Divergence in urinary 8-iso-PGF(2alpha) (iPF(2alpha)-III, 15-F(2t)-IsoP) levels from gas chromatography-tandem mass spectrometry quantification after thin-layer chromatography and immunoaffinity column chromatography reveals heterogeneity of 8-iso-PGF(2alpha). Possible methodological,mechanistic and clinical implications

Free radical-catalysed oxidation of arachidonic acid esterified to lipids leads to the formation of the F(2)-isoprostane family which may theoretically comprise up to 64 isomers. We have previously shown that the combination of TLC and GC-tandem MS (referred to as method A) allows for the accurate and highly specific quantification of 8-iso-PGF(2alpha) (iPF(2alpha)-III, 15-F(2t)-IsoP) in human urine. Immunoaffinity column chromatography (IAC) with immobilized antibodies raised against 8-iso-PGF(2alpha) (i.e. 15(S)-8-iso-PGF(2alpha)) has been shown by others to be highly selective and specific for this 8-iso-PGF(2alpha) isomer when quantified by GC-MS. In the present study we established IAC for urinary 8-iso-PGF(2alpha) for subsequent quantification by GC-tandem MS (referred to as method B). This method was fully validated and found to be highly accurate and precise for urinary 15(S)-8-iso-PGF(2alpha). 8-iso-PGF(2alpha) was measured in urine of 10 young healthy humans by both methods. 8-iso-PGF(2alpha) was determined to be 291+/-102 pg/mg creatinine by method A and 141+/-41 pg/mg creatinine by method B. Analysis of the combined through and wash phases of the IAC step, i.e. of the unretained compounds, by method A showed the presence of non-immunoreactive 8-iso-PGF(2alpha) at 128+/-55 pg/mg creatinine. This finding suggests that urinary 8-iso-PGF(2alpha) is heterogenous, with 15(S)-8-iso-PGF(2alpha) contributing by approximately 50%. PGF(2alpha) and other 8-iso-PGF(2alpha) isomers including 15(R)-8-iso-PGF(2alpha) are not IAC-immunoreactive and are chromatographically separated from 15(S)-8-iso-PGF(2alpha). We assume that ent-15(S)-8-iso-PGF(2alpha) is also contributing by approximately 50% to urinary 8-iso-PGF(2alpha). This finding may have methodological, mechanistic and clinical implications.     

Urinary excretion of three nucleic acid oxidation adducts and isoprostane F(2)alpha measured by liquid chromatography-mass spectrometry in smokers, ex-smokers, and nonsmokers

To assess novel liquid chromatography/mass spectrometric methods for measuring oxidative damage to nucleic acids and lipids, we compared urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG), 5-hydroxymethyl-2'-deoxyuridine (5-OHmU), and 8-hydroxyguanosine (8-OxoG), and an isoprostane, 8-iso-prostaglandin F(2)alpha (IsopF(2)alpha) in 234 healthy men (n = 113) and women (n = 121), 80 current smokers, 96 never-smokers), and 58 ex-smokers (no tobacco use for 3 years). The 8-OHdG and 8-OxoG did not differ significantly by group; 5-OHmU was higher in smokers, compared with ex- (p <.003) and never- (p <.0001) smokers and in ex- vs. never-smokers (p =.014) at, respectively, 13.5 +/- 0.7, 11.3 +/- 1.0, and 8.7 +/- 0.3 microg/g creatinine. IsopF(2)alpha was higher in smokers, compared with ex- (p =.007) and never-smokers (p <.0001) and in ex- vs. never- smokers (p =.002) at, respectively, 1.1 +/- 0.10; 0.74 +/- 0.07, and 0.51 +/- 0.04 microg/g creatinine. There were significant correlations among all three nucleic acid adducts and between IsopF(2)alpha and both 5-OHmU and 8-OHdG. Many smokers and ex-smokers had high levels of either 5-OHmU excretion or IsopF(2)alpha excretion, but not both. We conclude that 5-OHmU and IsopF(2)alpha are more discriminating of oxidative stress from tobacco smoke than the other two compounds measured. Whether characteristic patterns of excretion of these indicators forecast differential disease risk should be explored in future research.     

Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and genetic polymorphisms in breast cancer patients

Reactive oxygen species (ROS) causes damage to DNA, but the role of ROS in breast carcinoma is still not clear. The objective of this study was to measure the urinary 8-OHdG levels of breast cancer patients at each stage of carcinogenesis and assess its association with the development of breast cancer. Sixty patients with malignant breast tumors were matched with 60 control subjects of the same ages in this case control study. Urinary 8-OHdG levels were significantly higher among breast cancer patients than among the control subjects, after making adjustments for confounders such as smoking, coffee consumption and use of oral contraceptives. The breast cancer patients were divided into three groups based on the stages of their cancer; urinary 8-OHdG levels decreased with each stage of breast carcinoma. Using multiple regression and logistic models adjusted for other covariates, urinary 8-OHdG levels significantly correlated with the development of breast cancer. However, it was found that breast cancer was not significantly influenced by CYP1A1, CYP1M1 or NAT2 polymorphisms. In conclusion, it was found that oxygen radical generation occurred within carcinoma cells, but the role of polymorphism of specific genes in the development of breast cancer should be evaluated.     

Urinary excretion of prostaglandin E2 and prostaglandin F2alpha in potassium-deficient rats

Potassium-deficiency was induced in rats by dietary deprivation of potassium. The animals became polyuric and urine osmolality decreased more then three-fold compared to controls. Urinary excretion of prostaglandin E2 (PGE2) and prostaglandin F2alpha (PGF2alpha) did not increase during 2 weeks of potassium depletion. Partial inhibition of renal prostaglandin synthesis by meclofenamate did not increase the urine osmolality after water deprivation. These results make unlikely the hypothesis that the polyuria of potassium-deficiency, is the result of enhanced renal synthesis of prostaglandins with subsequent antagonism of the hydro-osmotic effect of vasopressin. Male animals consistently excreted less PGE2 than female animals.     

Urinary excretion of immunoreactive prostaglandin F2 alpha in healthy children and adults

The 24-hours urinary excretion of immunoreactive prostaglandin F2 alpha (U-iPGF2 alpha) in normal children on a free diet was not significantly different in 30 boys (aged 3-15 years; geometric mean 589 ng/24 h) compared to 27 girls (aged 4-14 years; mean 473 ng/24 h). In both sexes this excretion rose with age until adolescence where it reached a plateau. In normal adults the men had significantly higher (p less than 0.001) excretions of U-iPGF2 alpha than the women; also body weight and urinary creatinine excretion were higher in men (p less than 0.001). In the children, as well as in the total population, U-iPGF2 alpha correlated best with body weight (r = 0.44 and r = 0.48 respectively; p less than 0.001) and the urinary creatinine excretion (r = 0.53 and 0.57 respectively; p less than 0.001); both body weight and urinary creatinine excretion are reflections of total body development. After the correction for urinary creatinine excretion or for body weight, the sex difference in the adult U-iPGF2 alpha totally disappeared.     

Quantification of the major urinary metabolite of 15-F2t-isoprostane (8-iso-PGF2alpha) by a stable isotope dilution mass spectrometric assay

The isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical-catalyzed peroxidation of arachidonic acid. The first series of IsoPs characterized contained F-type prostane rings analogous to PGF2alpha. One F-ring IsoP, 15-F2t-IsoP (8-iso-PGF2alpha) has been shown to be formed in abundance in vivo and to exert potent biological activity. As a means to assess the endogenous production of this compound, we developed a method to quantify the major urinary metabolite of 15-F2t-IsoP, 2,3-dinor-5,6-dihydro-15-F2t-IsoP (2,3-dinor-5, 6-dihydro-8-iso-PGF2alpha), by gas chromotography/negative ion chemical ionization mass spectrometry. This metabolite was chemically synthesized and converted to an 18O2-labeled derivative for use as an internal standard. After purification, the compound was analyzed as a pentafluorobenzyl ester trimethylsilyl ether. Precision of the assay is +/-4% and accuracy is 97%. The lower limit of sensitivity is approximately 20 pg. Levels of the urinary excretion of this metabolite in 10 normal adults were found to be 0. 39 +/- 0.18 ng/mg creatinine (mean +/- 2 SD). Substantial elevations in the urinary excretion of the metabolite were found in situations in which IsoP generation is increased and antioxidants effectively suppressed metabolite excretion. Levels of 2,3-dinor-5, 6-dihydro-15-F2t-IsoP were not affected by cyclooxygenase inhibitors. Thus, this assay provides a sensitive and accurate method to assess endogenous production of 15-F2t-IsoP as a means to explore the pathophysiological role of this compound in human disease.     

Urinary excretion of 5beta-pregnane-3alpha,20alpha,21-triol in human gestation

The steroids in urine from normal pregnant women have been studied. After extraction of conjugate steroids, solvolysis and enzymatic hydrolysis, the liberated steroids were separated by chromatography on Sephadex LH-20, and were analysed by gas-liquid chromatography and gas chromatography mass spectrometry. The following steroids were isolated and completely identified in the LH-20 fraction 7: 5beta-pregnane-3alpha,20alpha-diol, 5beta-pregnane-3alpha,17,20alpha-triol, 5beta-pregnane-3alpha,20alpha,21-triol and 5alpha-pregnane-3beta,16alpha,20alpha-triol. In addition, two metabolites tentatively identified as 5xi-pregnane-2xi,3xi,20xi-triol and 2xi,3xi,16xi-trihydroxy-5xi-pregnan-20-one, have not been reported as occcurring in urine from pregnant women. The 5beta-pregnane-3alpha,20alpha,21-triol was detected only in the third trimester of pregnancy and the urinary excretion values are between 320 and 650 microgram per 24 h. With the present data, it is not possible to establish the precursor(s) of this steroid. However, these results tentatively suggest that 5beta-pregnane-3alpha,20alpha,21-triol arises from foeto-placental unit.     

Kinetic studies of bioactive products nitric oxide and 8-iso-PGF(2alpha) in Burkholderia pseudomallei infected human macrophages, and their role in the intracellular survival of these organisms

The oxidative response of Burkholderia pseudomallei and Escherichia coli infected macrophages from normal and melioidosis subjects was determined by measuring the production of nitric oxide which is one of the reactive nitrogen intermediates, and the activation state of these macrophages was determined by measuring the generation of 8-iso-PGF(2alpha), a bioactive product of free radical induced lipid peroxidation. Macrophages obtained from the melioidosis patients generated significantly lower levels of nitric oxide and 8-iso-PGF(2alpha) compared to macrophages obtained from the normal subjects (P<0.001). The reduced efficiency of the oxygen dependent microbicidal mechanism in macrophages of melioidosis patients may be one of the survival strategies developed by B. pseudomallei to remain viable intracellularly.     

Urinary excretion of uric acid,allantoin, and 8-OH-Deoxyguanosine in uricase-knockout mice

ABSTRACT

Although uricase-knockout (Uox KO) mice are reported to develop uric acid (UA) nephropathy, those that mature without severe nephropathy could be useful for research into purine metabolism in humans. In this study, we measured the urinary excretion of creatinine, UA, allantoin, and 8-hydroxy-2′-deoxyguanosine (8-OHdG) collected from Uox KO mice housed in metabolic cages. UA and allantoin were determined using liquid chromatography–mass spectrometry and creatinine and 8-OHdG were measured with a commercial kit. Uox KO mice excreted significantly higher levels of UA than wild-type mice (C57BL/6), while the excretion of allantoin was significantly lower. Urinary allantoin was detected in Uox KO mice despite a lack of uricase, which is the same as in humans. In contrast to the elevated levels of UA, the daily excretion of 8-OHdG, an oxidative stress marker, was lower in Uox KO mice. UA is thought to act as an anti-oxidizing agent in humans; thus, these results show that Uox KO mice are potential animal models for research into human purine metabolism.     

LDL-apheresis decreases plasma levels and urinary excretion of 8-epi-PGF2alpha

Isoprostanes (IP) generated during free radical catalyzed oxidation injury have been claimed as a reliable indicator of oxidative stress in vivo. In particular, they are formed during LDL-oxidation. Vascular content, plasma levels and urinary excretion of IP were reported to be elevated in hypercholesterolemia. We therefore assessed the values of the IP 8-epi-PGF2alpha in plasma and urine in nine patients (7 males, 2 females) suffering from severe heterozygous hypercholesterolemia before and after LDL-apheresis as well as during the interval. LDL-apheresis caused a significant (P<0.01) drop in 8-epi-PGF2alpha in plasma and urine. The respective values in smokers (n = 4) were significantly (P<0.01) higher as compared to non-smokers. No sex difference was seen. Together with the findings of a parallel decrease in oxidized LDL, these data show a significant benefit of LDL-apheresis reducing in vivo oxidation injury. This benefit may at least partly contribute to the clinical improvement seen in the patients treated.     

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.     

Urinary excretion of digoxin-like factor (DLF) and ADH during DOCA-salt and Goldblatt 2 kidney-1 clip hypertension development

Urinary digoxin-like factor, ADH, sodium and potassium excretion and urine osmolality were studied during the development of two pathogenically different models of hypertension, DOCA-salt (low-renin) and Gold-blatt 2 kidney-1 clip (renin-dependent). Urinary digoxin-like factor was increased in rats that were given saline (NaCl 1%) to drink, uninephrectomized-salt and DOCA-salt rats, with no significant differences between the two groups urinary ADH was elevated in DOCA-salt rats during the study, compared with uninephrectomized-salt rats. Urinary digoxin-like factor and urinary ADH were not significantly modified in Goldblatt 2 kidney-1 clip and sham-operated rats. In addition, positive correlations between digoxin-like factor urinary excretion and urinary ADH and also with sodium urinary excretion were found. These data suggest that: a) digoxin-like factor and ADH could play a role in the pathogenesis of DOCA-salt but not in Goldblatt 2 kidney-1 clip hypertension. b) A common mechanism may stimulate ADH and digoxin-like factor simultaneously. c) Digoxin-like factor plays a role in the control of urinary sodium excretion.     

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.     

Urinary excretion of 2,7, 8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman is a major route of elimination of gamma-tocopherol in humans

Little is known of the post-absorptive, metabolic fate of gamma-tocopherol, the major form of vitamin E in North American diets. The objective of this study was to determine the extent of urinary excretion of 2,7, 8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), a recently identified metabolite of gamma-tocopherol. A method for measurement of urinary gamma-CEHC was developed, using gas chromatography-mass spectrometry (GC-MS) with a deuterated internal standard, 2,7,8-trimethyl-2-(beta-carboxyethyl)-(3, 4-2H2)-6-hydroxychroman (d2-gamma-CEHC). This standard was synthesized by dehydrogenation of 6-acetyl-gamma-CEHC followed by deuteration of the resulting 3,4-double bond. The use of d2-gamma-CEHC resulted in accurate determinations of the concentration of d0-gamma-CEHC in human urine. Urine samples containing added d2-gamma-CEHC were treated with beta-glucuronidase, extracted with an organic solvent, and analyzed by GC-MS. Analysis of 24-h urine pools from healthy subjects revealed gamma-CEHC concentrations, normalized against creatinine, ranging from 2.5 to 31.5 micromol/g creatinine, or a total of 4.6 to 29.8 micromol per day. These results correspond to 2-12 mg gamma-tocopherol excreted daily as gamma-CEHC in the urine. Given an estimated mean intake of gamma-tocopherol of 20 mg/day, catabolism of gamma-tocopherol to gamma-CEHC, followed by glucuronide conjugation and urinary excretion, is a major pathway for elimination of gamma-tocopherol in humans.     

Urinary excretion of oestrone conjugates and gonadotrophins during pregnancy in the Goeldi's monkey (Callimico goeldii)

Oestrone conjugate and LH/CG were measured in the urine of 4 Goeldi's monkeys during 6 pregnancies. The gestational length was a mean of 148.8 days from the post-partum LH/CG peak to parturition. CG was first detected a mean of 18.8 days after the LH/CG peak and values remained elevated for a mean of 44.8 days. Three different gonadotrophin assays were used to detect LH/CG: the mouse in-vitro interstitial cell bioassay, a mixed heterologous LH RIA, and a monkey CG RIA. The mouse in-vitro interstitial cell bioassay was useful for measuring both the LH peak which occurred post partum and the CG concentrations during pregnancy. However, both immunoassays were inconsistent in measuring LH due to poor cross-reactivity or lack of specificity; CG concentrations were measurable. Oestrone conjugates became elevated at the time of the LH/CG peak and concentrations continued to increase throughout pregnancy, reaching peak levels before parturition. The postpartum interval, pregnancy and parturition can therefore be monitored in the Goeldi's monkey by the use of urinary assays: those for bioactive LH and immunoreactive oestrone conjugates to determine the post-partum LH peak and those for immunoreactive LH/CG and immunoreactive oestrone conjugates to follow pregnancy and parturition.