Specific analysis in plasma and urine of 2,3-dinor-5, 6-dihydro-isoprostane F(2alpha)-III, a metabolite of isoprostane F(2alpha)-III and an oxidation product of gamma-linolenic acid

F(2)-isoprostanes (iPs) are free radical-catalyzed isomers of prostaglandin F(2alpha). Circulating and urinary iPs have been used as indices of lipid peroxidation in vivo. Utilizing an (18)O-labeled homologous internal standard, we developed a gas chromatography/mass spectrometry assay for the 2,3-dinor-5,6-dihydro (dinor-dihydro) metabolite of iPF(2alpha)-III. Although urinary excretion of iPF(2alpha)-III reflects systemic lipid peroxidation, the metabolite is more abundant (median of 877 (range of 351-1831) versus 174 (range of 56-321) pg/mg of creatinine; p < 0.01) than the parent iP in urine and can be measured in plasma. Metabolite analysis may be preferable in plasma due to the abundance of arachidonic acid as a source of ex vivo lipid peroxidation. Also, iPF(2alpha)-III may be formed in blood samples in a cyclooxygenase-dependent manner by platelets ex vivo. By contrast, the metabolite is not formed by aggregated platelets (0.71 +/- 0.08 versus 0.65 +/- 0.09 pg/ml). Although the metabolite/parent ratio is altered in cirrhosis, urinary dinor-dihydro-iPF(2alpha)-III is elevated and increases further during reperfusion following orthoptic liver transplantation. In addition to its formation as an iPF(2) metabolite, analysis of gamma-linolenic acid autooxidation products and the compound present in freeze-thawed plasma suggests that gamma-linolenic acid may also be an important source of dinor-dihydro-iPF(2alpha)-III.     

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.     

The effect of iron overload on urinary excretion of immunoreactive prostaglandin E2

The effect of in vivo lipid peroxidation on the excretion of immunoreactive prostaglandin E2 (PGE2) in the urine of rats was studied. Weanling, male Sprague-Dawley rats were fed a vitamin E-deficient diet containing 10% tocopherol-stripped corn oil (CO) or 5% cod liver oil (CLO) with or without 40 mg dl-alpha-tocopheryl acetate/kg. To induce a high, sustained level of lipid peroxidation, some rats were injected intraperitoneally with 100 mg of iron as iron dextran after 10 days of feeding. Iron overload stimulated in vivo lipid peroxidation in rats, as measured by the increase in expired ethane and pentane. Dietary vitamin E reversed this effect. Rats fed the CLO diet excreted 9.5-fold more urinary thiobarbituric acid-reactive substances (TBARS) than did rats fed the CO diet. Iron overload increased the excretion of TBARS in the urine of rats fed the CO diet, but not in urine of rats fed the CLO diet. Dietary vitamin E decreased TBARS in the urine of rats fed either the CO or the CLO diet. Iron overload decreased by 40% the urinary excretion of PGE2 by rats fed the CO diet, and dietary vitamin E did not reverse this effect. Iron overload had no statistically significant effect on urinary excretion of PGE2 by rats fed the CLO diet. A high level of lipid peroxidation occurred in iron-treated rats, as evidenced by an increase in alkane production and in TBARS in urine in this study, and by an increase in alkane production by slices of kidney from iron-treated rats in a previous study [V. C. Gavino, C. J. Dillard, and A. L. Tappel (1984) Arch. Biochem. Biophys. 233, 741-747]. Since PGE2 excretion in urine was not correlated with these effects, lipid peroxidation appears not to be a major factor in renal PGE2 flux.     

Epidermal growth factor in blood and urine of athyreotic adults

The total contribution of the thyroid to epidermal growth factor (EGF) levels in blood and urine was determined in 18 patients cured from thyroid cancer at the time of thyroxine treatment and 6 weeks off it in the state of athyroidism. As compared to the euthyroid state, the athyrodism was associated with a drop in the urinary EGF excretion from 24.8 +/- 8.4 to 11.8 +/- 4.6 ng/mg creatinine (p less than 0.0001) and some increase in the EGF level in blood serum from 159.5 +/- 55.0 to 191.2 +/- 41.0 pM (p = 0.023). We conclude that the thyroid affects the synthesis and/or blood delivery of EGF and is responsible for approximately one half of its urinary excretion.     

Urinary platelet-activating factor excretion is elevated in non-insulin dependent diabetes mellitus.

Proteinuria is currently considered a very sensitive predictor of diabetic nephropathy, but 20-25% of all diabetic patients with negative Albustix reaction excrete higher than normal (< 20 mg/24 h) amounts of albumin in their urine. It is our hypothesis that platelet-activating factor (PAF), a potent glycerophospholipid that acts as a chemical mediator for a wide spectrum of biological activities, including increased vascular permeability, may be produced in significant amounts during periods preceding microalbuminuria. In this study, we compared urinary PAF excretion in Mexican-American subjects who were diagnosed with non-insulin dependent diabetes mellitus (NIDDM) with their healthy control counterparts. The age of the NIDDM subjects (45.9 +/- 2.1 years) was not significantly different from the healthy control group, which was 39.4 +/- 2.7 years (P < 0.0672). The NIDDM subjects (body mass index, 29.9 +/- 1.1 compared to 26.1 +/- 0.9 kg/m2 in healthy controls) were characterized by significantly increased (P < 0.05) fasting plasma glucose (192 +/- 11 vs. 97 +/- 4 mg/dl in healthy controls), fasting insulin (20.9 +/- 2.4 vs. 12.3 +/- 1.6 microU/ml), fasting C-peptide (2.93 +/- 1.26 vs. 1.48 +/- 0.51 ng/ml), and hemoglobin A1c (10.3 +/- 0.7 vs. 5.6 +/- 0.3%), respectively. The urine output for the NIDDM and control subjects were 1942 +/- 191 ml/24 h and 1032 +/- 94 ml/24 h, respectively, and urinary albumin excretion (UAE) rates were estimated to be 38 +/- 7 micrograms/min and 11 +/- 1 micrograms/min, respectively. The NIDDM subjects produced significantly increased levels of urinary PAF (2606.3 +/- 513.1 ng/24 h compared with 77.9 +/- 14.1 ng/24 h in controls (or 1706.3 +/- 420.8 ng/ml compared with 85.4 +/- 17.8 pg/ml of urine, in NIDDM and control subjects, respectively). We found that urinary PAF excretion was significantly correlated with microalbumin excretion (r = 0.7) especially at UAE rates greater than 30 mg/day and more importantly, some NIDDM patients with negative Albustix reaction (i.e. normal UAE) produced significantly more PAF, suggesting that PAF excretion may precede microalbuminuria and that subtle injury to the kidneys are present in NIDDM long before overt albuminuria ensues, urinary PAF measurements could potentially therefore serve as a sensitive indicator of renal injury in diabetes mellitus. These results lend further credence to our hypothesis that PAF may be the biochemical compound linking the various members of the insulin resistance syndrome.     

Determination of estradiol-17-sulfate in human urine by a direct radioimmunoassay: urinary levels throughout the menstrual cycle

The measurement of urinary estradiol-17-sulfate concentration by direct radioimmunoassay was established. The urinary estradiol-17-sulfate levels measured by the radioimmunoassay correlated well with those determined by high-performance liquid chromatography equipped with electrochemical detector. Estradiol-17-sulfate concentrations in early morning urine of six healthy adult men was 8.2 +/- 2.0 ng/mL, or 5.7 +/- 1.8 ng/mg creatinine. The urinary levels in women throughout the menstrual cycle showed a characteristic three-peak excretion pattern: the first and the second peaks appeared just after and three days before the urinary LH peak, and the third peak appeared a few days before menstruation.     

Increased urinary 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine excretion in thalassemia patients: markers for lipid peroxidation-induced DNA damage

Thalassemic diseases including homozygous beta-thalassemia and beta-thalassemia/Hb E (beta-Thal/Hb E) are prevalent in Southeast Asia. Iron overload is a common complication in beta-thalassemia patients which induces intracellular oxidative stress and lipid peroxidation (LPO). LPO end products generate miscoding etheno adducts in DNA which after their repair are excreted in urine. We investigated whether urinary levels of 1,N6-ethenodeoxyadenosine (epsilondA) and 3,N4-ethenodeoxycytidine (epsilondC) can serve as putative cancer risk markers in beta-Thal/Hb E patients. epsilondA and epsilondC levels were assayed in collected urine samples by immunoprecipitation-HPLC-fluorescence and 32P-postlabeling TLC, respectively. Mean epsilondA (fmol/micromol creatinine) levels in urine of beta-Thal/Hb E patients ranged from 4.8 to 120.4 (33.8+/-3.9; n=37) and were 8.7 times higher compared to asymptomatic controls (1.4-13.8; 3.9+/-0.8; n=20). The respective epsilondC levels ranged from 0.15 to 32.5 (5.2+/-1.3; n=37) and were increased some 13 times over controls (0.04-1.2; 0.4+/-0.7; n=20). epsilondC levels were correlated positively with NTBI (r=0.517; P=0.002), whereas epsilondA showed only a trend (r=0.257; P=0.124). We conclude that the strongly increased urinary excretion of etheno adducts indicates elevated LPO-induced DNA damage in internal organs such as the liver. These highly promutagenic lesions may contribute to the increased risk of thalassemia patients to develop hepatocellular carcinoma.     

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.     

An improved method for the measurement of urinary and plasma F2-isoprostanes using gas chromatography-mass spectrometry

We have developed an improved method for the measurement of F2-isoprostanes using stable isotope dilution capillary gas chromatography/electron capture negative ionization mass spectrometry (GC-ECNI-MS). The F2-isoprostane family consists of a series of chemically stable prostaglandin F2 (PGF2)-like compounds generated during peroxidation of arachidonic acid in phospholipids. There is evidence that measurement of F2-isoprostanes represents a reliable and useful index of lipid peroxidation and oxidant stress in vivo. Furthermore, 8-epi-PGF2alpha, which is one of the more abundant F2-isoprostanes, is biologically active, being a potent mitogen and vasoconstrictor of rat and rabbit lung and kidney, as well as a partial agonist of platelet aggregation. Measurement of F2-isoprostanes in biological samples is complex and has involved methods which utilize multiple chromatographic steps, including separation by thin-layer chromatography, leading to poor sample recovery. We now present an improved method for the measurement of plasma and urinary F2-isoprostanes using a combination of silica and reverse-phase extraction cartridges, high-performance liquid chromatography (HPLC), and GC-ECNI-MS. Different approaches to the derivatization of the F2-isoprostanes prior to GC-ECNI-MS are also addressed. The overall recovery of F2-isoprostanes is improved (approx 70% for urine) and the within and between assay reproducibility is 6.7% (n = 23) and 3.7% (n = 3), respectively. The mean urinary excretion of F2-isoprostanes in eight healthy males was 365 +/- 5 pmol/mmol creatinine and in three smokers 981 +/- 138 pmol/mmol creatinine. The mean total (free + esterified) plasma F2-isoprostane concentration was 952 +/- 38 pmol/liter, with a within and between assay reproducibility of 8% (n = 13) and 5.6% (n = 3), respectively. This improved method for the measurement of F2-isoprostanes represents a significant advance in terms of the rapidity and yield in the purification of biological samples. The inclusion of HPLC separation enables improved analysis of F2-isoprostanes by GC-MS. This methodology will assist in defining the role of F2-isoprostanes as in vivo markers of oxidant stress in clinical and experimental settings.     

Quantification of 8-iso-prostaglandin-F(2alpha) and 2,3-dinor-8-iso-prostaglandin-F(2alpha) in human urine using liquid chromatography-tandem mass spectrometry

Quantification of 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) has been suggested to be a reliable indicator of lipid peroxidation that may be related to in vivo free radical generation, oxidative damage, and antioxidant deficiency. We have developed a LC-MS/MS method to quantify 8-iso- PGF(2alpha) and its dinor metabolite, 2,3-dinor-8-iso-prostaglandin F(2alpha) (2,3-dinor-8-iso-PGF(2alpha)), in human urine samples. After an initial purification step using an automated C18 solid phase extraction procedure, the urine sample was injected directly into a liquid chromatography (LC) system and detected with tandem mass spectrometry. The detection limit of the assay was 9 pg for 8-iso-PGF(2alpha) and 3 pg for 2,3-dinor-8-iso-PGF(2alpha) with both inter- and intraday variations of less than 12%. The inaccuracies were less than 3% for both analytes at three different levels. The urinary excretion rate of 2,3-dinor-8-iso-PGF(2alpha) was higher than that of 8-iso-PGF(2alpha), and changed in proportion to the parent compound (R = 0.70, n = 60). Values obtained with this method showed good linear correlation to duplicate 8-iso-PGF(2alpha) measurements performed with GCMS (R = 0.97, n = 15). The mean excretion rates of 8-iso-PGF(2alpha) and 2,3-dinor-8-iso-PGF(2alpha) were significantly higher in smokers than in nonsmokers (0.53 +/- 0.37 vs. 0.25 +/- 0.15 microg/g creatinine, p = 0.002 for 8-iso-PGF(2alpha) and 8.9 +/- 3.8 vs. 4.6 +/- 2.6 microg/g creatinine, p = 0.003 for 2,3-dinor-8-iso-PGF(2alpha), respectively). The excellent accuracy, reproducibility, and high throughput of this method should permit it to be used in large clinical studies and standard clinical laboratories.     

The source of urinary epidermal growth factor in humans

To clarify the source of human urine EGF, we studied EGF renal clearance in 20 healthy, young adult subjects. Immunoreactive EGF was measured hourly in EDTA plasma, heparin plasma, serum and urine of 12 males and 8 females during a 3 h study period. Plasma and urine creatinine and creatinine clearance were measured and calculated hourly. Mean (and SEM) creatinine clearance was similar in males and females (118 +/- 12 vs 105 +/- 6 ml/min). EGF was not detectable in plasma, whereas relatively high levels were measured in serum (2.5 +/- 0.25 vs 1.5 +/- 0.18 ng/ml in males and females respectively p less than 0.05). Urine EGF excretion averaged 1641 +/- 233 ng/h in males and 1507 +/- 191 ng/h in females (p greater than 0.05). A significant correlation was observed between urine creatinine and urine EGF concentrations in both male (r = 0.98, p less than 0.01) and female (r = 0.94, p less than 0.01) subjects. EGF immunoreactivity in urine and serum eluted from G-75 sephadex columns similarly to recombinant 6000 Mr hEGF. Urine excretion of EGF approximated 1.5 micrograms/h or 25 ng/mg creatine. The high concentrations of EGF found in urine in the face of non-detectable levels of EGF in plasma favor the hypothesis that EGF in urine is derived from kidney synthesis and secretion. The significant positive correlation between urine creatinine and urine EGF suggests a functional correlation between glomerular filtration and the process of tubular EGF excretion.     

An imbalance between prostacyclin and thromboxane in relation to cerebral blood flow in neonates with maternal preeclampsia.

OBJECTIVE: A disturbance of prostacyclin (PGI2) and thromboxane A2 (TXA2) balance has been reported in preeclampsia. However, little is known about the concentrations of these prostanoids in neonates born to preeclamptic pregnant women. The purpose of this study is to determine whether the PGI2 and TXA2 concentrations are altered and whether the prostanoid balance correlates to the cerebral blood flow in neonates born to preeclampsia. METHODS: Spontaneously voided urine samples were collected from 20 neonates of normotensive and 16 neonates of preeclamptic women during the first 24 h after birth. We measured by radioimmunoassay the concentrations of urinary 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) and 11-dehydro-thromboxane B2 (11-dehydro-TXB2), respectively. Blood flow velocity in the middle cerebral artery was studied by pulsed Doppler ultrasonography in the neonates between 17 and 38 h after birth. RESULTS: There was no significant difference between the urinary 6-keto-PGF1alpha in the neonates of mothers with and without preeclampsia (median, 5.3 vs. 3.6 ng/mg of creatinine). In contrast, the urinary 11-dehydro-TXB2 and the ratio of 11-dehydro-TXB2 to 6-keto-PGF1alpha in the neonates of mothers with preeclampsia were significantly lower as compared with the neonates without preeclampsia, respectively (13.7 vs. 20.6 ng/mg of creatinine and 3.0 vs. 5.2, median). The resistance index in the middle cerebral artery was significantly reduced in the neonates with preeclampsia than without preeclampsia (0.67 +/- 0.01 vs. 0.74 +/- 0.02, mean +/- SEM). CONCLUSIONS: There was an association between maternal preeclampsia and the imbalance in the neonatal urinary excretion of PGI2 and TXA2 metabolites. This imbalance may contribute to the regulation of cerebral blood flow.     

Human urinary epidermal growth factor: effects of age, sex and pregnancy

Urinary concentrations of immunoreactive human epidermal growth factor (hEGF) were determined by specific homologous radioimmunoassay in 169 healthy men (aged 20-69 years), 275 healthy women (20-8 years). healthy women (20-68 years) and 413 pregnant women (20-39 years). Relative hEGF concentrations in urine (micrograms/g creatinine) decreased significantly in both sexes between 24 and 64 years of age. The relative concentrations of hEGF in urine were significantly higher in women than in men at ages 20-69 years. The mean values of relative urinary hEGF concentrations in pregnant women in their twenties and thirties (30.0 +/- 0.7 micrograms/g creatinine and 29.6 +/- 1.2 micrograms/g creatinine) were significantly higher than those in age-matched nonpregnant women (27.3 +/- 1.8 micrograms/g creatinine and 22.8 +/- 0.7 micrograms/g creatinine). Among the trimesters, it was highest in the 2nd trimester of women in the twenties and thirties (33.4 +/- 1.3 micrograms/g creatinine and 31.7 +/- 1.9 micrograms/g creatinine). The significance of the increased urinary excretion of hEGF (micrograms/g creatinine) in pregnancy is not known. Further studies are required to find a source of hEGF in urine and a possible relation between increased hEGF excretion and fetoplacental growth and development.     

Regulation of prostaglandin biosynthesis by nitric oxide is revealed by targeted deletion of inducible nitric-oxide synthase

We investigated the effects of targeted deletion of the inducible NO synthase (iNOS) gene on the formation of prostaglandins in vivo and ex vivo. Peritoneal macrophages were obtained from control and iNOS-deficient mice, and prostaglandin E(2) (PGE(2)) was quantified after stimulation with gamma-interferon and lipopolysaccharide to induce COX-2. Total nitrate and nitrite production was completely abolished in cells from iNOS-deficient animals compared with control cells. PGE(2) formation by cells from iNOS-deficient animals was decreased compared with cells from control animals 80% at 12 h (0.85 +/- 0.90 ng/10(6) cells versus 15.4 +/- 2.1 ng/10(6) cells, p < 0.01) and 74% at 24 h (9.4 +/- 4.3 ng/10(6) cells versus 36.8 +/- 4.1 ng/10(6) cells, p < 0.01). COX-2 protein expression was not significantly different in cells from control or knockout animals. Levels of PGE(2) in the urine of iNOS-deficient mice were decreased 78% (0.24 +/- 0.14 ng/mg of creatinine versus 1.09 +/- 0.66 ng/mg of creatinine, p < 0.01) compared with control animals. In addition, the levels of urinary F(2)-isoprostanes, an index of endogenous oxidant stress, were significantly decreased in iNOS-deficient animals. In contrast, the levels of thromboxane B(2) derived from platelets allowed to aggregate ex vivo were significantly increased in iNOS-deficient mice compared with wild-type mice. These studies support the hypothesis that NO and/or NO-derived species modulate cyclooxygenase activity and eicosanoid production in vivo.     

Effect of dietary calcium on renal prostaglandins.

The present study was designed to clarify the possible role of renal prostaglandins (PGs) on blood pressure (BP) regulation during calcium (Ca) restriction or supplementation. Twelve normotensive women with a mean age of 21.2 years participated in the study. After 1 week of normal Ca intake (mean +/- SE, 536 +/- 2 mg/day), a low-Ca diet (163 +/- 1 mg/day) was given for a further 1 week. Additional asparagine Ca (3 g as Ca/day) was also given to half of the subjects. BP, heart rate, and serum total and ionized Ca concentrations were measured at the end of each period. Levels of Ca, sodium, PGE2, 6-keto-PGF1 alpha and thromboxane (TX) B2 excreted into urine were also determined. The plasma level of ionized Ca was significantly increased without any change in total Ca in both groups. Low and high Ca intake decreased and increased urinary Ca excretion by 28% and 56%, respectively. BP was not altered after Ca deprivation or loading. However, urinary PGE2 excretion was significantly augmented from 668.9 +/- 68.1 to 959.7 +/- 183.1 ng/day by Ca loading, whereas Ca deprivation decreased PGE2 excretion (695.4 +/- 108.1 to 513.2 +/- 55.2 ng/day). No changes were observed in 6-keto-PGF1 alpha or TXB2 urinary excretion. These results suggest that renal PGE2 synthesis is stimulated or decreased by 1-week Ca loading or deprivation, indicating a possible antihypertensive role of renal PGE2 during high-Ca intake in hypertensives.     

Liquid chromatography-based determination of urinary free and total N(epsilon)-(carboxymethyl)lysine excretion in normal and diabetic subjects

We propose a specific, reproducible and sensitive HPLC method for the determination of N(epsilon)-(carboxymethyl)lysine (CML) excreted in urine. Total CML was measured in acid hydrolysates of urine samples, while free CML was measured in acetonitrile-deproteinised urine samples using a RP-HPLC method with ortho-phtaldialdehyde (OPA)-derivatisation and fluorescence detection suited for automation. We compared the CML excretion of 51 non-proteinuric patients with diabetes mellitus (DM) (age 57+/-14 years, HbA1c 8.0+/-1.8%) to 42 non-diabetic controls (C) (age 45+/-17 years). The urinary excretion of total CML in diabetic patients was increased by approximately 30% (DM: 0.58+/-0.21; C: 0.45+/-0.14 microM/mmol creatinine; P<0.001). While urinary excretion of free CML was not significantly different, excretion of bound CML was increased (DM: 0.36+/-0.17; C: 0.27+/-0.14; P<0.05) in diabetic patients. CML excretion was correlated with protein and albumin excretion, but did not correlate with HbA1c, duration of DM or diabetic complications such as neuropathy or retinopathy. Furthermore, no age-dependent change of total CML excretion was found, while free CML excretion was lower in younger subjects. The specific and sensitive determination of CML by RP-HPLC of its OPA-derivative is well suited for automation and better than that of less defined glycoxidation products (AGEs).     

Determination of urinary 8-epi-prostaglandin F(2alpha) using liquid chromatography-tandem mass spectrometry: increased excretion in diabetics

Liquid chromatography-tandem mass spectrometry (LC/MS-MS) was applied to the quantitative analysis of urinary 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)) level. 8-Epi-PGF(2alpha) and its internal standard, [(2)H(4)]-8-epi-PGF(2alpha), were extracted from urine by using a solid phase extraction cartridge and loaded to LC/MS-MS in selected reaction monitoring (SRM) mode. The standard curve showed good linearity in the range of 40 pg to 10 ng (r = 0. 997). The accuracy of the added 8-epi-PGF(2alpha) ranged from 96.8 to 104.9% with a mean +/- SD of 99.5+/-2.5%. The average level +/- SD of urinary 8-epi-PGF(2alpha) in 13 healthy volunteers (five women and eight men, 31+/-7.4 years old) was 429.4+/-149.6 pg/mg creatinine. The level of seven patients with noninsulin dependent diabetes mellitus (two women and five men, 40+/-13.6 years old), 630.9+/-275.6 pg/mg creatinine, was statistically higher than that of healthy volunteers (P<0.05). This finding suggested that diabetics are in a highly oxidative condition. This simple and rapid LC/MS-MS method can be used to elucidate the pathophysiological feature of diabetes or for monitoring the curative effect.     

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.     

A reduction in alcohol consumption is associated with reduced plasma F2-isoprostanes and urinary 20-HETE excretion in men

There is considerable evidence that chronic moderate-to-high alcohol consumption increases blood pressure. The mechanisms by which this occurs are not clear. Alcohol consumption can induce oxidative stress and cytochrome P450 (CYP450) isoforms that are associated with oxidative stress and may influence vascular tone. To study the role of such mechanisms we examined whether reducing alcohol intake in moderate-to-heavy drinkers (40-110 g/day) resulted in changes in urinary excretion of 20-HETE, a CYP450 metabolite of arachidonic acid, and plasma and urinary F(2)-isoprostanes as markers of lipid peroxidation. After a 4-week run-in period during which healthy men maintained their usual drinking pattern they were randomized to a two-way crossover intervention study. In each of the 4-week treatment periods subjects either substituted their usual alcohol intake with a 0.9% alcohol beer or maintained their usual alcohol intake. Plasma and urinary F(2)-isoprostanes and urinary 20-HETE were measured by gas chromatography mass spectrometry, and serum gamma-glutamyl transpeptidase (gamma-GT) was measured as a biomarker of alcohol consumption, at the end of each study period. Sixteen healthy men age 51.0+/-2.7 years and with a BMI of 26.4+/-0.61 kg/m(2) completed the study. The reductions in alcohol intake (72.4+/-5.0 vs 7.9+/-1.6 g/day, p<0.001) and serum gamma-GT (geometric mean 24.4 U/L (95% CI 19.7, 30.2) vs 18.6 U/L (95% CI 15.5, 22.2, p<0.01) were accompanied by a significant fall in blood pressure as well as urinary 20-HETE excretion (158+/-23 vs 109+/-19 pmol/mmol creatinine, p<0.001) and plasma F(2)-isoprostanes (3438+/-158 vs 2929+/-145 pmol/L, p=0.01). A substantial reduction in alcohol consumption in healthy men lowered plasma F(2)-isoprostanes and urinary 20-HETE. Increased oxidative stress and 20-HETE production may be linked, at least in part, to the pathogenesis of alcohol-related hypertension.     

Glomerular filtration rate and blood pressure monitoring in awake baboons

Minimally invasive techniques were used to collect urine with an external catheter together with automated intermittent monitoring of arterial blood pressure in awake male baboons. Using endogenous creatinine, 24-hour creatinine clearances were measured for 2 to 3 consecutive days in four intact and in four uninephrectomized baboons. Despite large differences in urinary volume and sodium excretion, reproducibility of 24-hour creatinine clearances was within 15% in 15 of 19 studies obtained from 6 of 8 animals. Arterial blood pressure was monitored intermittently at 30 to 60 minute intervals over 24 hours with a Dinamap monitor and recorder. Mean blood pressure averaged 71 +/- 4.4 to 89 +/- 5.5 mm Hg in different animals. Blood pressure tended to be lower at night than during the day. In separate studies using 15 to 60 minute urine collection periods, inulin clearance was compared in awake and in anesthetized animals with endogenous or exogenous creatinine clearance measured simultaneously. The clearance of creatinine systematically exceeded the clearance of inulin, even in intact animals with a normal serum creatinine. The creatinine-to-inulin clearance ratio averaged 1.16 +/- 0.03 at a serum concentration of 0.7 to 0.8 mg/dl; 1.27 +/- 0.03 at a serum creatinine of 1.0 to 1.1 mg/dl and 1.56 +/- 0.04 at a serum creatinine greater than 10 mg/dl. All values exceed unity significantly (p less than 0.001). Thus, renal function, including inulin clearance, can be measured in awake baboons. Duplicate or triplicate 24-hour urine collections are needed to assess the reliability of creatinine excretion. However, creatinine clearance overestimates glomerular filtration rate, as it does in humans.