Frank-Mathias Gutzki

Urinary 8-iso-Prostaglandin F2α as a Risk Marker in Patients With Coronary Heart Disease A Matched Case-Control Study

Background—Oxidative stress is involved in the pathophysiology of atherosclerosis, diabetes mellitus, hypertension, obesity, and cigarette smoking, all of these being risk factors for coronary heart disease (CHD). We tested the hypothesis that risk factors of CHD are associated with abundant systemic oxidative stress. Methods and Results—We conducted a case-control study with 93 CHD patients and 93 control subjects frequency-matched by age and sex. Urinary excretion of the F2-isoprostane 8-iso-prostaglandin (PG) F2&agr; and its major urinary metabolite, 2,3-dinor-5,6-dihydro-8-iso-PGF2&agr;, were measured by gas chromatography–tandem mass spectrometry. Body mass index, systolic blood pressure, and C-reactive protein were elevated in CHD patients (P <0.01). Urinary 8-iso-PGF2&agr; and 2,3-dinor-5,6-dihydro-8-iso-PGF2&agr; also differed, from 77 (interquartile range, 61–101) to 139 (93–231) pmol/mmol creatinine and from 120 (91–151) to 193 (140–275) pmol/mmol in control subjects and case subjects, respectively (P <0.001). 8-iso-PGF2&agr; and its metabolite were highly correlated (Spearman’s &rgr;=0.664, P <0.001). HDL cholesterol was diminished in CHD patients (P <0.001). All of these characteristics predicted CHD in univariate analysis. In a multivariate model, the odds ratios were increased only for 8-iso-PGF2&agr; (≥131 pmol/mmol, P <0.001) and C-reactive protein (>3 mg/L, P <0.01), ie, by 30.8 (95% CI, 7.7–124) and 7.2 (1.9–27.6), respectively. 8-iso-PGF2&agr; was found to be a novel marker in addition to known risk factors of CHD, ie, diabetes mellitus, hypercholesterolemia, hypertension, and smoking. Urinary excretion of 8-iso-PGF2&agr; correlated with the number of risk factors for all subjects (P <0.001 for trend). Conclusions—8-iso-PGF2&agr; is a sensitive and independent risk marker of CHD.

Divergence in urinary 8-iso-PGF2α (iPF2α-III, 15-F2t-IsoP) levels from gas chromatography–tandem mass spectrometry quantification after thin-layer chromatography and immunoaffinity column chromatography reveals heterogeneity of 8-iso-PGF2α: Possible methodological, mechanistic and clinical implications

Abstract Free radical-catalysed oxidation of arachidonic acid esterified to lipids leads to the formation of the F2-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-PGF2α (iPF2α-III, 15-F2t-IsoP) in human urine. Immunoaffinity column chromatography (IAC) with immobilized antibodies raised against 8-iso-PGF2α (i.e. 15(S)-8-iso-PGF2α) has been shown by others to be highly selective and specific for this 8-iso-PGF2α isomer when quantified by GC–MS. In the present study we established IAC for urinary 8-iso-PGF2α 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-PGF2α. 8-iso-PGF2α was measured in urine of 10 young healthy humans by both methods. 8-iso-PGF2α 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-PGF2α at 128±55 pg/mg creatinine. This finding suggests that urinary 8-iso-PGF2α is heterogenous, with 15(S)-8-iso-PGF2α contributing by ∼50%. PGF2α and other 8-iso-PGF2α isomers including 15(R)-8-iso-PGF2α are not IAC-immunoreactive and are chromatographically separated from 15(S)-8-iso-PGF2α. We assume that ent-15(S)-8-iso-PGF2α is also contributing by ∼50% to urinary 8-iso-PGF2α. This finding may have methodological, mechanistic and clinical implications.

Quantification of endocannabinoids in biological systems by chromatography and mass spectrometry: A comprehensive review from an analytical and biological perspective☆

The endocannabinoids anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2AG) are physiologically occurring, biologically active compounds on CB(1) and CB(2) receptors with multiple physiological functions. AEA and 2AG have been identified and quantified in many mammalian biological fluids and tissues, such as human plasma, adipocytes, tissues and tissue microdialysates, at concentrations in the picomolar-to-nanomolar range under basal conditions. In this article, recently published chromatographic and mass spectrometric analytical methods, i.e., HPLC with fluorescence or ultraviolet detection, LC-MS, LC-MS/MS, GC-MS and GC-MS/MS, are reviewed and discussed, notably from the quantitative point of view. We focus on and emphasize the particular importance of blood sampling, sample storage and work-up including solvent and solid-phase extraction and derivatization procedures, matrix-effects, and stability of analytes. As 2AG spontaneously isomerizes to its CB(1)/CB(2) receptors biologically inactive 1-arachidonoyl glycerol (1AG) by acyl migration, this phenomenon and its particular importance for accurate quantification of 2AG are discussed in detail. Due to the electrical neutrality of AEA and 2AG their solvent extraction by toluene offers the least matrix-effect and minimum isomerization. LC-MS/MS is the most frequently used analytical technique for AEA and 2AG. At present, the utility of the GC-MS/MS methodology seems to be limited to AEA measurement in human plasma, bronchoalveolar liquid (BAL) and microdialysate samples. Despite great instrumental advances in the LC-MS/MS methodology, sampling and sample treatment remains one of the most crucial analytical steps in 2AG analysis. Extension of the LC-MS/MS methodology, for instance to microdialysate and BAL samples from clinical studies, is a big analytical challenge in endocannabinoid analysis in clinical settings. Currently available LC-MS/MS and GC-MS/MS methods should be useful to investigate the metabolism of AEA and 2AG beyond hydrolysis, i.e., by β- and ω-oxidation pathways.

Assessment of nitric oxide synthase activity in vitro and in vivo by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric method for the determination of nitric oxide synthase activity is described. The method is based on the gas chromatographic-mass spectrometric measurement of L-[15N2]arginine-derived [15N]nitrite as its pentafluorobenzyl derivative in the negative-ion chemical ionization mode. Application of the method to the analysis of [15N]nitrite formation by purified neuronal nitric oxide synthase revealed K(M) values of 3.1 microM by Hanes and 4.6 microM by Lineweaver-Burk for L-[15N2]arginine. The corresponding Vmax values were 0.204 and 0.228 micromol [15N]nitrite min(-1) mg(-1) NOS, respectively. N(G)-Nitro-L-arginine and N(G),N(G)-dimethylarginine (asymmetric dimethylarginine) were identified by this method as the most potent enzyme inhibitors. Nitric oxide synthase activity was also assessed in vivo by i.v. injection of L-[15N2]arginine in a rat and determination of plasma [15N]nitrite and [15N]nitrate. The assay described in this work allows for accurate, specific and highly sensitive determination of nitric oxide synthase activity in vitro and in vivo.

Circulating and excretory nitrite and nitrate as indicators of nitric oxide synthesis in humans : methods of analysis

Nitric oxide (NO) is produced in various cells from l-arginine by the catalytical action of NO synthases (NOS). The main metabolic fate of NO includes oxidation to nitrate by oxyhemoglobin in red blood cells and autoxidation to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in plasma, serum, and urine can be used to assess NO synthesis in humans. Circulating nitrite reflects constitutive endothelial NOS activity. Excretory nitrate indicates systemic NO production. Nitrite and nitrate can be measured in plasma, serum, and urine of humans by various analytical methods which are based on different analytical principles. These methods include colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis, and mass spectrometry. The present article gives an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human plasma, serum, and urine in the framework of clinical studies and discusses their significance.

Tandem mass spectrometric quantification of 8-iso-prostaglandin F2α and its metabolite 2,3-dinor-5,6-dihydro-8-iso-prostaglandin F2α in human urine

Whole body synthesis of F2-isoprostanes, a family of cyclooxygenase-independent eicosanoids formed by free-radical catalysed peroxidation, should be best assessed by quantifying their urinary metabolites. Two methods for the quantitative determination of F2-isoprostane metabolites in human urine performing either thin-layer chromatography (TLC) (method A) or high-performance liquid chromatography (HPLC) (method B) prior to GC–tandem MS are described. Method A allows for simultaneous quantification of 8-iso-PGF2α, one prominent member of the F2-isoprostane family, and its major urinary metabolite, 2,3-dinor-5,6-dihydro-8-iso-PGF2α. Mean excretion was found to be 223 and 506 pg/mg creatinine of 8-iso-PGF2α and 2,3-dinor-5,6-dihydro-8-iso-PGF2α, respectively (n=14). A tight correlation existed between the urinary excretion of these two isoprostanes (r=0.86). Method B enables quantification of dinor-dihydro metabolites of various F2-isoprostanes including 8-iso-PGF2α. 2,3-Dinor-5,6-dihydro-8-iso-PGF2α was found to be an abundant dinor-dihydro F2-isoprostane metabolite. Validity of method A was proven by a combination of HPLC with TLC prior to GC–tandem MS analysis. A correlation was observed between the urinary concentrations of 2,3-dinor-5,6-dihydro-8-iso-PGF2α measured by GC–MS and GC–tandem MS (r=0.84).

GC-MS determination of creatinine in human biological fluids as pentafluorobenzyl derivative in clinical studies and biomonitoring: Inter-laboratory comparison in urine with Jaffé, HPLC and enzymatic assays.

In consideration of its relatively constant urinary excretion rate, creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous biomolecules. Assays based on the reaction of creatinine and picric acid first reported by Jaffé in 1886 still belong to the most frequently used laboratory approaches for creatinine measurement in urine. Further analytical methods for creatinine include HPLC-UV, GC-MS, and LC-MS and LC-MS/MS approaches. In the present article we report on the development, validation and biomedical application of a new GC-MS method for the reliable quantitative determination of creatinine in human urine, plasma and serum. This method is based on the derivatization of creatinine (d(0)-Crea) and the internal standard [methyl-trideutero]creatinine (d(3)-Crea) with pentafluorobenzyl (PFB) bromide in the biological sample directly or after dilution with phosphate buffered saline, extraction of the reaction products with toluene and quantification in 1-μl aliquots of the toluene extract by selected-ion monitoring of m/z 112 for d(0)-Crea-PFB and m/z 115 for d(3)-Crea-PFB in the electron-capture negative-ion chemical ionization mode. The limit of detection of the method is 100 amol of creatinine. In an inter-laboratory study on urine samples from 100 healthy subjects, the GC-MS method was used to test the reliability of currently used Jaffé, enzymatic and HPLC assays in clinical and occupational studies. The results of the inter-laboratory study indicate that all three tested methods allow for satisfactory quantification of creatinine in human urine. The GC-MS method is suitable for use as a reference method for urinary creatinine in humans. In serum, creatine was found to contribute to creatinine up to 20% when measured by the present GC-MS method. The application of the GC-MS method can be extended to other biological samples such as saliva.

Simultaneous UPLC-MS/MS quantification of the endocannabinoids 2-arachidonoyl glycerol (2AG), 1-arachidonoyl glycerol (1AG), and anandamide in human plasma: minimization of matrix-effects, 2AG/1AG isomerization and degradation by toluene solvent extraction.

Analysis of the endocannabinoid (EC) systems key molecules 2-arachidonoyl glycerol (2AG) and arachidonoyl ethanolamide (anandamide, AEA) is challenging due to several peculiarities. 2AG isomerizes spontaneously to its biologically inactive analogue 1-arachidonoyl glycerol (1AG) by acyl migration and it is only chromatographically distinguishable from 1AG. Matrix-effects caused primarily by co-extracted phospholipids may further compromise analysis. In addition, 2AG and 1AG are unstable under certain conditions like solvent evaporation or reconstitution of dried extracts. We examined effects of different organic solvents and their mixtures, such as toluene, ethyl acetate, and chloroform-methanol, on 2AG/1AG isomerisation, 2AG/1AG stability, and matrix-effects in the UPLC-MS/MS analysis of 2AG and AEA in human plasma. Toluene prevented, both, 2AG isomerisation to 1AG and degradation of 2AG/1AG during evaporation. Toluene extracts contain only 2% of matrix-effect-causing plasma phospholipids compared to extracts from the traditionally used solvent mixture chloroform-methanol. Toluene and all other tested organic solvents provide comparable 2AG and AEA extraction yields (60-80%). Based on these favourable toluene properties, we developed and validated a UPLC-MS/MS method with positive electrospray ionization (ESI+) that allows for simultaneous accurate and precise measurement of 2AG and AEA in human plasma. The UPLC-MS/MS method was cross-validated with a previously described fully-validated GC-MS/MS method for AEA in human plasma. A close correlation (r(2)=0.821) was observed between the results obtained from UPLC-MS/MS (y) and GC-MS/MS (x) methods (y=0.01+0.85x). The UPLC-MS/MS method is suitable for routine measurement of 2AG and AEA in human plasma samples (1 mL) in clinical settings as shown by quality control plasma samples processed over a period of 100 days. The UPLC-MS/MS method was further extended to human urine. In urine, AEA was not detectable and 2AG was detected in only 3 out of 19 samples from healthy subjects at 160, 180 and 212 pM corresponding to 12.3, 14.5 and 9.9 pmol/mmol creatinine, respectively.

Lack of oxidative stress during sustained therapy with isosorbide dinitrate and pentaerythrityl tetranitrate in healthy humans: a randomized, double-blind crossover study.

The mechanisms by which tolerance to organic nitrates develops are still poorly understood. Enhanced oxidative stress, i.e., increased free radical production following organic nitrate administration, has been recently suggested as a possible mechanism. A randomized, double-blind, crossover study assessed in 18 healthy young volunteers at baseline and 1 and 5 days after oral administration with therapeutically relevant doses of isosorbide dinitrate (ISDN, 30 mg TID) or pentaerythrityl tetranitrate (PETN, 80 mg TID) the effect on two index parameters of oxidative stress in vivo, i.e., urinary 8-iso-prostaglandin (PG)F2&agr; and circulating 3-nitrotyrosine and their major urinary metabolites, 2,3-dinor-5,6-dihydro-8-iso-PGF2&agr; and 3-nitro-4-hydroxyphenylacetic acid. In addition, urinary cGMP and serum and urinary nitrate and nitrite were determined. All parameters were quantified by gas chromatography–mass spectrometry or gas chromatography–tandem mass spectrometry except for cGMP, which was analyzed by radioimmunoassay. Serum and urinary nitrite levels increased significantly following 5-day administration of ISDN and PETN. Neither urinary excretion of 8-iso-PGF2&agr; and plasma 3-nitrotyrosine nor their respective metabolites changed significantly after ISDN or PETN administration. There were no significant differences between ISDN and PETN regarding these parameters. Urinary cGMP increased significantly only after ISDN. This study is compatible with a stimulation of cGMP by ISDN, but neither ISDN nor PETN enhances systemic oxidative stress in healthy volunteers.

Specific and rapid quantification of 8-iso-prostaglandin F2α in urine of healthy humans and patients with Zellweger syndrome by gas chromatography–tandem mass spectrometry

8-iso-Prostaglandin F2alpha (8-iso-PGF2alpha) is currently discussed as a potential index parameter of oxidative stress in vivo. We describe in this article a fully validated gas chromatographic-tandem mass spectrometric method for the quantitative determination of 8-iso-PGF2alpha in human urine. The method is highly specific and requires a single thin-layer chromatographic step for sample purification. Inter- and intraday imprecision were below 8%. Mean inaccuracy was 5.3% for added levels of 8-iso-PGF2alpha up to 2000 pg/ml of urine. We measured highly elevated excretion of 8-iso-PGF2alpha in the urine of children with peroxisomal beta-oxidation deficiency, i.e. Zellweger syndrome, (63.3+/-16.6 ng/mg creatinine) compared to that of healthy children (0.51+/-0.16 ng/mg creatinine) (mean+/-S.D., both n=5). The method could be useful for diagnosing Zellweger syndrome and for investigating the utility of 8-iso-PGF2alpha as a novel marker for oxidative stress in vivo in man.