Neopterin in renal cell carcinoma: inhalational administration of interleukin-2 is not accompanied by a rise of urinary neopterin.

Inhalational administration of interleukin-2 (IL-2) is effective in controlling renal cell carcinoma (RCC) lung metastases with minimal toxicity. Neopterin is an indicator of systemic immune activation in metastatic cancer and is increased after systemic IL-2 administration. Urinary neopterin was investigated in 13 patients with metastatic RCC and 18 controls. In seven patients, urinary neopterin was followed before and after treatment with inhalational IL-2. Neopterin was measured by high-performance liquid chromatography and creatinine was determined by Jaffé reaction. Urinary neopterin was significantly increased in patients with metastatic RCC compared to controls (257 +/- 263 micromol/mol creatinine vs. 110 +/- 41 micromol/mol creatinine; Mann-Whitney U-test, p < 0.05). Median survival was significantly longer in patients with urinary neopterin <173 micromol/mol creatinine compared to patients with neopterin > or = 173 micromol/mol creatinine (698 vs. 245 days; log-rank test, p < 0.05). No significant increase was observed after inhalational IL-2 therapy (147 +/- 101 vs. 153 +/- 54 micromol/mol creatinine). We conclude that urinary neopterin is increased in patients with metastatic RCC, and higher neopterin concentrations are indicative of poor prognosis. The absence of an increase in urinary neopterin after inhalational IL-2 therapy is in accord with the lack of significant systemic toxicity.     

Accurate quantification of dimethylamine (DMA) in human urine by gas chromatography-mass spectrometry as pentafluorobenzamide derivative: evaluation of the relationship between DMA and its precursor asymmetric dimethylarginine (ADMA) in health and disease

Dimethylamine [DMA, (CH(3))(2)NH)] is abundantly present in human urine. Main sources of urinary DMA have been reported to include trimethylamine N-oxide, a common food component, and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is excreted in the urine in part unmetabolized and in part after hydrolysis to DMA by dimethylarginine dimethylaminohydrolase (DDAH). Here we describe a GC-MS method for the accurate and rapid quantification of DMA in human urine. The method involves use of (CD(3))(2)NH as internal standard, simultaneous derivatization with pentafluorobenzoyl chloride and extraction in toluene, and selected-ion monitoring of m/z 239 for DMA and m/z 245 for (CD(3))(2)NH in the electron ionization mode. GC-MS analysis of urine samples from 10 healthy volunteers revealed a DMA concentration of 264+/-173 microM equivalent to 10.1+/-1.64 micromol/mmol creatinine. GC-tandem MS analysis of the same urine samples revealed an ADMA concentration of 27.3+/-15.3 microM corresponding to 1.35+/-1.2 micromol/mmol creatinine. In these volunteers, a positive correlation (R=0.83919, P=0.0024) was found between urinary DMA and ADMA, with the DMA/ADMA molar ratio being 10.8+/-6.2. Elevated excretion rates of DMA (52.9+/-18.5 micromol/mmol creatinine) and ADMA (3.85+/-1.65 micromol/mmol creatinine) were found by the method in 49 patients suffering from coronary artery disease, with the DMA/ADMA molar ratio also being elevated (16.8+/-12.8). In 12 patients suffering from end-stage liver disease, excretion rates of DMA (47.8+/-19.7 micromol/mmol creatinine) and ADMA (5.6+/-1.5 micromol/mmol creatinine) were found to be elevated, with the DMA/ADMA molar ratio (9.17+/-4.2) being insignificantly lower (P=0.46). Between urinary DMA and ADMA there was a positive correlation (R=0.6655, P<0.0001) in coronary artery disease, but no correlation (R=0.27339) was found in end-stage liver disease.     

Effect of 5 alpha-pregnan-3 alpha-ol-20-one on nitric oxide biosynthesis and plasma volume in rats

Plasma 5 alpha-pregnan-3 alpha-ol-20-one (pregnan) levels and nitric oxide (NO) biosynthesis increase during pregnancy. These factors have independently been implicated in the control of blood pressure and volume. We wished to determine whether pregnan might be responsible both for the increase in NO biosynthesis and for the increase in plasma volume observed during pregnancy. Virgin female Long-Evans rats were implanted with indwelling cannulas and maintained on a low nitrate/nitrite diet. After the rats recovered from surgery, 500 microg of pregnan or vehicle were given daily for 2 days. NO biosynthesis and plasma volume were measured in conscious animals before and after treatment. Pregnan caused a significant increase in NO biosynthesis (1.9 +/- 0.8 micromol/24 h, n = 10) compared with the vehicle-treated control group (0.3 +/- 0.4 micromol/24 h, n = 10, P < 0.05). Similarly, there was a significant increase in plasma volume in the pregnan-treated group (0.7 +/- 0.2 ml/100 g, n = 11) compared with the vehicle-treated control group (0.2 +/- 0.1 ml/100 g, n = 11, P < 0.05). These results confirm that pregnan can mimic pregnancy by its ability to increase both NO biosynthesis and plasma volume.     

Oxoproline kinetics and oxoproline urinary excretion during glycine- or sulfur amino acid-free diets in humans

L-5-oxoproline (L-5-OP) is an intermediate in glutathione synthesis, possibly limited by cysteine availability. Urinary 5-OP excretion has been proposed as a measure of glycine availability. We investigated whether 5 days of dietary sulfur amino acid (SAA-free) or glycine (Gly-free) restriction affects plasma kinetics of 5-OP and urinary excretion of L- and D-5-OP in 6 healthy men. On day 6, L-5-[1-(13)C]oxoproline and [3,3-(2)H(2)]cysteine were infused intravenously for 8 h (3 h fast/5 h fed). In a control study (adequate amino acid mixture), plasma oxoproline fluxes were 37.8 +/- 13.8 (SD) and 38.4 +/- 14.8 micromol x kg(-1) x h(-1); oxidation accounted for 85% of flux. Cysteine flux was 47.9 +/- 8.5 and 43.2 +/- 8.5 micromol x kg(-1) x h(-1) for fast and fed phases, respectively. Urinary excretion of L- and D-5-OP was 70 +/- 34 and 31.1 +/- 13.3 micromol/mmol creatinine, respectively, during days 3-5, and 46.4 +/- 13.9 and 22.4 +/- 8.3 micromol/mmol over the 8-h tracer study. The 5-OP flux for the Gly-free diet was higher (P = 0. 018) and tended to be higher for the SAA-free diet (P = 0.057) when compared with the control diet. Oxidation rates were higher on the Gly-free (P = 0.005) and SAA-free (P = 0.03) diets. Cysteine fluxes were lower on the the Gly-free (P = 0.01) and the SAA-free diets (P = 0.001) compared with the control diet. Rates of L-5-OP excretion were unchanged by withdrawal of SAA or Gly for 5 days but increased on day 6 (P = 0.005 and P = 0.019, respectively). Thus acute changes in the dietary availability of SAA and Gly alter oxoproline kinetics and urinary 5-OP excretion.     

组胺对肺动脉内皮细胞一氧化氮合酶基因表达的影响

本实验研究了组胺对原代培养的肺动脉内皮细胞一氧化氮合酶(nitric oxidCsynthase,NOS)基因表达的影响及分子机制。采用RT-PCR和免疫印迹技术分别检测mRNA和蛋白质的表达水平,用荧光素酶报告基因实验检测eNOS基因转录起始点上游长1.6-kb的启动子活性,用硝酸还原酶法检测NO的产量。结果发现,组胺增强eNOS表达,呈浓度和时间依赖性,10μmol/L组胺处理肺动脉内皮细胞24h可使eNOS mRNA和蛋白质的表达达到高峰,eNOS mRNA水平为正常对照组的160.8±12.2%(P<0.05),蛋白质水平为正常对照组的136.2±11.2%(P<0.05)。特异性CaMK Ⅱ抑制剂KN-93可抑制组胺的这一效应,表明组胺可通过激活CaMK Ⅱ增强肺动脉内皮细胞eNOS基因的表达。报告基因实验表明,10μmol/L组胺处理24h后肺动脉内皮细胞eNOS基因启动子的活性增强,为正常对照组的148.2±33.7%(P<0.05)。组胺可使肺动脉内皮细胞产生NO增加。这些结果表明组胺在转录水平增强肺动脉内皮细胞eNOS基因的表达,并使细胞产生NO增加,这可能是组胺调节肺血管张力的机制之一。CaMK Ⅱ可能是组胺增强肺动脉内皮细胞eNOS基因表达的途径之一。     

Mixed exhaled nitric oxide and plasma nitrites and nitrates in newborn infants.

Plasma nitrite (NO2-) and nitrate (NO3-) are the stable end-products of endogenous nitric oxide (NO) metabolism. NO is present in the exhaled air of humans, but it is not clear if exhaled NO may be an indicator of the systemic endogenous NO production. The aims of the study were to determine the levels of exhaled NO and plasma NO2-/NO3- in healthy term and preterm newborns, and to assess if exhaled NO correlates with plasma NO2-/NO3- at birth. After the stabilization of the newborn, we measured by chemiluminescence the concentration of NO in the mixed expired breath of 133 healthy newborns. Measurement of exhaled NO was repeated after 24 and 48 hours. Plasma NO2-/NO3- levels at birth were measured by the Griess reaction. NO concentrations were 8.9 (CI 8.1-9.8) parts per billion (ppb), 7.7 (CI 7.2-8.3) ppb and 9.0 (CI 8.4-9.6) ppb at birth, 24 and 48 hours, respectively. At birth, exhaled NO was inversely correlated with gestational age (p=0.008) and birth weight (p<0.001). Plasma NO2-/NO3- level was 27.30 (CI 24.26-30.34) micromol/L. There was no correlation between exhaled NO and plasma NO2-/NO3- levels at birth (p=0.88). We speculate that the inverse correlation between exhaled NO and gestational age and birth weight may reflect a role of NO in the postnatal adaptation of pulmonary circulation. At birth, exhaled NO does not correlate with plasma NO2-/NO3- and does not seem to be an index of the systemic endogenous NO production.     

Oxidative stress in very low birth weight infants as measured by urinary 8-OHdG

Very low birth weight (VLBW) infants can be subjected to oxidative stress in the course of intensive care. We measured 8-hydroxydeoxyguanosine (8-OHdG), a biomarker of oxidative stress, and estimated the degree of oxidative stress in such infants. We also examined if the administered oxygen was related to oxidative stress. Urine samples of 50 Japanese VLBW infants [birth weights: 956.3+/-277.6g, and gestational ages: 28.0+/-2.6 weeks (mean +/- SD)] were collected on various postnatal days and 8-OHdG levels were determined using an ELISA kit. Sixteen term infants served as normal controls. As body weights at sampling increased, the average levels of urinary 8-OHdG decreased. 8-Hydroxydeoxyguanosine levels were: infants under 1000g, 29.5+/-16.4 micromol/mol creatinine (n = 24); 1000-1500g, 23.8+/-14.9 (n = 12); over 1500g, 16.1+/-8.5 (n = 14); and control, 10.9+/-7.2 (n = 16). Significant differences were found between <1000g group and > or = 1500g group (p = 0.0030), <1000g group and control (p < 0.0001), and 1000-1500g group and control (p = 0.0108). Also as postconceptional age at sampling increased, the average levels of 8-OHdG decreased. 8-Hydroxydeoxyguanosine levels were: infants before 252 days (36 weeks) of postconception: 27.4+/-15.5 micromol/mol creatinine (n = 34); after 252 days, 18.2+/-12.5 (n = 16). Differences between <252 days group and control (p < 0.0001), and <252 days group and > or = 252 days groups (p = 0.0253) were statistically significant. Among the three groups based on ambient oxygen concentration (21%, 22-29%, and > or = 30%) there was no significant difference (p = 0.417). The more premature the infants were, the more intense was the oxidative stress, hence, it is the prematurity rather than the administered oxygen which causes oxidative stress in VLBW infants. Drury et al. ["Urinary 8-hydroxydeoxyguanosine in infants and children" Free Radic. Res. 28 (1998) 423-4281 measured urinary 8-OHdG of 28 infants (24-40 weeks gestation) and found no gestation or birthweight related differences. This discrepancy seemed to be because of difference in birth weights and sampling period of the subjects.     

Urinary N tau-methylimidazole acetic acid excretion in respiratory disease

N tau-methylimidazole acetic acid (N tau-MIAA) is the principal urinary metabolite of histamine. The basal urinary excretion rate of N tau-MIAA was determined as 0.117 +/- 0.008 (SE) mg/h, with a renal clearance for N tau-MIAA of 273 +/- 27 ml/min implying active secretion. After subpharmacological infusion of histamine (50 ng.kg-1.min-1 over 2 h) in five volunteers that increased plasma histamine from 0.28 +/- 0.04 to 0.71 +/- 0.15 ng/ml, urinary excretion of N tau-MIAA over 8 h was increased by less than 17% compared with a control saline infusion. Urinary N tau-MIAA excretion in normal controls (273 +/- 14 micrograms/mmol creatinine) was similar to that observed in patients with severe acute asthma (253 +/- 22 micrograms/mmol), antigen-induced bronchoconstriction (269 +/- 21 micrograms/mmol), seasonal allergic rhinitis (304 +/- 31 micrograms/mmol), and clinically stable bronchiectasis (270 +/- 22 micrograms/mmol). In contrast, large increases in metabolite excretion (greater than 7,000 micrograms/mmol creatinine) were observed in a patient with systemic mastocytosis where very high plasma histamine levels were recorded (greater than 500 ng/ml) and marked systemic hemodynamic effects occurred. We conclude that urinary N tau-MIAA will only be increased in pathologies where sustained hyperhistaminemia occurs and that increased local histamine production in the lung or the upper airway does not cause a measurable change in the basal urinary excretion of this metabolite.     

Muscle soreness-induced reduction in force generation is accompanied by increased nitric oxide content and DNA damage in human skeletal muscle

We examined the effect of exercise-induced muscle soreness on maximal force generation, tissue nitric oxide (NO) and 8-hydroxydeoxyguanosine (8-OHdG) content in human skeletal muscle. Female volunteers were assigned to control (C) and muscle soreness (MS) groups (n = 6 in each). MS group performed 200 eccentric muscle actions of the rectus femoris to induce muscle soreness. Maximal force generation was measured 24 h before and after exercise in both groups. Needle biopsy samples were assayed for NO content with electron spin resonance spectroscopy after ex vivo spin trapping, and 8-OHdG content were measured with an enzyme-linked immuno assay. Maximal force decreased by 11+/-5.4% (p < .05) 24 h after exercise in MS group. Muscle soreness increased NO and 8-OHdG contents from their control values of 0.39+/-0.08 arbitrary units and 0.035+/-0.004 pmol/micromol DNA to 0.96+/-0.05 (p < .05) arbitrary units and 0.044+/-0.005 (p < .05) pmol/micromol DNA, respectively. This is the first demonstration that muscle soreness-induced decrease in maximal force generation is a result of an increase in muscular NO content and associated with enhanced formation of 8-OHdG in human skeletal muscle.     

Nephrotoxicity of cyclosporin A in hereditary hypertriglyceridemic rats

It has been suggested that cyclosporin A (CsA) nephrotoxicity can be reduced by the concomitant administration of omega-3 fatty acids or vitamin E. The present study was designed to establish whether the effect of the above substances can also be demonstrated in rats with hereditary hypertriglyceridemia (HTG) whose sensitivity to the nephrotoxic effect is greater than in control AVN rats. CsA administration at a dose of 10 mg/kg/day to HTG rats resulted in a significant rise (p<0.001) in serum levels of creatinine (from 66.0+/-7.6 to 108.4+/-11.6 micromol/l) and urea (from 8.3+/-0.7 to 22.3+/-18 mmol/l) which was not found in AVN rats. The baseline values of systolic blood pressure (SBP) were significantly higher in HTG rats. However, in both strains CsA administration was associated with a similar SBP increase which was not prevented by omega-3 fatty acids (EPAX) or vitamin E administration. Concomitant administration of CsA with EPAX at a dose of 600 mg/kg b.w./day in HTG rats prevented the rise in the serum levels of creatinine (65.4+/-14.7 micromol/l) and reduced the increase in the serum urea levels (11.9+/-7.6 mmol/l). Concomitant administration of CsA and vitamin E (at a dose of 25 mg/kg/day) also reduced the increase (p<0.05) in the serum levels of creatinine (70.7+/-14.3 micromol/l) and urea (9.8+/-3.4 mmol/l) compared to the effects elicited by the administration of CsA alone (p<0.05). Administration of CsA alone or in combination with EPAX or vitamin E did not have a marked effect on diuresis, proteinuria, urinary osmolality, urinary excretion of urea, creatinine and potassium. Under all experimental conditions, the rate of urinary excretion of sodium in HTG rats was significantly lower (p<0.01) than in AVN rats. The results obtained support the assumption that omega-3 fatty acids and vitamin E at the doses used reduce CsA nephrotoxicity in rats with hereditary hypertriglyceridemia whose sensitivity to the nephrotoxic effect of CsA is significantly higher than in AVN rats.     

Dimethylarginine dimethylaminohydrolase-2 overexpression improves impaired nitric oxide synthesis of endothelial cells induced by glycated protein.

Nitric oxide (NO) synthesis is modulated by dimethylarginine dimethylaminohydrolase (DDAH) via metabolizing asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor. This study investigated whether glycosylated bovine serum albumin (GBSA) could impair NO synthesis by inhibition of DDAH expression and activity, and whether DDAH2 overexpression could reverse the impaired NO synthesis induced by GBSA in endothelial cells. Overexpression of DDAH2 gene was established by liposome-mediated gene transfection in ECV304 endothelial cell line. Cells were incubated with 1.70 mmol/L GBSA for 48h. And the expressions of DDAH1 and DDAH2, gene activities of DDAH and NOS in cells, as well as concentrations of ADMA and NO in media were assayed. The activity of DDAH and expression of DDAH2 gene but not DDAH1 gene were inhibited in endothelial cells after exposure to GBSA, whereas the concentrations of ADMA were increased concomitantly with the decrease of NOS activity in cells and NO production in media. Overexpression of DDAH2 gene could prevent the inhibition of DDAH activity induced by GBSA (0.55+/-0.02 vs 0.42+/-0.02U/g pro; n=3; P<0.05), decrease ADMA concentration (0.59+/-0.04 vs 1.13+/-0.11 micromol/L; n=3; P<0.01), and increase NOS activity and NO production (53.77+/-3.40 vs 34.59+/-2.57 micromol/L; P<0.05) compared with untransfected cells treated with GBSA. These results suggest that decreased DDAH activity and subsequent elevated endogenous ADMA are implicated in the inhibition of NO synthesis induced by GBSA, and overexpression of DDAH2 gene can prevent these changes in DDAH/ADMA/NOS/NO pathway of endothelial cells exposed to GBSA.     

Interaction between endogenously produced carbon monoxide and nitric oxide in regulation of renal afferent arterioles

Heme oxygenases (HO-1 and HO-2) catalyze the conversion of heme to carbon monoxide (CO), iron, and biliverdin. CO causes vasorelaxation via stimulation of soluble guanylate cyclase (sGC) and/or activation of calcium-activated potassium channels. Because nitric oxide (NO) exerts effects via the same pathways, we tested the interaction between CO and NO on rat afferent arterioles (AAs) using the blood-perfused juxtamedullary nephron preparation. AAs were superfused with either tricarbonyldichlororuthenium (II) dimer, known as CO releasing molecule (CORM-2), 10 micromol/l CO solution, or 15 micromol/l chromium mesoporphyrin (CrMP, HO inhibitor). AAs were also superfused with 1 mmol/l N(omega)-nitro-L-arginine (L-NNA) to inhibit NO synthase (NOS) or 10 micromol/l 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one to inhibit sGC, and then CrMP was superfused during NOS inhibition or sGC inhibition. Treatment with 150 and 300 micromol/l CORM-2 or with CO (10 micromol/l) significantly dilated AAs (22.0 +/- 0.9 and 22.8 +/- 0.9 vs. 18.3 +/- 0.9 microm, n = 5, P < 0.05; and 26.0 +/- 1.4 vs. 18.8 +/- 0.7 microm, n = 5, P < 0.05). In untreated vessels, HO inhibition did not alter AA diameter (17.5 +/- 0.7 vs. 17.2 +/- 0.6 microm, n = 7, P > 0.05); however, during inhibition of NO production, which constricted arterioles to 14.6 +/- 1.2 microm, n = 6, P < 0.05, concurrent HO inhibition led to further vasoconstriction (11.7 +/- 1.6 microm, n = 6, P < 0.05). CORM-2 attenuated the L-NNA-induced vasoconstriction. Inhibition of sGC caused significant constriction (15.7 +/- 0.4 vs. 18.8 +/- 0.4 microm, n = 6, P < 0.05). HO inhibition during sGC inhibition did not cause further change in AAs (15.5 +/- 0.7 microm, n = 6). We conclude that endogenously produced CO does not exert a perceptible influence on AA diameter in the presence of intact NO system; however, when NO production is inhibited, CO serves as an important renoprotective reserve mechanism to prevent excess afferent arteriolar constriction.     

一氧化氮供体对过氧化氢引起的心肌细胞损伤的保护作用

关于一氧化氮(NO)对心肌细胞是否具有保护作用目前尚存在争议,为探讨NO对过氧化氢(H2O2)引起的心肌细胞损伤是否具有保护作用及其可能的机制,实验将体外培养的新生大鼠心肌细胞分为3组(1)阴性对照组(Normal组);(2)H2O2组H2O2(0.1mmol/L)与心肌细胞共育4h;(3)S-亚硝基-N-乙酰青霉胺(SNAP)+H2O2组NO供体SNAP(0.5mmol/L)处理心肌细胞10min后,加入H2O2与心肌细胞共育4 h.用流式细胞术检测心肌细胞凋亡率,心肌细胞损伤程度以心肌细胞存活率和乳酸脱氢酶(lactate dehydrogenase,LDH)活性来表示,同时检测心肌细胞超氧化物歧化酶(superoxide dismutase,SOD)活性和丙二醛(MDA)含量.通过激光共聚焦显微术检测在不同处理条件下心肌细胞胞内钙的变化.结果表明,正常心肌细胞LDH活性和细胞存活率分别为631.4±75.6 U/L和93.1±6.2%,细胞凋亡率为0;H2O2处理细胞后可使细胞LDH活性显著增高(1580.5±186.7 U/L,P＜0.01),细胞存活率明显下降(58.3±7.6%,P＜0.01),流式细胞仪检测到大量心肌细胞凋亡,凋亡率为26.4±5.7%;SOD活性较正常细胞19.67±0.85 NU/ml显著下降,为14.73±1.68 NU/m(P＜0.01),MDA含量较正常细胞6.95±0.83μmol/L显著增高,为15.35±3.49μmol/L(P＜0.01).SNAP预处理细胞可显著提高心肌细胞存活率(79.7±9.3%,P＜0.01),降低LDH活性和细胞凋亡率(分别为957.8±110.9 U/L和9.1±3.3%,P＜0.01);并提高细胞抗氧化能力,表现为较H2O2处理组的SOD活性增高(21.36±3.11 NU/ml,P＜0.01),MDA含量下降(9.12±1.47 μmol/L,P＜0.01).激光共聚焦显微镜检测结果表明,H2O2可升高细胞内钙,而SNAP则可降低细胞内钙,SNAP预处理细胞后可取消H2O2升高细胞内钙的作用.上述结果提示,NO供体SNAP可对抗H2O2对心肌细胞的损伤,其机制与提高心肌细胞抗氧化损伤能力和对抗H2O2引起的细胞内钙超载有关.     

Shift in metabolic substrate uptake by the heart during development of alloxan-induced diabetes

Inhibition of endothelial nitric oxide (NO) synthase (eNOS) is associated with an increase in glucose uptake by the heart. We have already shown that Type I diabetes also causes a decrease in eNOS protein expression and altered NO control of both coronary vascular resistance and oxygen consumption. Therefore, we predict that the increase in plasma glucose and the reduction in eNOS during diabetes together would result in a large increase in cardiac glucose uptake. Arterial (A) and coronary sinus (C) plasma levels of glucose, free fatty acid (FFA), beta-hydroxybutyric acid (beta-HBA), and lactate were measured, and myocardial uptake was calculated before and at week 1, 2, 3, and 4 of alloxan-induced diabetes. The heart of healthy dogs consumed FFA (19.2 +/- 2.6 microeq/min) and lactate (19.7 +/- 3.4 micromol/min). Dogs in the late stage of diabetes (at week 4) had elevated arterial beta-HBA concentrations (1.6 +/- 0.7 micromol/l) that were accompanied by an increased beta-HBA uptake (0.3 +/- 0.2 micromol/min). In contrast, myocardial lactate (-4.8 +/- 3.0 micromol/min) and FFA uptake (2.5 +/- 1.9 microeq/min) were significantly reduced in diabetic animals. Despite a marked hyperglycemia (449 +/- 25 mg/dl), the heart did not take up glucose (-7.9 +/- 4.1 mg/dl). Our results indicate significant changes in the myocardial substrate utilization in dogs only in the late stage of diabetes, at a time when myocardial NO production is already decreased.     

Revascularization decreases 8-isoprostaglandin F2alpha excretion in chronic lower limb ischemia

8-Isoprostaglandin F2alpha is one of a series of isoprostanes formed by free radical catalysed peroxidation of arachidonic acid. Urinary 8-isoprostaglandin F2alpha is a new marker which reflects oxidative stress in vivo and can be utilized as a diagnostic tool to assess the extent of oxidative stress in various disease states associated with lipid peroxidation. Increased levels of 8-isoprostaglandin F2alpha in cardiac ischemia/reperfusion provide evidence for oxidative stress during coronary perfusion. In animal studies, the restoration of blood flow after lower limb ischemia is followed by reperfusion syndrome. In this study we investigated whether lower limb ischemia/reperfusion is associated with oxidative stress, as reflected by urinary levels of 8-isoprostaglandin F2alpha. Ten patients (mean age 72 years, range 61-82 years) suffering from chronic lower limb ischemia and 10 healthy volunteers (mean age 69 years, range 60-79 years) participated in the study. In all patients, diagnostic angiography had revealed stenosis or occlusion either in the aortoiliac or femoropopliteal region. Surgical revascularization consisted of femoropopliteal reconstruction, femorofemoral reconstruction, aortobifemorial reconstruction, or femoral endartectomy. Urine samples from patients were collected a day before surgery and in the second postoperative day. Urinary 8-isoprostaglandin F2alpha was extracted on a C2 silica cartridge and determinated by radioimmunoassay. After revascularization, 8-isoprostaglandin F2alpha excretion (pg/micromol creatinine, mean +/- SD) was decreased by 2.5-fold (preoperative 48.9 +/- 8.9, postoperative 19.1 +/- 9.5, P < 0.001). The postoperative values were similar to the concentrations measured in healthy volunteers (18.0 +/- 11.0). All revascularizations were successful, and the increase in ankle-brachial index (preoperative 0-0.6, postoperative 0.4-0.8) revealed improved blood flow in the ischemic lower limb. We suggest that, as assessed by the quantitation of urinary 8-isoprostaglandin F2alpha, chronic lower limb ischemia is associated with increased oxidative stress, which is decreased by revascularization.     

Purine loss after repeated sprint bouts in humans.

The influence of the number of sprint bouts on purine loss was examined in nine men (age 24.8 +/- 1.6 yr, weight 76 +/- 3.9 kg, peak O(2) consumption 3.87 +/- 0.16 l/min) who performed either one (B1), four (B4), or eight (B8) 10-s sprints on a cycle ergometer, 1 wk apart, in a randomized order. Forearm venous plasma inosine, hypoxanthine (Hx), and uric acid concentrations were measured at rest and during 120 min of recovery. Urinary inosine, Hx, and uric acid excretion were also measured before and 24 h after exercise. During the first 120 min of recovery, plasma inosine and Hx concentrations, and urinary Hx excretion rate, were progressively higher (P < 0.05) with an increasing number of sprint bouts. Plasma uric acid concentration was higher (P < 0.05) in B8 compared with B1 and B4 after 45, 60, and 120 min of recovery. Total urinary excretion of purines (inosine + Hx + uric acid) was higher (P < 0. 05) at 2 h of recovery after B8 (537 +/- 59 micromol) compared with the other trials (B1: 270 +/- 76; B4: 327 +/- 59 micromol). These results indicate that the loss of purine from the body was enhanced by increasing the number of intermittent 10-s sprint bouts.     

Role of nitric oxide in the regulation of glucose kinetics in response to endotoxin in dogs.

The purpose of the present in vivo study was to determine the role of nitric oxide (NO) in the regulation of glucose metabolism in response to endotoxin by blocking NO synthesis with N(G)-monomethyl-L-arginine (L-NMMA). In five dogs, the appearance and disappearance rates of glucose (by infusion of [6,6-(2)H(2)]glucose), plasma glucose concentration, and plasma hormone concentrations were measured on five different occasions: saline infusion, endotoxin alone (E coli, 1.0 microg/kg i.v.), and endotoxin administration plus three different doses of primed, continuous infusion of L-NMMA. Endotoxin increased rate of appearance of glucose from 13.7 +/- 1.6 to 23.6 +/- 3.3 micromol x kg(-1) x min(-1) (P < 0.05), rate of disappearance of glucose from 13.9 +/- 1.1 to 24.8 +/- 3.1 micromol x kg(-1) x min(-1) (P < 0.001), plasma lactate from 0.5 +/- 0.1 to 1.7 +/- 0.1 mmol/l (P < 0.01), and counterregulatory hormone concentrations. L-NMMA did not affect the rise in rate of appearance and disappearance of glucose, plasma lactate, or the counterregulatory hormone response to endoxin. Plasma glucose levels were not affected by endotoxin with or without L-NMMA. In conclusion, in vivo inhibition of NO synthesis by high doses of L-NMMA does not affect glucose metabolism in response to endotoxin, indicating that NO is not a major mediator of glucose metabolism during endotoxemia in dogs.     

Long-term dietary L-arginine supplementation attenuates proteinuria and focal glomerulosclerosis in experimental chronic renal transplant failure.

Glomerular endothelial nitric oxide synthase expression is decreased in humans during acute rejection and chronic renal transplant failure (CRTF). This may contribute to vascular damage through changes in the renal hemodynamics and enhanced endothelial adhesion of leukocytes and platelets. Dietary supplementation of L-arginine may increase endothelial NO production, thereby protecting the vascular wall and improving renal hemodynamics. We tested the hypothesis that long-term L-arginine supplementation attenuates the development of CRTF in an experimental model for renal transplantation. In the Fisher 344 to Lewis rat model for renal transplantation, renal function and histology of untreated rats was compared with rats receiving L-arginine in the drinking water (10g/L), starting 2 days before transplantation. Every 4 weeks systolic blood pressure was measured and serum and urine were collected for measurement of nitrite and nitrate (NO(x)), creatinine, and proteinuria. At 34 weeks the histological renal damage was assessed by scoring focal glomerulosclerosis and measurement of alpha-smooth muscle actin (alpha-SMA) expression. Urinary NO(x) was significantly increased in treated animals. Proteinuria was significantly lower in L-arginine-treated animals from week 24 onward (p<0.05). Plasma creatinine and creatinine clearance did not differ between the groups. The focal and segmental glomerulosclerosis (FGS) score (max 400) at week 34 was also significantly lower in treated rats arbitrary U (20+/-21 vs 61+/-67 arbitrary U; p<0.05). The expression of alpha-SMA was lower in L-arginine-treated rats than in untreated rats (1.93+/-0.8% area surface vs 3.64+/-2.5% area surface). In conclusion, in this experimental model for CRTF, L-arginine administration significantly reduced FGS and proteinuria, without affecting renal function. Our data suggest that dietary L-arginine supplementation attenuates progression of CRTF and may therefore be an additional therapeutic option in human renal allograft recipients.     

Effects of exercise training of 8 weeks and detraining on plasma levels of endothelium-derived factors, endothelin-1 and nitric oxide, in healthy young humans

Vascular endothelial cells produce nitric oxide (NO), which is a potent vasodilator substance and has been proposed as having antiatherosclerotic property. Vascular endothelial cells also produce endothelin-1 (ET-1), which is a potent vasoconstrictor peptide and has potent proliferating activity on vascular smooth muscle cells. Therefore, ET-1 has been implicated in the progression of atheromatous vascular disease. Because exercise training has been reported to produce an alteration in the function of vascular endothelial cells in animals, we hypothesized that exercise training influences the production of NO and ET-1 in humans. The purpose of the present study was to examine whether chronic exercise could influence the plasma levels of NO (measured as the stable end product of NO, i.e., nitrite/nitrate [NOx]) and ET-1 in humans. Eight healthy young subjects (20.3 +/- 0.5 yr old) participated in the study and exercised by cycling on a leg ergometer (70% VO2max for 1 hour, 3-4 days/week) for 8 weeks. Venous plasma concentrations of NOx and ET-1 were measured before and after (immediately before the end of 8-week exercise training) the exercise training, and also after the 4th and 8th week after the cessation of training. The VO2max significantly increased after exercise training. After the exercise training, the plasma concentration of NOx significantly increased (30.69 +/- 3.20 vs. 48.64 +/- 8.16 micromol/L, p < 0.05), and the plasma concentration of ET-1 significantly decreased (1.65 +/- 0.14 vs. 1.23 +/- 0.12 pg/mL, p < 0.05). The increase in NOx level and the decrease in ET-1 level lasted to the 4th week after the cessation of exercise training and these levels (levels of NOx and ET-1) returned to the basal levels (the levels before the exercise training) in the 8th week after the cessation of exercise training. There was a significant negative correlation between plasma NOx concentration and plasma ET-1 concentration. The present study suggests that chronic exercise causes an increase in production of NO and a decrease in production of ET-1 in humans, which may produce beneficial effects (i.e., vasodilative and antiatherosclerotic) on the cardiovascular system.     

A simple, accurate, and sensitive assay method of dehydroepiandrosterone sulfate: application for quantitative determination in human breast cyst and duct fluids

Radioimmunoassay (RIA) is the most prevalent method for measuring small amounts of hormones, peptides, and other compounds in human body fluids. The method, however, has several problems, such as cross reactions or non-specific reactions of the antibody used. In order to establish an improved method for assaying dehydroepiandrosterone sulfate (DHEAS) and cholesterol, which are the largest components of human breast cyst and duct fluids, we describe a simple, accurate, and sensitive method using high-performance liquid chromatography (HPLC). The samples were treated with cholesterol oxidase for quantitation of dehydroepiandrosterone (DHEA) and free cholesterol, and the respective oxidized substances, 4-androstene-3,17-dione and 4-cholesten-3-one, were extracted with n-hexane. The extracts were analyzed by straight phase HPLC. Effluents were monitored by measuring absorption at 240 nm, where a newly introduced chromophoric group, an alpha,beta-unsaturated ketone, showed intense absorption (epsilon = 16,000). When the total amount of DHEA (DHEAS plus DHEA) was measured, the sample had been solvolyzed by sulfatase beforehand. The amounts of DHEAS were quantified by comparing the amounts of DHEA before and after solvolysis. Levels of free cholesterol, DHEAS, and DHEA in human breast cyst fluids (n = 30) were 1.77 +/- 1.12 mmol/dl, 8.27 +/- 10.24 micromol/dl, and 0.02 +/- 0.02 micromol/dl (means +/- SD), respectively. The levels of sterol and steroid measured in breast duct fluids that were turbid, brown, dark green, or milky in color (n = 9) (mean levels, 3.20 +/- 2.97 mmol/dl for free cholesterol and 14.77 +/- 13.75 micromol/dl for DHEAS) were significantly (P < 0.01) higher than the levels in clear or serous breast fluids (n = 21) (mean levels, 0.14 +/- 0.13 mmol/dl for free cholesterol and 0.04 +/- 0.07 micromol/dl for DHEAS).