period of exercise, a greater (median (range)) volume of the CE drink was emptied from the stomach on the Walking trial (247 mL (102-361 mL)) than on the Soccer trial (84 mL (17-230 mL); P = 0.022). In the second exercise period, however, gastric emptying rates were not different (P = 0.16) between the Walking trial (247 mL (104-363 mL)) and Soccer trial (148 mL (17-293 mL)). CONCLUSION: As indicated by heart rate, the overall exercise intensity of an indoor soccer match appears to be moderate. The pattern of activity during a match is intermittent with periods of low level activity punctuated by short bursts of high-intensity sprinting. The present study demonstrates that the intensity of activity during this type of exercise is sufficient to slow gastric emptying.

Effect of intermittent high-intensity exercise on gastric emptying in man.

PURPOSE: The effect on gastric emptying of brief intermittent high-intensity sprints and a moderate-intensity steady-state cycle exercise was studied. METHODS: Eight healthy male subjects were each studied at rest (R), during steady-state exercise at a constant power output corresponding to 66% of their VO(2max) (C66), during intermittent high-intensity exercise at a power output averaging 66% of their VO(2max) (I66), and during intermittent high-intensity exercise at a power output averaging 75% of their VO(2max) (I75). Gastric emptying was measured using the double-sampling gastric aspiration technique. Subjects ingested 600 mL of a 6% carbohydrate-electrolyte solution immediately before exercise or seated rest. RESULTS: The volume of test solution in the stomach was less at all time points on trial I66 than on trial I75 (P = 0.023). The rate of gastric emptying, expressed as the median (range) time (minutes) taken to empty half the test meal volume (t(1/2)), was not different on trials R (20 (7--30)) and C66 (21 (7--49)), and was faster than on trial I75 (62 (27--100); P = 0.003 and P = 0.005, respectively). Median t(1/2) was faster on trial R than on trial I66 (30 (15--74) min; P = 0.019), but no difference was detected between C66 and I66 or between I66 and I75. However, over the initial 30 min period after ingestion, the median (range) volume of test drink delivered to the duodenum was faster (P < 0.01) on trials R (387 (296--541) mL) and C66 (389 (165--584) mL) than on trials I66 (331 (191--494) mL) or I75 (249 (79-335) mL). CONCLUSION: The data demonstrate that gastric emptying of liquids is slowed during brief intermittent high-intensity exercise compared with rest or steady-state moderate exercise.

Structural insights into the hydrolysis of cellular nitric oxide synthase inhibitors by dimethylarginine dimethylaminohydrolase.

Nitric oxide synthase is inhibited by asymmetric NG-methylated derivatives of arginine whose cellular levels are controlled in part by dimethylarginine dimethylaminohydrolase (DDAH, EC 3.5.3.18). Levels of asymmetric NG,NG-dimethylarginine (ADMA) are known to correlate with certain disease states. Here, the first structure of a DDAH shows an unexpected similarity to arginine:glycine amidinotransferase (EC 2.1.4.1) and arginine deiminase (EC 3.5.3.6), thus defining a superfamily of arginine-modifying enzymes. The identification of a Cys-His-Glu catalytic triad and the structures of a Cys to Ser point mutant bound to both substrate and product suggest a reaction mechanism. Comparison of the ADMA-DDAH and arginine-amidinotransferase complexes reveals a dramatic rotation of the substrate that effectively maintains the orientation of the scissile bond of the substrate with respect to the catalytic residues. The DDAH structure will form a basis for the rational design of selective inhibitors, which are of potential use in modulating NO synthase activity in pathological settings.

Comparison of water turnover rates in men undertaking prolonged cycling exercise and sedentary men.

Total body water (TBW) and water turnover rates (WTR) of six competitive male cyclists (CG) and six age-matched sedentary men (SG) were determined using deuterium oxide dilution and elimination. During the 7 day study, individuals in the CG cycled daily outside on average 50 (range 12-146) km at an average speed of 29 km.h(-1), while the SG did no regular exercise. During the study, the weather was cool (10 [4-18]degrees C), mainly cloudy but dry. Daily average (median [range]) nude body mass remained essentially the same in the CG (77.25 [76.54-77.54] kg) and SG (65.04 [64.45-65.44] kg). Expressed as a percentage of body mass, median TBW of the CG (70.1 [65.5-73.9]%) was greater than that of the SG (63.5 [52.7-71.0]% ). Average median WTR was faster in the CG (47 [42-58] ml.kg.d(-1)) than the SG (36 [29-50] ml.kg.d(-1)). The average median daily urinary loss was similar in the CG (27 [22-33]ml.kg.d(-1)) and SG (29 [24-31]ml.kg.d(-1)). Calculated non-renal daily water loss was faster in the CG (19 [13-35] ml.kg.d(-1)) than the SG (6 [5-22] ml.kg.d(-1)), but there was no relationship between the average distance cycled daily and the WTR. This study demonstrates that WTR are faster in individuals undertaking prolonged exercise than in sedentary men, and that the difference was due to the almost three times greater non-renal water losses that the exercising group incurred. This suggests that exercise-induced increases in respiratory water loss and sweat rate are major factors in water loss even in cool environments.

Chromosomal localization, gene structure, and expression pattern of DDAH1: comparison with DDAH2 and implications for evolutionary origins.

Endogenously produced asymmetrically methylated arginine residues are competitive inhibitors of all three isoforms of nitric oxide synthase (NOS). The enzyme dimethylarginine dimethylaminohydrolase (DDAH) specifically hydrolyzes these asymmetrically methylated arginine residues to citrulline and methylamines. Previously we have proposed that regulation of asymmetric methylarginine concentration by DDAH may provide a novel mechanism for the regulation of NOS activity in vivo. Recently we reported the cloning of human DDAH and identified a novel human DDAH isoform (DDAH I and DDAH II, respectively). Here we report that the DDAH1 gene maps to chromosome 1p22 and confirm that DDAH2 maps to the MHC III region of chromosome 6p21.3. Extensive analysis of the distribution of DDAH1 and DDAH2 mRNA in 50 human tissues indicates differential expression of DDAH isoforms in brain regions, in immune cells, and during development. DDAH2 expression predominates in highly vascularized tissues that express the endothelial NOS isoform and in immune tissues that can express iNOS. Whereas DDAH2 is expressed at relatively high levels in all fetal tissues examined, DDAH1 expression varies little between fetal and adult tissues. The chromosomal localization of the DDAHs is consistent with gene duplication, and consistent with this, comparison of the gene structures indicates that the intron/exon organization is highly conserved. Phylogenetic analysis of DDAH sequences from diverse species suggests that DDAH gene duplication occurred prior to the emergence of bony fish some 400 million years ago. Overall the data suggest that DDAH2 may be the more ancient of the two genes.

Stay on Your Feet Safety Walks Group.

The Safety Walks Group is an initiative that evolved from the Stay on Your Feet Program. The strategies used in this program target both behavioural and environmental change and are based on the five areas for action under the Ottawa Charter (WHO, 1986) and Jakarta Declaration (WHO, 1997). The Safety Walks Group addresses the issue of public hazards via the use of a standard checklist covering pedestrian areas, business houses and accommodation. The project provided a forum for seniors to be proactive, working with the authorities to address the issue of public hazards and make the environment safer.

Intracellular localization of dimethylarginine dimethylaminohydrolase overexpressed in an endothelial cell line.

Methylarginines are endogenous inhibitors of nitric oxide synthase (NOS) and have been implicated in the regulation of the nitric oxide pathway in health and disease. Cellular concentrations of free methylarginines are determined in part by the activity of dimethylarginine dimethylaminohydrolase (DDAH). There are two isoforms of DDAH which have distinct tissue distributions with some relationship to NOS isoforms. We have determined the intracellular localization of both DDAH isoforms by overexpression of epitope-tagged DDAH in an immortalized endothelial cell line. Immunofluorescence confocal microscopy and immunoblotting indicate that both isoforms are predominantly cytosolic with no specific association with organelles or the plasma membrane. These data suggest that the key role for DDAH may be to ensure that under normal conditions the levels of methylarginines are kept low throughout the whole cell.

Improved gastric emptying rate in humans of a unique glucose polymer with gel-forming properties.

BACKGROUND: The energy density of a nutrient drink is one of the main factors that affect the gastric emptying of the solution, while osmolality and viscosity are thought to have only a minimal influence. METHOD: The rate of gastric emptying of two isoenergetic carbohydrate solutions with different osmolality and viscosity was determined using a double sampling gastric aspiration technique. Six healthy male subjects were studied on two occasions using approximately 550 ml of a solution containing 13.5% of carbohydrate either in the form of a mixture of monomeric glucose and short chain glucose oligomers (G-drink) or of long chain glucose polymers composed of 78% amylopectin and 22% amylose (C-drink). RESULT: The half emptying time (t(1/2), median and range) for the viscous, markedly hypotonic (62 mosmol/kg) C-drink was faster (17.0 (6.2-31.4) min) than for the moderately hypertonic (336 mosmol/kg) G-drink (32.6 (25.2-40.7) min). The amount (median and range) of carbohydrate delivered to the small intestine was greater during the first 10 min after ingestion of C-drink (31.8 (15.8-55.9) g) than after ingestion of G-drink (14.3 (6.8-22.2) g). However, there was no difference in the blood glucose (P = 0.73) or serum insulin (P = 0.38) concentration at any time point after ingestion of the two test drinks. CONCLUSION: The results of this study show that the carbohydrate present in C-drink, although it has the propensity to form a gel, empties from the stomach faster than that of an isoenergetic carbohydrate solution (G-drink) without potentiating increased circulating blood glucose or insulin levels.

Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases.

Methylarginines inhibit nitric oxide synthases (NOS). Cellular concentrations of methylarginines are determined in part by the activity of dimethylarginine dimethylaminohydrolase (DDAH; EC 3.5.3. 18). We have cloned human DDAH and identified and expressed a second novel DDAH isoform (DDAH I and II respectively). DDAH I predominates in tissues that express neuronal NOS. DDAH II predominates in tissues expressing endothelial NOS. These results strengthen the hypothesis that methylarginine concentration is actively regulated and identify molecular targets for the tissue and cell-specific regulation of methylarginine concentration.