Isotope Dilution Gas Chromatography-mass Spectrometry Research Articles

Preparation and value assignment of standard reference material 968e fat-soluble vitamins, carotenoids, and cholesterol in human serum

Standard Reference Material 968e Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum provides certified values for total retinol, γ- and α-tocopherol, total lutein, total zeaxanthin, total β-cryptoxanthin, total β-carotene, 25-hydroxyvitamin D(3), and cholesterol. Reference and information values are also reported for nine additional compounds including total α-cryptoxanthin, trans- and total lycopene, total α-carotene, trans-β-carotene, and coenzyme Q(10). The certified values for the fat-soluble vitamins and carotenoids in SRM 968e were based on the agreement of results from the means of two liquid chromatographic methods used at the National Institute of Standards and Technology (NIST) and from the median of results of an interlaboratory comparison exercise among institutions that participate in the NIST Micronutrients Measurement Quality Assurance Program. The assigned values for cholesterol and 25-hydroxyvitamin D(3) in the SRM are the means of results obtained using the NIST reference method based upon gas chromatography-isotope dilution mass spectrometry and liquid chromatography-isotope dilution tandem mass spectrometry, respectively. SRM 968e is currently one of two available health-related NIST reference materials with concentration values assigned for selected fat-soluble vitamins, carotenoids, and cholesterol in human serum matrix. This SRM is used extensively by laboratories worldwide primarily to validate methods for determining these analytes in human serum and plasma and for assigning values to in-house control materials. The value assignment of the analytes in this SRM will help support measurement accuracy and traceability for laboratories performing health-related measurements in the clinical and nutritional communities.

Trace analysis of acidic pharmaceutical residues in waters with isotope dilution gas chromatography–mass spectrometry via methylation derivatization

Acidic pharmaceutical residues are pollutants of emerging concern and are generally monitored by HPLC–MS/MS. However, due to the limited separation efficiency of HPLC column and lack of suitable mass transition for confirmation analysis, some interference may not be separated completely and differentiated from ibuprofen, which may cause the results with interference, especially in sample with complex matrix. The objective of this study is to develop a sensitive and reliable method for the determination of acidic pharmaceutical residues in water samples by GC–MS with better resolution by using methylation derivatization and isotope dilution techniques. TMSDM, a mild reagent, was used as the derivatization reagent coupling with the isotope dilution technique, for the first time, to improve the precision and accuracy of the analytical method to determine the pharmaceutical residues in water. The MDLs for the five acidic organic compounds: ibuprofen, gemfibrozil, naproxen, ketoprofen and diclofenac were from 0.7 to 1.1ng/L, with recoveries ranging from 93 to 110%. Alternative to the HPLC–MS/MS method, the developed GC–MS protocols provides an additional option for the analysis of acidic pharmaceutical residues in water, with better separation efficiency in reducing interferences from complicated sample matrix, for determination of ibuprofen residues.

Quantification of carcinogenic 4- to 6-ring polycyclic aromatic hydrocarbons in human urine by solid-phase microextraction gas chromatography–isotope dilution mass spectrometry

Polycyclic aromatic hydrocarbons (PAHs) are pollutants found in living and working environments. The aim of this study was to develop a solid-phase microextraction (SPME) gas chromatography (GC)-isotope dilution mass spectrometry method for the quantification of 10 four- to six-ring PAHs in urine samples. Seven of the selected PAHs have been classified as carcinogenic. Under the final conditions, analytes were sampled with a 100-μm polydimethylsiloxane SPME fibre for 60 min at 80 °C and desorbed in the injection port of the GC at 270 °C. Fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, indeno[1,2,3-cd]pyrene and benzo[ghi]perylene were separated using a highly arylene-modified phase capillary column and quantified by MS using eight deuterated PAHs as surrogate internal standards. Limits of quantification (LOQ) were in the 0.5- to 2.2-ng/L range. Validation showed linear dynamic ranges up to 340 ng/L, inter- and intra-run precisions <20%, and accuracies within 20% of spiked concentrations. Matrix effect evaluation and the use of control charts to monitor process performances showed that the isotope dilution approach allowed for the control of bias sources. Urinary PAHs were above or equal to LOQ, depending on different compounds, in 58-100% (min-max), 40-100% and 5-39% of samples from coke oven workers (n = 12), asphalt workers (n = 10) and individuals not occupationally exposed to PAHs (n = 18), respectively. Chrysene was the most abundant PAH determined with median levels of 62.6, 6.9 and <0.6 ng/L, respectively. These results show that the method is suitable for quantifying carcinogenic PAHs in specimens from individuals with different levels of PAH exposure.

Validation of an isotope dilution gas chromatography–mass spectrometry method for analysis of 7-oxygenated campesterol and sitosterol in human serum

High dose daily intake of plant sterols decreases the uptake of cholesterol in the intestine by competitive mechanisms and thus leads to reduced serum levels of total and LDL-cholesterol. By this, the commercialization of plant sterol enriched ‘functional food’ products is rapidly increasing. Subjects using these kinds of diet present a duplication of their serum plant sterol levels after long-term intake. In analogy to cholesterol, plant sterols such as campesterol and sitosterol can be oxidized to oxyphytosterols and these may counteract the primary anti-atherosclerotic action of cholesterol lowering. In order to investigate the whole spectrum of the consequences following high plant sterol intake a highly sensitive and specific isotope dilution gas chromatography–mass spectrometry method for the analysis of 7-oxygenated campesterol/sitosterol in trace amounts in human serum is presented in this paper. The validation was based on limits for detection and quantification, recovery, precision and minimization of autoxidation during work-up. Our results show an overall coefficient of variation ≤10% for the precision. The lowest limits for detection and quantification for 7α-hydroxy-campesterol were 7 pg/mL and 23 pg/mL, respectively. Data for overall sum recovery ranged from 92% to 115%. We practically used this method for analysis of oxyphytosterols simultaneously with plant sterol concentrations in serum from healthy volunteers. Sixteen subjects were treated with plant sterol enriched margarine (3 g/day) for 28 days. The results showed a significant increase of the oxyphytosterol 7β-hydroxy-sitosterol from 1.19 ± 0.54 (before intake) to 2.24 ± 1.24 ng/mL (mean ± SD; +86.7%; P = 0.007) after intake of the margarine. There was a highly significant correlation between the serum levels of campesterol and the sum of 7-oxygenated campesterol ( R 2 = 0.915; P < 0.001) and sitosterol and the sum of 7-oxygenated sitosterol ( R 2 = 0.915; P < 0.001). We can conclude from this study that the analytic method is well suited for detection of OPS, even at trace amounts.

Simultaneous determination of selenomethionine enantiomers in biological fluids by stable isotope dilution gas chromatography–mass spectrometry

A method for the stereoselective determination of d- and l-enantiomers of selenomethionine in mouse plasma was developed using gas chromatography–mass spectrometry with selected-ion monitoring (GC–MS-SIM). dl-[ 2H 3, 82Se]selenomethionine was used as analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. Selenomethionine enantiomers in mouse plasma were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N-acylation with optically active (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. Quantification was performed by SIM of the molecular-related ions of the diastereomers on the chemical ionization mode. The intra- and inter-day precision for d- and l-selenomethionine spiked to mouse plasma gave good reproducibility with relative standard deviation of 3% and 3% for d-selenomethionine and 6% and 3% for l-selenomethionine, respectively. The estimated amounts were in good agreement with the actual amounts spiked, the intra- and inter-day relative error being 5% and 2% for d-selenomethionine and 2% and 1% for l-selenomethionine, respectively. The present method is sensitive enough to determine pharmacokinetics of selenomethionine enantiomers.

Proposed Serum Cholesterol Reference Measurement Procedure by Gas Chromatography–Isotope Dilution Mass Spectrometry

Our purpose was to establish a mass spectrometry reference measurement procedure (RMP) for cholesterol to use in the CDC's standardization programs. We explored a gas chromatography-isotope dilution mass spectrometry (GC-IDMS) procedure using a multilevel standard calibration curve to quantify samples with varying cholesterol concentrations. We calibrated the mass spectrometry instrument by isotope dilution with a pure primary standard reference material and an isotopically enriched cholesterol analog as the internal standard (IS). We diluted the serum samples with Tris-HCl buffer (pH 7.4, 0.05 mol/L, 0.25% Triton X-100) before analysis. We used 17 serum pools, 10 native samples, and 2 standard reference materials (SRMs). We compared the GC-IDMS measurements with the CDC's modified Abell-Levy-Brodie-Kendall (AK) RMP measurements and assessed method accuracy by analyzing 2 SRMs. We evaluated the procedure for lack of interference by analyzing serum spiked with a mixture of 7 sterols. The mean percent bias between the AK and the GC-IDMS RMP was 1.6% for all samples examined. The mean percent bias from NIST's RMP was 0.5% for the SRMs. The total %CVs for SRM 1951b levels I and II were 0.61 and 0.73%, respectively. We found that none of the sterols investigated interfered with the cholesterol measurement. The low imprecision, linear response, lack of interferences, and acceptable bias vs the NIST primary RMP qualifies this procedure as an RMP for determining serum cholesterol. The CDC will adopt and implement this GC-IDMS procedure for cholesterol standardization.

Concentration ratios of free-morphine to free-codeine in femoral blood in heroin-related poisoning deaths

The concentrations of free-morphine (Mo), free-codeine (Co) and 6-monoacetyl morphine (6-MAM) were determined in femoral blood in N = 747 heroin-related deaths. The opiates were determined by isotope dilution gas chromatography-mass spectrometry after solid-phase extraction. The median blood concentrations of 6-MAM, free-morphine and free-codeine were 0.01 mg/L, 0.24 mg/L and 0.02 mg/L, respectively. The mean and median Mo/Co concentration ratios were 13.2 and 11.0, respectively with a range from 0.2 to 124. Despite the fact that all victims had taken heroin, there were eight cases (1.1%) with a Mo/Co ratio less than one and 18 cases (2.4%) with a ratio less than two. The free-morphine concentration in blood did not depend on the Mo/Co ratio; median 0.29 mg/L (Mo/Co < 2.0) and median 0.25 mg/L (Mo/Co ratio > 2.0). By contrast, the concentration of free-codeine in blood was highly dependent on the Mo/Co ratio; median 0.75 mg/L (Mo/Co < 1.0) and median 0.30 mg/L (Mo/Co ratio < 2.0). A Mo/Co ratio in post-mortem (PM) femoral blood >1.0 is compelling evidence that the deceased had taken illicit heroin. However, finding a low Mo/Co ratio (<1.0 or <2.0) does not preclude use of heroin because such low ratios are possible if a person had co-ingested heroin along with use or abuse of codeine medication.

Cholestenoic Acid Is an Important Elimination Product of Cholesterol in the Retina: Comparison of Retinal Cholesterol Metabolism with That in the Brain

Accumulating evidence indicates a link between cholesterol and age-related macular degeneration. Yet, little is known about cholesterol elimination from the retina and retinal pigment epithelium (RPE), the two layers that are damaged in this blinding disease. Several different pathways of enzymatic cholesterol removal exist in extraocular tissues. The authors tested whether metabolites from these pathways could also be quantified in the bovine and human retina and RPE. For comparison, they measured cholesterol oxidation products in two regions of the bovine and human brain and in the bovine liver and adrenal glands. Sterol quantification was carried out by isotope dilution gas chromatography-mass spectrometry. Bovine tissues were used first to optimize analytical procedures and to investigate postmortem changes in oxysterol concentrations. Then human specimens were analyzed for oxysterol concentrations. Qualitatively, oxysterol profiles were similar in the bovine and human tissues. In the human retina and RPE, the authors could not detect 27-hydroxycholesterol but unexpectedly found that its oxidation product, 5-cholestenoic acid, is the most abundant oxysterol, varying up to threefold in different persons. 24S-Hydroxysterol and pregnenolone were also present in the retina, but at much lower quantities and without significant interindividual variability. In the brain, the predominant oxysterol was 24S-hydroxycholesterol. The oxysterol profile of the retina suggests that all known pathways of cholesterol elimination in extraocular organs are operative in the retina and that they likely vary depending on specific cell type. However, overall oxidation to 5-cholestenoic acid appears to be the predominant mechanism for cholesterol elimination from this organ.

Results of the recalibration of creatinine measurement with the modular Beckman Coulter® Jaffe creatinine method

Serum creatinine is important for detecting the beginning of a decline in kidney function. The Beckman Coulter Jaffe reagents for measuring creatinine have been standardized to the internationally accepted reference method: isotope dilution mass spectrometry (IDMS). The impact of this recalibration on the Beckman Coulter modular or cup (stat) Jaffe method is studied. Recalibrated Jaffe (calibrator set points IDMS traceable) and classic National Institute of Standards and Technology (NIST) creatinine methods (traceable to NIST 914a) were compared with an enzymatic method (Sentinel, traceable to NIST SRM 967). All measurements were performed on Synchron DxC 800 systems. Imprecision of the routine methods was studied using the Clinical and Laboratory Standards Institute (CLSI) protocols and laboratory quality survey. Thirteen plasma pools, with concentrations < 354 mmol/L, were analyzed with a gas chromatography isotope dilution mass spectrometry (GC-IDMS) method and routine methods. Total error of 8.2% based on biological variability, set on the GC-IDMS values and acceptance criteria (bias < 5%, imprecision < 8% at concentrations ≥ 88.4 mmol/L and a maximum 10% increase in the relative error of estimated glomerular filtration rate (eGFR) of the National Kidney Disease Educational Program (NKDEP) were used for evaluating analytical performance of the routine methods studied. After recalibration of the Jaffe method, median bias (mmol/L) decreased from 12.4 (95% CI: 9.1-21.2) to 7.3 (95% CI: 1.5-10.5). Imprecision of the Jaffe method is in agreement with the claim of the manufacturer, namely < 9 mmol/L or < 3% below or above 292 mmol/L. Below creatinine of 88.4 mmol/L, imprecision of the recalibrated Jaffe and enzymatic methods is between 4.1% and 6.9%, and 5.0% and 7.1%, respectively, and significantly different (p = 0.02 for both the Jaffe and enzymatic methods) from the goal, based on biological variability, of 2.7%. For the adult pools, all recalibrated Jaffe and enzymatic results fall within the total error of 8.2%. In the pediatric samples, one-third of the recalibrated Jaffe and three of the six enzymatic results fall within this total error goal. Recalibration significantly reduced bias of the Jaffe method. For pediatric samples, recalibrated Jaffe results do not comply with either the imprecision goal or the total error based on biological variability. Adult recalibrated Jaffe results are in compliance with the goals based on biological variability and with the acceptance criteria from the NKDEP.

GC–MS analysis of bisphenol A in human placental and fetal liver samples

A method based on extraction with acetonitrile, followed by solid-phase extraction, derivatization with acetic anhydride, and isotope dilution gas chromatography–mass spectrometry (GC–MS) analysis was applied to determine levels of free and conjugated BPA in human tissues. β-Glucuronidase was used to de-conjugate the glucuronized BPA in the samples. The method was validated using various animal organ meat samples including pork liver and kidney, beef and calf liver, chicken liver and heart; recoveries were from 85% to 112% at two spiking levels. The average method limit of quantification (LOQ) was estimated at 0.77ng/g for placenta samples and 1.2ng/g for fetal liver samples based on 10 times the signal to noise ratio. BPA was detected in all animal tissue samples, with concentrations ranging from 1.8ng/g in beef and calf livers to 17.1ng/g in pork kidney. The method was used successfully to determine both free and conjugated BPA levels in human placental and fetal liver tissue samples. BPA was detected in 86% of the placental samples; concentrations of free BPA in the positive samples ranged from 0.60ng/g to as high as 64ng/g with an average of 9.5ng/g and a median of 3.0ng/g, and conjugated BPA was as high as 7.8ng/g. BPA was also detected in most of the fetal liver samples (57%); concentrations of free BPA in the positive samples ranged from 1.3 to 27ng/g with an average of 8.5ng/g and a median of 3.2ng/g. Conjugated BPA was also detected in most of the liver samples analysed for total BPA, ranging from 0.64 to 20ng/g with an average of 3.9ng/g and a median of 1.5ng/g. This study, while primarily designed as a method validation, has demonstrated that BPA can be detected in human fetal liver samples as early as the third month of fetal life. Further work will be conducted to validate these preliminary findings.

Analysis of serum bile acids by isotope dilution-mass spectrometry to assess the performance of routine total bile acid methods

WEQAS, one of the largest EQA (External Quality Assessment) providers in the UK, offers a bile acid EQA scheme, with linear serum pools containing cholic acid, deoxycholic acid and chenodeoxycholic acid, reflecting levels observed in obstructive cholestasis. Total bile acids are currently measured routinely by non-specific enzymatic methods. Traceability of results to the SI unit utilizing reference target values is the preferred method of comparison of returned results where available, ensuring the transfer of accuracy from definitive methods to routine methods. Target values have been assigned to EQA material utilizing isotope dilution gas chromatography mass spectrometry (ID-GCMS). The methodology was based on published routine methods and adapted for use as a ID-GCMS target method. The total bile acid target value was reported as the sum of the three major bile acids measured: cholic acid, deoxycholic acid and chenodeoxycholic acid. The produced target values have been used to assess the performance of total bile acid methods. A degree of variability was observed between the third-generation enzyme-formazan methods and the fifth-generation thio-NADH methods. Additionally, the Sentinel results showed a positive bias in comparison to their peer formazan method group. The use of ID-GCMS target results provides a common comparison for resumed results in EQA schemes, highlighting any method differences. Thus can then aid in the harmonisation of results observed for each of the different methods.

Equivalent lipid oxidation profiles in advanced atherosclerotic lesions of carotid endarterectomy plaques obtained from symptomatic type 2 diabetic and nondiabetic subjects

Type 2 diabetes (T2D) increases the risk for cardiovascular disease and is thought to be associated with increased oxidative stress, a contributor to atherogenesis. Surprisingly, however, there is little direct evidence that T2D-associated oxidative stress results in increased lipid oxidation and/or decreased antioxidant capacity in human atherosclerotic lesions. The aim of this study was to measure vascular lipid oxidation and antioxidants in T2D. The arterial content of oxidized lipid and antioxidants in carotid endarterectomy specimens obtained from diabetic and normoglycemic patients was determined using high-performance liquid chromatography (HPLC), stable isotope dilution gas chromatography–mass spectrometry (GC/MS), and gas–liquid chromatography techniques. The concentrations of hydroxyoctadecanoic acid, F 2-isoprostanes, and 7-ketocholesterol, as well as α-tocopherol, ascorbate, and urate were not different in the two patient groups, whether expressed per unit protein or as a ratio per parent compound. Unexpectedly, a significant decrease in the level of arterial lipid hydroperoxide was found in diabetic patients. Our results do not support the notion that advanced atherosclerotic lesions from T2D patients contain more oxidized lipids than corresponding lesions from nondiabetic subjects.

Ultrasonication extraction and gel permeation chromatography clean-up for the determination of polycyclic aromatic hydrocarbons in edible oil by an isotope dilution gas chromatography–mass spectrometry

An analytical method for the determination of US EPA priority pollutant 16 polycyclic aromatic hydrocarbons (PAHs) in edible oil was developed by an isotope dilution gas chromatography–mass spectrometry (GC–MS). Extraction was performed with ultrasonication mode using acetonitrile as solvent, and subsequent clean-up was applied using narrow gel permeation chromatographic column. Three deuterated PAHs surrogate standards were used as internal standards for quantification and analytical quality control. The limits of quantification (LOQs) were globally below 0.5 ng/g, the recoveries were in the range of 81–96%, and the relative standard deviations (RSDs) were lower than 20%. Further trueness assessment of the method was also verified through participation in international cocoa butter proficiency test (T0638) organised by the FAPAS with excellent results in 2008. The results obtained with the described method were satisfying ( z ≤ 2). The method has been applied to determine PAH in real edible oil samples.

Modifications to the NIST reference measurement procedure (RMP) for the determination of serum glucose by isotope dilution gas chromatography/mass spectrometry

The definitive method (DM), now known as the reference measurement procedure (RMP), for the analysis of glucose in serum was originally published in 1982 by the National Institute of Standards and Technology (NIST). Over the years the method has been subject to a number of modifications to adapt to newer technologies and simplify sample preparation. We discuss here an adaptation of the method associated with serum glucose measurements using a modified isotope dilution gas chromatography/mass spectrometry (ID-GC/MS) method. NIST has used this modified method to certify the concentrations of glucose in SRM 965b, Glucose in Frozen Human Serum, and SRM 1950, Metabolites in Human Plasma. Comparison of results from the revised method with certified values for existing Standard Reference Materials (SRMs) demonstrated that these modifications have not affected the quality of the measurements, giving both good precision and accuracy, while reducing the sample preparation time by a day and a half.

Accurate analysis of urea in milk and milk powder by isotope dilution gas chromatography–mass spectrometry

A high order method for measuring urea concentrations in milk and milk powder was developed. The method can be applied to certify the concentration of urea in some new milk and milk powder CRMs. This high accurate method for analysis of milk is valuable given the inherent challenges associated with the complexity of the sample matrix. A measurement procedure based on gas chromatography/isotope dilution mass spectrometry (GC/IDMS) was developed. Samples were pre-treated with acetonitrile to remove proteins and the method was applied to determine urea concentrations in milk and milk powder. Excellent precision was obtained, with within- and between-set coefficients of variation of 0.15–0.46 and 0.18–0.65%, respectively. The measurement uncertainty is evaluated. The method can trace to mass.

Quantification of the 2-Deoxyribonolactone and Nucleoside 5′-Aldehyde Products of 2-Deoxyribose Oxidation in DNA and Cells by Isotope-Dilution Gas Chromatography Mass Spectrometry: Differential Effects of γ-Radiation and Fe2+−EDTA

The oxidation of 2-deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC-MS) method for the rigorous quantification of two major 2-deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1'-oxidation and the nucleoside 5'-aldehyde of 5'-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC-MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5'-aldehyde lesions. Further, the well-defined 2-deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin gamma(1)(I), was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5'-aldehyde per 10(6) nt per microM in accord with its established minor 1'- and major 5'-oxidation chemistry. Calicheamicin unexpectedly caused 1'-oxidation at a low level of 10 2-deoxyribonolactone per 10(6) nt per microM in addition to the expected predominance of 5'-oxidation at 560 nucleoside 5'-aldehyde per 10(6) nt per microM. The two hydroxyl radical-mediated DNA oxidants, gamma-radiation and Fe(2+)-EDTA, produced nucleoside 5'-aldehyde at a frequency of 57 per 10(6) nt per Gy (G-value 74 nmol/J) and 3.5 per 10(6) nt per microM, respectively, which amounted to 40% and 35%, respectively, of total 2-deoxyribose oxidation as measured by a plasmid nicking assay. However, gamma-radiation and Fe(2+)-EDTA produced different proportions of 2-deoxyribonolactone at 7% and 24% of total 2-deoxyribose oxidation, respectively, with frequencies of 10 lesions per 10(6) nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 10(6) nt per microM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5'-aldehyde of 9.7 and 73 lesions per 10(6) nt, respectively. Gamma-irradiation of the cells caused increases of 0.045 and 0.22 lesions per 10(6) nt per Gy, respectively, which represents a approximately 250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-deoxyribose oxidation products generated by gamma-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-deoxyribose in DNA, but the large differences between gamma-radiation and Fe(2+)-EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry.

Bromotyrosines in sputum proteins and treatment effects of terbutaline and budesonide in asthma

Inhaled corticosteroids are widely used in the treatment of persistent asthma, usually combined with inhaled beta2-agonists. Previous research suggests that short-acting beta2-agonists (SABAs) may downregulate the anti-inflammatory effects of inhaled corticosteroids, thereby increasing asthma morbidity. To determine whether 3-bromotyrosine and 3,5-dibromotyrosine levels, specific markers of eosinophil activation, reflect treatment effects on airway inflammation of inhaled corticosteroids and SABAs and support previous conclusions. Levels of 3-bromotyrosine and 3,5-dibromotyrosine were measured in sputum supernatants using stable isotope dilution gas chromatography-mass spectrometry in a randomized, placebo-controlled, crossover study of treatment with terbutaline, budesonide, and their combination in patients with persistent asthma. Thirty-four individuals were randomized, and 28 completed the study. Treatment with budesonide lowered median 3-bromotyrosine levels compared with treatment with placebo, terbutaline, and budesonide-terbutaline (0.24 vs 0.64, 0.62, and 0.43 3-bromotyosine/tyrosine [mmol/mol]; P < .05) and lowered median 3,5-dibromotyrosine levels compared with placebo and terbutaline treatments (0.04 vs 0.11 and 0.07 3,5-dibromotyrosine/ tyrosine [mmol/mol], P < .05). Unlike eosinophil numbers, 3-bromotyrosine and 3,5-dibromotyrosine levels did not increase with terbutaline treatment compared with placebo treatment but were significantly raised when terbutaline was added to budesonide treatment. 3-Bromotyrosine levels correlated significantly with eosinophil cationic protein levels in all groups. 3-Bromotyrosine and 3,5-dibromotyrosine levels reflect treatment effects in asthma and support previous findings that SABAs impair the anti-inflammatory effects of inhaled corticosteroids. In addition to eosinophil numbers and eosinophil cationic protein levels, these modified tyrosine residues provide useful information about the inflammatory state of the airways.

Development of a Candidate Reference Measurement Procedure for the Analysis of Cortisol in Human Serum Samples by Isotope Dilution-Gas Chromatography-Mass Spectrometry

A candidate reference measurement procedure involving isotope dilution coupled with gas chromatography-mass spectrometry (GC-MS) has been developed and critically evaluated. An isotopically labeled internal standard, cortisol-d(2), was added to a serum sample. After equilibration, solid-phase extractions (SPE) for sample preparation and derivatization with heptafluorobutyric anhydride (HFBA) were performed for GC-MS analysis. The limit of detection (LOD) and the limit of quantification (LOQ) were 5 and 20 ng g(-1), respectively. The recovery of the added cortisol ranged from 99.8 to 101.0%. Excellent precision was obtained with a within-day variation (RSD) of 0.7% for GC-MS analysis. The accuracy of the measurement was evaluated by comparing of results of this reference measurement procedure on lyophilized human serum reference materials for cortisol (European Reference Materials (ERM)-DA 192) as Certified Reference Materials (CRMs). The results of this method for total cortisol agreed with the certified values within some uncertainty. This method, which demonstrates simply, easy, good accuracy, high precision, and is free from interferences from structural analogues, qualifies as a reference measurement procedure.

A gas chromatography–mass spectrometry method for the quantitation of N-nitrosoproline and N-acetyl- S-allylcysteine in human urine: Application to a study of the effects of garlic consumption on nitrosation

Biomarkers in urine can provide useful information about the bioactivation of chemical carcinogens and can be used to investigate the chemoprotective properties of dietary nutrients. N-Nitrosoproline (NPRO) excretion has been used as an index for endogenous nitrosation. In vitro and animal studies have reported that compounds in garlic may suppress nitrosation and inhibit carcinogenesis. We present a new method for extraction and sensitive detection of both NPRO and N-acetyl- S-allylcysteine from urine. The latter is a metabolite of S-allylcysteine, which is found in garlic. Urine was acidified and the organic acids were extracted by reversed-phase extraction (RP–SPE) and use of a polymeric weak anion exchange (WAX–SPE) resin. NPRO was quantified by isotope dilution gas chromatography–mass spectrometry (GC–MS) using [ 13C 5]NPRO and N-nitrosopipecolinic acid (NPIC) as internal standards. This method was used to analyze urine samples from a study that was designed to test whether garlic supplementation inhibits NPRO synthesis. Using this method, 2.4 to 46.0 ng NPRO/ml urine was detected. The method is straightforward and reliable, and it can be performed with readily available GC–MS instruments. N-Acetyl- S-allylcysteine was quantified in the same fraction and detectable at levels of 4.1 to 176.4 ng/ml urine. The results suggest that 3 to 5 g of garlic supplements inhibited NPRO synthesis to an extent similar to a 0.5-g dose of ascorbic acid or a commercial supplement of aged garlic extract. Urinary NPRO concentration was inversely associated with the N-acetyl- S-allylcysteine concentration. It is possible that allyl sulfur compounds found in garlic may inhibit nitrosation in humans.

Impact of rosiglitazone and glyburide on nitrosative stress and myocardial blood flow regulation in type 2 diabetes mellitus

Cardiovascular disease, the leading cause of death in patients with type 2 diabetes mellitus (T2DM), is usually preceded by endothelial dysfunction and altered myocardial blood flow (MBF) regulation. Hyperglycemia, oxidative-nitrosative stress, systemic inflammation, and insulin resistance are implicated in the pathogenesis of abnormal MBF regulation, myocardial ischemia, and apoptosis. However, the impact of oral antihyperglycemic therapy on myocardial perfusion is controversial. Our objective was to explore the effect of rosiglitazone and glyburide on nitrosative stress and MBF regulation in subjects with T2DM. [ 13N]ammonia positron emission tomography and cold pressor testing were used in 27 diabetic subjects (mean age, 49 ± 11 years; glycohemoglobin, 7% ± 1.5%) randomized to either rosiglitazone 8 mg/d or glyburide 10 mg/d for 6 months. Isotope dilution gas chromatography–mass spectrometry was used to quantify plasma 3-nitrotyrosine, a stable marker of reactive nitrogen species. At 6 months, there were no significant differences between groups in the mean glycohemoglobin, blood pressure, or plasma lipids. Rosiglitazone significantly reduced plasma nitrotyrosine, high-sensitivity C-reactive protein, and von Willebrand antigen ( P < .03 for all) and significantly increased plasma adiponectin ( P < .05). No significant changes in these parameters were observed with glyburide. Treatment with glyburide, but not rosiglitazone, resulted in a significant deterioration in both resting and stress MBF. Rosiglitazone, but not glyburide, ameliorated markers of nitrosative stress and inflammation in subjects with T2DM without impairing myocardial perfusion.