Simplified assay for the quantification of 2,3-dinor-6-ketoprostaglandin F1α by gas chromatography—mass spectrometry

Endogenous prostacyclin production is best assessed by the measurement of its excreted metabolites, of which a major one is 2,3-dinor-6-ketoprostaglandin F_1α (2,3-dinor-6-keto-PGF_1α). Gas chromatographic—mass spectrometric (GC—MS) assays have been developed for this compound but are cumbersome and time-consuming. We now report a modified assay for the measurement of 2,3-dinor-6-keto-PGF_1α employing GC—MS in which sample preparation time is markedly shortened by replacing a number of extraction steps with reversed-phase column extraction and by modifying derivatization procedures. Precision of the assay is ± 5% and the accuracy is 98%. The lower limit of detection in urine is approximately 15 pg/mg creatinine. Normal urinary levels of this metabolite were found to be 141 ± 54 pg/mg creatinine (mean ± S.D.). Urinary excretion of 2,3-dinor-6-keto-PGF_1α is markedly altered in situations associated with abnormalities of prostacyclin generation when quantified using this assay. Thus, this assay provides a sensitive and accurate method to assess endogenous prostacyclin production and to further explore the role of this compound in human health and disease.     

Determination of seven prostanoids in 1 ml of urine by gas chromatography—negative ion chemical ionization triple stage quadrupole mass spectrometry

In an isotope dilution assay, prostaglandin (PG) E₂, 6-keto-PGF_1α, thromboxane (Tx) B₂ and their metabolites PGE-M (11α-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid), 2,3-dinor-6-keto-PGF_1α, 2,3-dinor-TxB₂ and 11-dehydro-TxB₂ were determined in urine by gas chromatography—triple stage quadrupole mass spectrometry (GC—MS—MS). After addition of deuterated internal standards, the prostaglandins were derivatized to their methoximes and extracted with ethyl acetate—hexane. The sample was further derivatized to the pentafluorobenzylesters and purified by thin-layer chromatography (TLC). Three zones were scraped from the TLC plate. The prostanoid derivatives were converted to their trimethylsilyl ethers and the products were quantified by GC—MS—MS. In each run, two or three prostanoids were determined.     

Excretion of primary prostanoids and their metabolites during acute volume expansion

Simultaneous determination of urinary excretion rates of primary unmetabolized prostanoids and their enzymatic metabolites were performed by gas chromatography-mass spectrometry (GC/MS) or tandem mass spectrometry (GC/MS/MS). Changes in kidney function were induced by acute (4 h) volume expansion. Despite marked changes in urine flow, GFR, urinary pH, osmolality, sodium and potassium excretion, only a insignificant or transient rise in the enzymatic prostanoid metabolites (2,3-dinor-6-keto-PGF_1α, PGE-M, 2,3-dinor-TxB₂ and 11-dehydro-TxB₂) was observed. The excretion rates of the primary prostanoids were elevated in parallel with the rise in urine flow: PGE₂ rose (p < 0.05) from 14.2 ± 4.0 to 86.2 ± 20.7, PGF_2α from 60.0 ± 4.9 to 119.8 ± 24.0, 6-keto-PGF_2α from 7.2 ± 1.3 to 51.5 ± 17.0, and txB₂ from 11.2 ± 3.3 to 13.6 ± 3.6 ng/h/1.73 m² ( ) at the maximal urine flow. Except for 6-keto-PGF_1α and TxB₂, this rise in urinary prostanoid levels was only transient despite a sustained fourfold elevated urine flow. We conclude that urine flow rate acutely affect urine prostanoid excretion rates, however, over a prolonged peroid of time these effects are not maintained. The present data support the concept that urinary levels of primary prostanoids mainly reflect renal concentrations whereas those of enzymatic metabolites reflect systemic prostanoid activity. From the excretion pattern of TxB₂ one can assume that this prostanoid represents renal as well as systemic TxA₂ activity.     

Role of renal prostaglandins in the fall in urine osmolality after papillary micropuncture

The effect of micropuncture of the renal papilla through an intact ureter on urinary concetrating ability of rats was examined. Micropuncture of the renal papilla caused a fall in urine osmolality in the punctured kidney from 1718 ± 106 to 1035 ± 79 mosmol/kg·H₂O. In order to investigate the role of renal prostaglandins in this process, PGE₂ excretion was measured and found to increase from 63.4 ± 14.0 to 205.5 ± 57.1 pg/min. Urine osmolality and PGE₂ excretion from the contralateral kidney were not significantly altered. In animals given meclofenamate (2 mg/kg·hr), renal PGE₂ excretion was reduced to 22.3 ± 5.1 pg/min prior to micropuncture and it remained low at 8.9 ± 1.8pg/min after papillary micropuncture. Meclofenamate also blocked the fall in urine osmolality caused by micropuncture of the renal papilla, with urine osmolality averaging 1940 ± 122 before and 1782 ± 96 mosmol/kg·H₂O after the micropuncture. These results indicated that papillary micropuncture through an intact ureter increased renal PGE₂ excretion and that a rise in renal production of PGE₂ or some other prostanoid is associated with a fall in urine concentrating ability.     

Regulation of urinary thromboxane B2 in man: Influence of urinary flow rate and tubular transport

Thromboxane B₂ (TxB) is excreted in human urine, but the mechanism of renal excretion and the quantitative relationship of urinary TxB to the active parent compound, thromboxane A₂, of renal or extrarenal origin is not established. To determine the effects of vasoactive hormones, uricosuric agents and urinary flow rate on TxB excretion, urinary TxB was measured by radioimmunoassay and mass spectrometry, and renal metabolism of blood TxB was determined by radiochromatography of urine after i.v. [³H]-TxB infusions. Basal TxB was 6.7 ± 1.1 ng/h during an oral water load, and TxB fell with s.q. antidiuretic hormone (to 3.4 ± 0.4 ng/h, P<0.01) and with fluid restriction (to 2.6 ± 0.5 ng/hr, P=0.001) in parallel with urinary volume. Urinary excretion of unmetabolized [³H]-TxB also fell (by 56%) with fluid restriction, implicating altered metabolism rather than synthesis as the mechanism of the urinary flow effect. Angiotensin II infusions slightly reduced both TxB and urine volume, consistent with a flow effect. In contrast, probenecid did not alter urine volume, but increased urinary uric acid (by 244%), TxB (from 5.6 ± 0.9 to 11.1 ± 2.9 ng/h) and urinary excretion of blood [³H]-TxB (by 243%) by similar amounts (all P<0.05), suggesting that TxB is actively reabsorbed in the proximal tubule, similarly to uric acid. Thus, urinary excretion of TxB of renal and extrarenal origin is regulated by proximal and distal tubule factors.     

Biomarkers of exposure and potential harm in adult smokers of 3–7 mg tar yield (Federal Trade Commission) cigarettes and in adult non-smokers

Abstract

The paper reports levels of 24-h urine nicotine and five of its major metabolites (expressed as nicotine-equivalents) and blood carboxyhaemoglobin as biomarkers of exposure to particulate- and gas-phase cigarette smoke, respectively, from an exploratory pilot study of adult smokers of 3.0–6.9 mg tar delivery (Federal Trade Commission (FTC) method) cigarettes. On multiple occasions over 6 weeks, blood high-sensitivity C-reactive protein (hs-CRP), fibrinogen, HDL- and LDL-cholesterol, and 24-h urine 8-epi-prostaglandin F_2α (8-epi-PGF_2α) and 11-dehydro-thromboxane B₂ (11-dehydro-TxB₂) were also evaluated as biomarkers of potential harm. All the biomarkers examined, except for LDL-cholesterol, discriminated with high sensitivity and specificity between adult smokers and non-smokers overall. Except for HDL-cholesterol, all biomarker medians were greater in adult smokers than in non-smokers: urine nicotine-equivalents 64.514 versus??1 creatinine (p<0.001), carboxyhaemoglobin 4.0 versus 0.4% saturation (p<0.001), hs-CRP 0.27 versus 0.12 mg dl^?1 (p=0.05), fibrinogen 292 versus 248 mg dl^?1 (p<0.001), HDL-cholesterol 46 versus 53 mg dl^?1 (p=0.003), LDL-cholesterol 119 versus 109 mg dl^?1 (p=0.18), urine 8-epi-PGF_2α 1935 versus 1034 pg mg^?1 creatinine (p<0.001) and urine 11-dehydro-TxB₂ 973 versus 710 pg mg^?1 creatinine (p<0.001). All the biomarkers of exposure and most of the biomarkers of potential harm showed no time of sampling (by visit week) effect.     

Prostaglandin (PG) analysis in urine of humans and rats by different radioimmunoassays: Effect on PG-excretion by PG-synthetase inhibitors, laparotomy and furosemide

Thw radioimmunological (RIA) determination of prostaglandin (PG) E₂ and of PGF_2α in urine humans and rats is described in detail. After extraction and chromatography PGE₂ was determined by using a PGE specific antibody or by using either PGB or PGF_2α specific antibodies after the respective conversion procedures. The three different RIA procedures were compared to each other. PGF_2α was determined by a specific antibody to PGF_2α. Basal excretion of PGE₂ and of PGF_2α in healthy women on free diet was 9.3 ng/hour ± 0.96 and 18.3 ng/hour ± 2.5 respectively. Furosemide increased the excretion of PGE₂ and of PGF_2α in humans significantly, while PG-excretion rates decreased on indomethacin. In rat urine PGE₂ and PGE_2α increased markedly from 46.2 pg/min ± 9.3 and 27 ± 3.4 to 253.8 ± 43.3 and 108 ± 12.6 pg/min (per one kidney) in the anesthetized-laparotomized animal. This increase was abolished after giving two different PG synthetase inhibitors.     

Analysis of the thromboxane/prostacyclin balance in human urine by gas chromatography/selected ion monitoring: abnormalities in diabetics

We microanalyzed 2,3-dinor-6-keto-prostaglandin F_1α (2,3-dinor-6-keto-PGF_1α 1) and 11-dehydrothromboxane B₂ (11-dehydro-TXB₂, 2) in human urine. Samples containing a [²H₄]-analogue as an internal standard were extracted by chromatography using Sep Pak tC18 and silica gel. The compounds were then analysed by means of the lactone ring opening reaction and dimethylisopropylsilylation. The conversion of 1 to 1-methyl ester (ME)-propylamide (PA)-9,12,15-dimethylisopropylsilyl (DMIPS) ether derivative and of 2 to 1-ME-6-methoxime (MO)-9,12,15-tris-DMIPS ether derivative was followed by gas chromatography/selected ion monitoring (GC/SIM). Interfering substances from the urine matrix were eliminated during GC/SIM analysis using a DB-5 column. We were able to detect 1 (222–1031 pg/mg creatinine) and 2 (18–155 pg/mg creatinine) in human urine. Furthermore, the thromboxane/prostacyclin (IX/PGI) ratio in the urine of diabetics was higher than that of healthy volunteers. This method can be used to determine the TX/PGI balance in human urine.     

Smoking alters thromboxane metabolism in man

Chronic smoking is a major risk factor of atherosclerosis and coronary heart disease. The measurement of three major thromboxane A2 metabolites, 11-dehydrothromboxane B2, 2,3-dinorthromboxane B2 and thromboxane B2, in the urines of 13 apparently healthy smokers (average 39 years, range 27-56 years) showed significantly elevated excretion rates for all thromboxane A2 metabolites as compared to 10 apparently healthy age-matched non-smokers (average 37 years, range 26-56 years). Importantly, characteristic alterations in the thromboxane A2 metabolite pattern were found in the urines of smokers. The contribution of 2,3-dinorthromboxane B2 to total measured excretion of thromboxane A2 metabolites was 59.2% in smokers (404.0 +/- 53.0 pg/mg creatinine) versus 19.4% in non-smokers (85.2 +/- 8.3 pg/mg creatinine), that of 11-dehydrothromboxane B2 35.7% in smokers (673.2 +/- 88.9 pg/mg creatinine) as compared to 75.5% in non-smokers (332.6 +/- 30.9 pg/mg creatinine). The contribution of thromboxane B2 (57.5 +/- 7.7 pg/mg creatinine in smokers versus 21.9 +/- 1.5 pg/mg creatinine in non-smokers) was similar at 5.1%. The excretion of cotinine, the major urinary metabolite of nicotine that correlates well with the reported daily cigarette consumption (r = 0.97, P less than 0.0001), showed a good correlation to thromboxane A2 metabolite excretion (2,3-dinorthromboxane B2: r = 0.92, P less than 0.0001; 11-dehydrothromboxane B2; r = 0.87, P less than 0.0001).     

Determination of 9α,11β-prostaglandin F2 in human urine and plasma by gas chromatography—mass spectrometry

Sensitive and specific assay methods for 9α,11β-prostaglandin F₂ (9α,11β-PGF₂) by gas chromatography—mass spectrometry with electron impact ionization are described. The mass spectrometric assay for 9α,11β-PGF₂ was based on the use of the methyl ester—dimethylisopropylsilyl ether derivative, and pentadeuterated PGF_2α as a convenient internal standard. The calibration graph for 9α,11β-PGF₂ was linear from 5 pg to 100 ng for both the standard and spiked biological samples. The limit of detection was 50 pg/ml for urine and 25 pg/ml for plasma (signal-to-noise RATIO = 2.3). The method was applied to the determination of 9α,11β-PGF₂ in urine and plasma samples from patients with bronchial asthma.     

Structure and quantitative determination of the major urinary metabolite of thromboxane B2 in the guinea pig

2,3-Dinor-thromboxane B₂ was the major urinary metabolite of thromboxane B₂ in the guinea pig. The structure was assessed mainly by mass spectrometric analysis of a number of derivatives of the metabolite and by chemical degradation by oxidative ozonolysis. A method for quantitative determination of 2,3-dinor-thromboxane B₂ in guinea pig urine based on multiple ion analysis and octadeuterated 2,3-dinor-thromboxane B₂ as internal standard was developed. The basal excretion of the metabolite was 65 ± 36 (S.D.) ng/kg × 24 h (n = 19; range 19–140 ng). This level corresponded to an endogenous synthesis of 543 ± 300 ng of TXB₂. No increase in the excretion was seen after anaphylaxis, in contrast to what has earlier been reported for PGF_2α.     

Thromboxane receptor blockade improves cyclosporine nephrotoxicity in rats

Cyclosporine A (CyA) nephorotoxicity is associated with impaired renal hemodynamic funtion and increased production of the vasoconstrictor eicosanoid thromboxane A₂ (TxA₂). In CyA toxic rats, renal dysfunction cna be partially reversed by inhibitors of thromoboxane sysnthase. However, interpretation of these results is complicated since inhibitance of thromboxane synthase may cause accumulation of prostaglandin endoperoxides that can act as partial agonists at the TxA₂ receptor and may blunt the efficacy of treatment. Furthermore, these endoperoxides may be used as substrate for production of vasodilator prostaglandins causing beneficial effects on hemodynamics which are independent of thromboxane inhibition. To more specially examine the role of TxA₂ in CyA toxicity, we investigated the effects of the thromboxane receptor antagonist GR32191 on renal hemodynamics in a rat model of CyA nephrotoxicity. In this model, administration of CyA resulted in a significant decrease in glomerular filtration rate (GFR) 2.85±0.26 [CyA] vs 6.82±0.96 ml/min/kg [vehicle]; p<0.0005) and renal blood flow (RBF) (21.6±2.31 [CyA] vs 31.8±3.60 ml/min/kg [vehicle]; p<0.025). Renal vascular resistance (RVR) was significantly higher in rats given CyA compared to animals treated with CyA vehicle (5.32±0.55 [cyCyA] vs 3.54±0.24 mm Hg/min/ml/kg [vehicle]; p<0.05). These hemodynamic alterations were associated with a significant increase in urinary excretion of unmetabolized, “native” thromboxane B₂ (TxB₂ (103±18 [CyA] vs 60±16 pg/hour [vehicle]; p<0.05). Only minimal histomorphologic changes were apparent by light microscopic examination of kidneys from both CyA and vehicle treated animals. However, with immunoperoxidase staining, a significantly greater number of cells experssing the rat common leukocyte antigen was found in the renal interstitium of rats given CyA^*. There was no detectable increase in monocytes/macrophages in the kidneys of CyA toxic animals. In rats treated with CyA, intraarterial infusion of GR32191 at maximally tolerated doses significanlty increased GFR and RBD, and decreased RVR. Although both RBF and RVR were restored to levels not different from controls, GFR remained significantly reduced following administration of GR32191. These data suggest that the potent vasoconstrictor TxA₂ plays an important role in mediating renal dysfunction in CyA nephrotoxicity. However, other factors may be important in producing nephrotoxicity associated with CyA.     

Development of a competitive enzyme immunoassay for 17α-19-nortestosterone

Because 17β-19-nortestosterone and its esters are frequently used anabolic steroids in cattle in Europe, there is a need for an assay that can also detect certain metabolites. The enzyme immunoassay described here involves the detection and quantitation of the major metabolite 17α-19-nortestosterone in urine. The assay is based on the coating of an antibody raised in a rabbit against 17α-19-nortestosterone-3-carboxy-methyloxime—bovine serum albumin (17α-19-NT-3-CMO-BSA), the competitive incubation of 17α-19-NT and the 17α-19-nortestosterone-3-CMO—horseradish peroxidase label, followed by the detection of the blue colour developed by the action of the enzyme on tetramethylbenzidine. The 3-CMO conjugate of 17α-19-nortestosterone was used to produce an antibody with selective affinity for the 17α-epimer. For the optimization of the assay, different coatings and incubation conditions were tested. The standard curve ranged between 0.98 and 4000 pg per well, with a B/B₀ 50% of ± 65 pg per well. Colour was measured with a microtitre plate reader. The method was validated by means of certified blank and spiked cattle urine samples.     

Gas chromatographic—mass spectrometric assay to measure urinary N-methylhistamine excretion in man

An assay has been developed for N^τ-methylhistamine, a major metabolite of the autocoid histamine, based on gas chromatography—electron-capture negative-ion chemical ionisation mass spectrometry. N^τ-Methylhistamine was extracted from urine by cation-exchange chromatography and converted to its di-(3,5-bistrifluoromethylbenzoyl) derivative. The latter has good chromatographic properties and gives a negative-ion mass spectrum with the molecular ion (M, m/z 605) as base peak. A commercially available trideuterated analogue of N^τ-methylhistamine was used as internal standard. Basal urinary excretion of N^τ-methylhistamine in five normal subjects was found to be 0.21 ± 0.05 μmol/h (289 ± 74 μmol/mol of creatinine). This value was not significantly altered in these subjects following the infusion of a sub-pharmacological dose of histamine. In eight atopic volunteers, basal urinary excretion of N^τ-methyl-histamine was also not significantly changed following challenge with inhaled allergen.     

Paired analysis of thromboxane and prostacyclin metabolites in urine from healthy mothers and their children

Eicosanoids have been implicated in the adaptation of the fetal to the neonatal circulation, but biochemical support for an activation of their synthesis in relation to birth has not been presented. We addressed this by assessing in pairs the excretion of two metabolites of thromboxane A2, 11-dehydro-TxB2 (dTx) and 2,3-dinor-TxB2 (Tx-M), and one metabolite of prostacyclin, 2,3-dinor-6-keto-PGF1 a (PGI-M), in the first voided urine from 13 healthy term neonates and in the immediate pre-delivery urine from their respective mothers. The excretion of dTx was higher (p less than 0.01) in the neonates than in their mothers (7430 and 1330 pg/mg creatinine, respectively), and so was the excretion of Tx-M (3730 and 900 pg/mg creatinine, respectively, p less than 0.001). Also the excretion of PGI-M was higher (p less than 0.05) in the neonates than in their mothers (2550 vs. 1510 pg/mg creatinine). Amniotic fluid contained detectable levels of both Tx-M and PGI-M. These data indicate that activation of platelets takes place in the neonate after separation of the fetal and maternal vascular circuits. The possible physiological implication of such activation requires further studies.     

A method for measuring the unstable thromboxane A2: Radioimmunoassay of the derived mono-o-methyl-thromboxane B2

A radioimmunoassay was developed for a mono-O-methyl derivative of thromboxane B₂. The antibodies showed high specificity for this compound and cross reacted only 1.2% with thromboxane B₂ and less than 0.1% with prostaglandins and prostaglandin metabolites. The method had a sensitivity of 7 picog. The radioimmunoassay was employed in studies where thromboxane A₂ was generated in human platelets and immediately converted into mono-O-methyl thromboxane B₂ by treatment of the sample with a large volume of methanol. In some of the experiments, thromboxane B₂ was simultaneously measured by a separate radioimmunoassay. Using these two assays it was demonstrated that thromboxane A₂ could be detected only during the earlier stages of the platelet aggregation, whereas thromboxane B₂ rapidly reached a constant level. In a separate experiment, the half-life of thromboxane A₂ in buffer was found to be 32.5±2.5 (S.D.) sec at 37°C; the compound was more stable at lower temperatures. The for thromboxane A₂ was also considerably longer in plasma.     

Determination of leukotriene E4 in human urine using liquid chromatography–tandem mass spectrometry

A liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method was developed for the quantitation of urinary leukotriene E₄ (LTE₄). LTE₄ and its internal standard were extracted by solid-phase extraction and analysed using LC–MS–MS in the selected reaction monitoring (SRM) mode. A good linear response over the range of 10 pg to 10 ng was demonstrated. The accuracy of added LTE₄ ranged from 97.0% to 108.0% with a mean and SD of 100.6±2.4%. We detected LTE₄ (63.1±18.7 pg/mg creatinine, n=10) in healthy human urine. This method can be used to determine LTE₄ in biological samples.     

Determination of 6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α, thromboxane B2, 2,3-dinor-thromboxane B2, PGE2, PGD2 and PGF2α in human urine by gas chromatography—negative ion chemical ionization mass spectrometry

A method for quantification of 6-keto-PGF_1α, 2,3-dinor-6-keto-PGF_1α, TXB₂, 2,3-dinor TXB₂, PGE₂, PGD₂ and PGF_2α in human urine samples, using gas chromatography—negative ion chemical ionization mass spectrometry, is described. Deuterated analogues were used as internal standards. Methoximation was carried out in urine samples which were subsequently applied to phenylboronic acid cartridges, reversed-phase cartridges and thin-layer chromatography. The eluents were further derivatized to pentafluorobenzyl ester trimethylsilyl ethers for final quantification by gas chromatography—mass spectrometry. The overall recovery was 77% for tritiated 6-keto-PGF_1α and 55% for tritiated TXB₂. Urinary levels of prostanoids were determined in a group of six volunteers before and after intake of the thromboxane synthase inhibitor Ridogrel, and related to creatinine clearance.     

Monitoring of mycotoxin biomarkers in the Czech Republic

AFM₁ was determined in 72 (72%) samples of human urine, range 19-6064 pg/g creatinine, mean 367 pg/g creatinine, median 158 pg/g creatinine and 90% percentile 755 pg/g creatinine in 1997. AFM₁ was determined in 46 (43.8%) samples of human urine, range 21-19219 pg/g creatinine, mean 414 pg/g creatinine, median 96 pg/g creatinine and 90% percentile 415 pg/g creatinine in 1998. OTA was determined in 2077 (94.2%) samples of human serum, range 0.1–13.7 μg/L, mean 0.28 μg/L, median 0.2 μg/L and 90% percentile 0.5 μg/L in 1994–2002. OTA was determined in 12 (40%) samples of human kidneys, range 0.1–0.2 μg/kg, mean 0.07 μg/kg, and median 0.05 μg/kg in 2001. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004.     

Electron capture gas chromatographic detection of thromboxane B2

An electron capture gas chromatographic method is described for the detection of thromboxane B₂. Thromboxane B₂ is esterified with diazomethane, followed by treatment with pentafluorobenzylhydroxylamine hydrochloride and silylation with BSA. In pyridine, the free aldehyde form of the acetal ring is favored allowing rapid formation of a novel thromboxane B₂ pentafluorobenzyloxime. The method has been applied to detect thromboxane B₂ formation during aggregation of washed platelets. It must be emphasized that by ordinary analytical standards, the derivatization reproducibility from 50–375 nanograms is poor (±11% – ±42%); however, the improved selectivity of the method and its ability to detect nanogram levels of thromboxane B₂ make it a useful complement to commonly employed bioassay techniques.