Edzard Schwedhelm

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.

Dietary l-arginine and α-tocopherol reduce vascular oxidative stress and preserve endothelial function in hypercholesterolemic rabbits via different mechanisms

Vascular oxidative stress brought about by superoxide radicals and oxidized low-density lipoproteins (oxLDL) is a major factor contributing to decreased NO-dependent vasodilator function in hypercholesterolemia and atherosclerosis. We investigated whether chronic administration of L-arginine (2% in drinking water) or of alpha-tocopherol (300 mg/day) improves endothelium-dependent vasodilator function and systemic NO production, reduces vascular oxidative stress, and reduces the progression of atherosclerosis in cholesterol-fed rabbits with pre-existing hypercholesterolemia. Systemic NO production was assessed as urinary nitrate excretion; oxidative stress was measured by urinary 8-iso-PGF2alpha excretion in vivo, by lucigenin-enhanced chemiluminescence of isolated aortic rings ex vivo, and by copper-mediated LDL oxidation in vitro. Endothelium-dependent relaxation was almost completely abrogated in cholesterol-fed rabbits. Urinary nitrate excretion was reduced by 46+/-10%, and 8-iso-PGF2alpha excretion was increased by 61+/-18% as compared to controls (each P <0.05). Vascular superoxide radical release stimulated by PMA ex vivo was increased by 273+/-93% in this group, and the lag time of LDL oxidation was reduced by 35+/-6% (each P <0.05). Treatment with L-arginine and alpha-tocopherol reduced intimal lesion formation (by 68+/-6 and 4+/-11%, respectively; P <0.05) and improved endothelium-dependent relaxation. Both treatments also normalized urinary 8-iso-PGF2alpha excretion. L-Arginine increased urinary nitrate excretion by 43+/-13% (P <0.05) and reduced superoxide radical release by isolated aortic rings to control levels, which was unaffected by vitamin E treatment. By contrast, vitamin E dramatically increased the resistance of isolated LDL to copper-mediated oxidation in vitro by 178+/-7% (P <0.05), which was only marginally prolonged by L-arginine. Intimal thickening was reduced by both treatments. We conclude that both L-arginine and alpha-tocopherol reduce the progression of atherosclerotic plaques in cholesterol-fed rabbits. However, while L-arginine increases NO formation and reduces superoxide release, alpha-tocopherol antagonizes mainly oxLDL-related events in atherogenesis. Thus, both treatments reduce urinary isoprostane excretion and improve endothelium-dependent vasodilation via different mechanisms.

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.

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).

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.

Accurate quantification of basal plasma levels of 3-nitrotyrosine and 3-nitrotyrosinoalbumin by gas chromatography–tandem mass spectrometry

Measurement of 3-nitro-L-tyrosine (NO(2)Tyr) and protein-related 3-nitro-L-tyrosine in human plasma is associated with numerous methodological problems which result in highly divergent basal plasma levels often ranging within two orders of magnitude. Recently, we have described an interference-free GC-tandem MS-based method for NO(2)Tyr which yielded the lowest basal plasma NO(2)Tyr levels reported thus far. This method was extended to quantify protein-associated 3-nitrotyrosine and in particular 3-nitrotyrosinated albumin (NO(2)TyrALB) in human plasma. NO(2)TyrALB and albumin (ALB) were extracted from plasma by affinity column extraction and digested enzymatically at neutral pH. 3-Nitro- L-[2H(3)]tyrosine was used as internal standard. In plasma of 18 healthy young volunteers the molar ratio of NO(2)TyrALB to albumin-derived tyrosine (TyrALB), i.e. NO(2)TyrALB/TyrALB, was determined to be 1.55+/-0.54x1:10(6) (mean+/-SD). The plasma concentration of NO(2)TyrALB was estimated as 24+/-4 nM. The NO(2)Tyr plasma levels in these volunteers were determined to be 0.73+/-0.53 nM. In the same volunteers, NO(2)TyrALB/TyrALB, NO(2)TyrALB and NO(2)Tyr were measured 15 days later and the corresponding values were determined to be 1.25+/-0.58x1:10(6), 25+/-6 nM and 0.69+/-0.16 nM. For comparison, NO(2)Tyr and NO(2)TyrALB were measured in six plasma samples from healthy volunteers by GC-MS and GC-tandem MS. Different values were found for NO(2)Tyr, i.e. 5.4+/-2.8 versus 2.7+/-1.5 nM, and comparable values for NO(2)TyrALB/TyrALB, i.e. 0.5+/-0.2x1:10(6) versus 0.4+/-0.1x1:10(6), by these methods. The ratio of the values measured by GC-MS to those measured by GC-tandem MS were 2.9+/-3.1 for NO(2)Tyr and 1.2+/-0.2 for NO(2)TyrALB/TyrALB. The present GC-tandem MS method provides accurate values of NO(2)Tyr and NO(2)TyrALB in human plasma.

Methodological Considerations on the Detection of 3-Nitrotyrosine in the Cardiovascular System

To the Editor: Reactive nitrogen species (RNS; eg, •NO, •NO2, ONOO−, NO2Cl) react preferably with tyrosine (Tyr) and protein-associated tyrosine (TyrProt) to form 3-nitrotyrosine, ie NO2Tyr and NO2TyrProt, respectively.1 Therefore, detection of NO2Tyr and/or NO2TyrProt provides evidence for generation of RNS rather than specifically peroxynitrite (ONOO−).1 Besides this difficulty, Tarpey and Fridovich2 have recently discussed, in an article published in Circulation Research , the problematic measurement of NO2Tyr and NO2TyrProt, giving special attention to artifactual formation of NO2Tyr and NO2TyrProt by acidification of biological samples. This methodological pitfall is very important and well-recognized,3–10⇓⇓⇓⇓⇓⇓⇓ but it is not the sole methodological problem in 3-nitrotyrosine detection. Tarpey and Fridovich2 restricted their discussion exclusively to artifactual formation of 3-nitrotyrosine referring to Yi et al3 and Frost et …

Gas chromatographic-tandem mass spectrometric quantification of human plasma and urinary nitrate after its reduction to nitrite and derivatization to the pentafluorobenzyl derivative.

Gas chromatography-mass spectrometry (GC-MS) of nitrite as its pentafluorobenzyl derivative in the negative-ion chemical ionization mode is a useful analytical tool to quantify accurately and sensitively nitrite and nitrate after its reduction to nitrite in various biological fluids. In the present study we demonstrate the utility of GC-tandem MS to quantify nitrate in human plasma and urine. Our present results verify human plasma and urine levels of nitrite and nitrate measured previously by GC-MS.

Synthesis of 3-nitro-L-[2H3]tyrosine for use as an internal standard for quantification of 3-nitro-L-tyrosine by gas chromatography-mass spectrometry

3-Nitro-L-tyrosine is produced in vitro and in vivo by the nitration of tyrosine and tyrosine residues in proteins. Plasma 3-nitro-L-tyrosine has been suggested as an index of oxidative damage in living organisms that depend on nitrating species. For use in the quantitative determination of 3-nitro-L-tyrosine in biological fluids by gas chromatography-mass spectrometry (GC-MS) we describe the chemical synthesis of 3-nitro-L-[2H3] tyrosine by nitration of commercially available L-[2H4] tyrosine by nitrate and sulphuric acid. GC-MS analysis revealed a 2H isotopic purity of about 98%. The isolated reaction product did not contain any detectable amounts of L-[2H4] tyrosine. The utility of 3-nitro-L-[2H3]tyrosine as an internal standard for the quantitative analysis of 3-nitro-L-tyrosine by GC-MS is shown.