Isotope Dilution Gas Chromatography-mass Spectrometry Research Articles

Determination of singlet oxygen-specific versus radical-mediated lipid peroxidation in photosensitized oxidation of lipid bilayers: effect of beta-carotene and alpha-tocopherol.

Photosensitized oxidation reactions damage tissue by catalyzing the formation of oxyradicals and singlet oxygen. beta-Carotene is hypothesized to exert photoprotective effects by quenching singlet oxygen formed by Type II reactions and by scavenging free radicals formed by Type I reactions. beta-Carotene antioxidant mechanisms were studied in a phospholipid membrane model of photooxidation with a new isotope dilution gas chromatography-mass spectrometry (GC-MS) assay that quantitatively distinguishes singlet oxygen-mediated and radical-mediated lipid peroxidation. This assay measures 9- and 10-hydroxylinoleate methyl esters and was used to generate photooxidation profiles for the photosensitizers methylene blue, Rose Bengal, and tetraphenylporphine. These profiles indicate a shift from Type II to Type I photooxidation mechanisms in later stages of photooxidation. beta-Carotene (0.45 mol %) inhibited singlet oxygen-mediated lipid peroxidation at early stages of methylene blue-sensitized photooxidation. Production of radical-mediated products increased faster than singlet oxygen-mediated products at later stages. beta-Carotene-5,8-endoperoxide, a specific marker for singlet oxygen oxidation of beta-carotene in solution, was unstable under the incubation conditions and was not detected in this system. alpha-Tocopherol (0.45 mol %) was ineffective in inhibiting photosensitized lipid peroxidation, whereas 4.5 mol % alpha-tocopherol inhibited almost all radical-mediated lipid peroxidation as well as early-stage singlet oxygen-mediated lipid peroxidation. Cumene hydroperoxide stimulated radical-mediated lipid peroxidation, indicating that accumulation of hydroperoxides from Type II photooxidation may enhance Type I reactions. These data suggest that singlet oxygen quenching, rather than radical scavenging reactions, accounts for the photoprotective actions of beta-carotene.

Caloric Restriction Attenuates Dityrosine Cross-Linking of Cardiac and Skeletal Muscle Proteins in Aging Mice

Oxidative damage, particularly to proteins, has been widely postulated to be a major causative factor in the loss of functional capacity during senescence. The nature of the various mechanisms that may contribute to protein oxidation is only partially understood. In this study, concentrations of two markers for oxidative damage,o,o′-dityrosine ando-tyrosine, were determined using stable isotope dilution gas chromatography–mass spectrometry in four tissues of the mouse, namely heart, skeletal muscle, brain, and liver, during youth (4 months old), adulthood (14 months old), and old (30 months old) age. A comparison was made between mice that had access to unlimited calories with those that were restricted to 60% of the caloric intake of thead libitumregimen. Caloric restriction of this magnitude extends the average and maximum life span of mice by ∼40%.In vitrostudies demonstrated thato,o′-dityrosine was generated selectively in proteins exposed to tyrosyl radical.o-Tyrosine increased in proteins oxidized with hydroxyl radical, which also resulted in a variable increase ino,o′-dityrosine. In mice fedad libitum, levels ofo,o′-dityrosine increased with age in cardiac and skeletal muscle but not in liver or brain. In contrast,o-tyrosine levels did not rise with age in any of the tissues examined. These results suggest that tyrosyl radical-induced protein oxidation increases selectively with age in skeletal muscle and heart. Caloric restriction prevented the increase ino,o′-dityrosine levels in cardiac and skeletal muscle but did not influenceo-tyrosine levels in any of the four tissues. This selective increase ino,o′-dityrosine levels and its prevention by a life-prolonging caloric restriction regimen raise the possibility that oxidation of muscle proteins by tyrosyl radical contributes to the deterioration of cardiac and skeletal muscle function with advancing age.

Determination of total cholesterol in serum by liquid chromatography–isotope dilution mass spectrometry

We have developed a liquid chromatography-isotope dilution mass spectrometry procedure to quantify total cholesterol in serum. A particle-beam interface was used for coupling the liquid chromatograph and the mass spectrometer. After electron impact ionization the ions m/z = 386 and m/z = 389 were used for selective ion monitoring of cholesterol and the internal standard [25,26,27-(13)C]cholesterol. The sample preparation steps required for serum materials are alkaline hydrolysis and an extraction of the cholesterol into the cyclohexane phase. Imprecision for the determination of cholesterol in control materials is typically <1.0%. The deviation from the certified reference values was <0.75% for all control materials tested. A method comparison of the results obtained by this method with those obtained by gas chromatography-isotope dilution mass spectrometry for n = 28 pooled human sera derived from samples analyzed in our routine laboratory did not show differences >2.5%.

Creatinine measurement proficiency testing: assignment of matrix-adjusted ID GC-MS target values

The results of an external quality-assessment experiment for serum creatinine measurement are described. Fifty-one laboratories performed quintuplicate analyses during three different analytical runs on six lyophilized sera and two frozen human serum pools. Isotope dilution gas chromatography-mass spectrometry (ID GC-MS) target values were assigned to all the materials. Intralaboratory within- and between-run imprecision results were very similar for all the materials tested (CV < or = 2.20% and < or = 4.70%, respectively). The overall imprecision obtained was high (CV 6.5-20.0%) because of increased interlaboratory-intermethod variability. A significant positive bias (+ 9.2-+43.7%) was found for all the materials at lower creatinine concentration. By using two human sera at different concentrations, we could calculate the constant and the proportional calibration bias displayed by each peer group. The majority of the lyophilized materials showed a behavior divergent from the frozen pools, indicating matrix-related problems. We propose a new algorithm for calculating matrix bias correction factor instrument-reagent specific for each material.

Urinary isoflavonoid excretion in humans is dose dependent at low to moderate levels of soy-protein consumption

Soybeans contain isoflavones, which have been associated with many health benefits, including decreased cancer risk. The purpose of our study was to measure urinary isoflavonoid excretion in response to daily consumption of soy that contained 0-36 mg isoflavones--a lower range than used in previous studies--and to compare urinary isoflavonoid excretion between equol excreters and nonexcreters. Fourteen men and women aged 20-40 y participated in the study. Half of the subjects were identified previously as equol excreters and the other half as equol nonexcreters. This randomized, double-blind, crossover study consisted of four 9-d diet treatment periods. During each treatment period participants consumed a low-photoestrogen controlled diet and a beverage containing 0, 5, 10, or 20 g soy protein. Urine collected on the last 3 d of each treatment period was analyzed for isoflavonoid (equol, O-desmethylangolensin, genistein, and daidzein) and lignan (enterodiol and enterolactone) contents by using isotope-dilution gas chromatography-mass spectrometry. There was a highly linear dose response of urinary isoflavonoid excretion to soy consumption, which did not differ significantly between equol excreters and nonexcreters. There were no significant differences in lignan excretion between the two diet treatments. Our results indicate that urinary isoflavonoid excretion is dose dependent in humans at low to moderate levels of soy consumption.

Primary hyperoxaluria in an adult with renal failure, livedo reticularis, retinopathy, and peripheral neuropathy

We present the case of a young woman who developed renal failure of unknown cause, and after 2 months of maintenance hemodialysis developed livedo reticularis, retinopathy, and peripheral sensory neuropathy. The patient was subsequently shown to have primary oxalosis type I, a rare autosomal recessive error of metabolism characterized by accumulation of calcium oxalate crystals in the kidneys, eyes, skin, and other organs. Intravascular obstruction, caused by deposition of calcium oxalate crystals in cutaneous arterioles, is thought to be responsible for the ischemic livedo reticularis lesions observed in this patient. A method is described for measuring serum glycolate by isotope dilution gas chromatography-mass spectrometry (GC-MS). An approach to the diagnosis and management is also briefly mentioned.

3-Chlorotyrosine, a specific marker of myeloperoxidase-catalyzed oxidation, is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima.

Oxidation of LDL may be of pivotal importance in atherogenesis, but the mechanisms that promote oxidation in vivo remain poorly understood. We have explored the possibility that one pathway involves myeloperoxidase, a heme protein secreted by phagocytes. Myeloperoxidase is the only human enzyme known to generate hypochlorous acid (HOCl), a potent oxidizing agent, at physiological halide concentrations. LDL exposed to the complete myeloperoxidase-H2O2-Cl- system underwent chlorination of its protein tyrosyl residues. Treatment of LDL with reagent HOCl resulted in 3-chlorotyrosine formation, implicating HOCl as an intermediate in the enzymatic reaction pathway. In contrast, 3-chlorotyrosine was undetectable in LDL oxidized by hydroxyl radical, copper, iron, hemin, glucose, peroxynitrite, horseradish peroxidase, lactoperoxidase, or lipoxygenase. These results indicate that 3-chlorotyrosine is a specific marker for LDL oxidation by myeloperoxidase. To address the role of myeloperoxidase in promoting LDL oxidation in vivo, we used stable isotope dilution gas chromatography-mass spectrometry to quantify 3-chlorotyrosine in human aortic tissue and in LDL isolated from atherosclerotic lesions. The level of 3-chlorotyrosine in atherosclerotic tissue obtained during vascular surgery was sixfold higher than that of normal aortic intima. Moreover, the level of 3-chlorotyrosine was 30-fold higher in LDL isolated from atherosclerotic intima compared with circulating LDL. The detection of 3-chlorotyrosine in human atherosclerotic lesions indicates that halogenation reactions catalyzed by the myeloperoxidase system of phagocytes constitute one pathway for protein oxidation in vivo. These findings raise the possibility that the myeloperoxidase-H2O2-Cl- system plays a critical role in converting LDL into an atherogenic form.

Accurate and precise isotope dilution mass spectrometry method for determining glucose in whole blood

An accurate and precise method to determine glucose concentration in whole blood is presented. The method, based on isotope dilution gas chromatography-mass spectrometry (ID GC-MS), was developed to be used as a Reference Method for determining glucose concentration in capillary or venous whole blood. Blood samples and standards are pipetted manually with "microcap" micropipettes, which makes it possible to collect samples even at the patient's bedside. Glucose is quantified as its aldononitrile pentaacetate. [13C6]Glucose is used as an internal standard. Assay of Seronorm and Pathonorm L and H controls by ID GC-MS gave within-run CVs of 0.66%, 0.96%, and 0.92%, respectively. For whole blood with glucose concentrations in the low, normal, and high ranges, the within-run CVs were 1.27%, 0.91%, and 0.78%, respectively. The between-run CV for glucose calculated from 36 separate single analyses of Seronorm was 1.44%. In an accuracy assessment test of the HemoCue blood glucose analyzer, 140 capillary blood samples were measured in parallel after split-sampling. For all samples the HemoCue analyzer results had a mean bias of +2.0% compared with the ID GC-MS results.

Convenient chromatographic prepurification step before measurement of urinary cortisol by radioimmunoassay

We applied various prepurification protocols (extraction with different solvents, liquid/solid separation on bonded silica media, Celite, and Sephadex LH20 chromatography) with a range of commercially available RIA kits to measure cortisol in urine samples. We then compared the results with the concentrations measured by a HPLC method validated with reference to isotope dilution gas chromatography-mass spectrometry. We conclude that chromatography on a commercial, prepacked diol minicolumn (Waters Sep-Pak Vac RC) in combination with dichloromethane extraction is a convenient and very effective purification step before RIA of urinary cortisol in patients not receiving corticoid medication. We tested numerous steroids for interference and found that free polar cortisol derivatives (hydroxylated or hydrogenated) could only partially account for the overestimations routinely encountered when free urinary cortisol concentrations are measured by direct RIA.

Congener-specific method for the determination of ortho and non-ortho polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans in foods by carbon-column fractionation and gas chromatography-isotope dilution mass spectrometry

The chromatographic behavior of ortho and non-ortho polychlorinated biphenyls (PCBs) on supported carbon columns has been investigated and the structure-affinity relationship of activated carbon towards PCB molecules established. Optimisation of the parameters controlling the elution of PCB congeners through the carbon column led to the development of a solvent scheme for the efficient separation of (i) ortho substituted PCBs, (ii) non-ortho substituted PCBs and (iii) polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in three separate fractions. A method for the extraction, clean-up, fractionation and determination of ortho and non-ortho substituted PCBs by GC-isotope dilution MS was developed and validated by analysis of a certified reference material. Possible losses of PCBs during freeze-drying and interferences of aliphatic hydrocarbons during mass spectrometric determination of ortho substituted PCBs are also discussed.

Determination of theophylline in serum by high performance liquid chromatography/mass spectrometry with atmospheric pressure chemical-ionization (APCI HPLC/MS)

A method for the determination of theophylline (TH), without derivatization, in serum by isotope dilution mass spectrometry using labelled [1,3-15N2-2-13C]theophylline (LTH) as internal standard is described. After deproteinization, the analyte is directly injected into a high performance liquid chromatography — mass spectrometer operating with atmosphericpressure chemical-ionization (APCI HPLC/MS). The concentrations of TH in sera measured by APCI HPLC/MS are compared with results from gas chromatography — isotope dilution mass spectrometry (GC-ID/MS), high performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA). The accuracy, precision and recovery of the APCI HPLC/MS and GC-ID/MS methods are discussed. The coefficient of variation (CV) determined from duplicate samples was less than 2%. The detection limit was 10 ng/ml at a signal-to-noise ratio of 3:1.

Mass Spectrometric Quantification of Markers for Protein Oxidation by Tyrosyl Radical, Copper, and Hydroxyl Radical in Low Density Lipoprotein Isolated from Human Atherosclerotic Plaques

Lipoprotein oxidation has been implicated in the pathogenesis of atherosclerosis. However, the physiologically relevant pathways mediating oxidative damage have not yet been identified. Three potential mechanisms are tyrosyl radical, hydroxyl radical, and redox active metal ions. Tyrosyl radical forms o,o'-dityrosine cross-links in proteins. The highly reactive hydroxyl radical oxidizes phenylalanine residues to o-tyrosine and m-tyrosine. Metal ions oxidize low density lipoprotein (LDL) by poorly understood pathways. To explore the involvement of tyrosyl radical, hydroxyl radical, and metal ions in atherosclerosis, we developed a highly sensitive and quantitative method for measuring levels of o, o'-dityrosine, o-tyrosine, and m-tyrosine in proteins, lipoproteins, and tissue, using stable isotope dilution gas chromatography-mass spectrometry. We showed that o,o'-dityrosine was selectively produced in LDL oxidized with tyrosyl radical. Both o-tyrosine and o, o'-dityrosine were major products when LDL was oxidized with hydroxyl radical. Only o-tyrosine was formed in LDL oxidized with copper. Similar profiles of oxidation products were observed in bovine serum albumin oxidized with the three different systems. Applying these findings to LDL isolated from human atherosclerotic lesions, we detected a 100-fold increase in o,o'-dityrosine levels compared to those in circulating LDL. In striking contrast, levels of o-tyrosine and m-tyrosine were not elevated in LDL isolated from atherosclerotic tissue. Analysis of fatty streaks revealed a similar pattern of oxidation products; compared with normal aortic tissue, there was a selective increase in o,o'-dityrosine with no change in o-tyrosine. The detection of a selective increase of o,o'-dityrosine in LDL isolated from vascular lesions is consistent with the hypothesis that oxidative damage in human atherosclerosis is mediated in part by tyrosyl radical. In contrast, these observations do not support a role for free metal ions as catalysts of LDL oxidation in the artery wall.

The oxysterols cholest-5-ene-3β,4α-diol, cholest-5-ene-3β,4β-diol and cholestane-3β,5α,6α-triol are formed during in vitro oxidation of low density lipoprotein, and are present in human atherosclerotic plaques

Isolated human low density lipoprotein (LDL) was oxidized with either cupric ions or soybean lipoxygenase and linoleic acid. Cholesterol oxidation products (oxysterols) were determined by isotope dilution gas chromatography-mass spectrometry. A new cholestane-3,5,6-triol isomer, cholestane-3β,5α,6α-triol, which has not previously been recognized as a cholesterol autoxidation product, was found at similar concentrations as the well-known cytotoxic cholestane-3β,5α,6β-triol during both copper- and lipoxygenase-mediated LDL oxidation. Furthermore, two epimeric cholest-5-ene-3β,4-diols. were identified in the oxidized LDL at similar concentrations. These two isomers were also identified in human atherosclerotic tissue in a ratio of 1:1 at a concentration more than 10-times higher than in non-atherosclerotic vessels. In vitro oxidation of LDL under an 180 2 atmosphere revealed that molecular oxygen was the only source of the oxygen functions at C-4 in the cholest-5-ene-3β,4-diols. Taken together, these findings suggest that the cholest-5-ene-3β,4-diols in atherosclerotic plaques are formed by autoxidation.

Methylmalonic acid quantification by stable isotope dilution gas chromatography-mass spectrometry from filter paper urine samples

A specific method for quantification of methylmalonic acid (MMA) from urine samples dried onto filter paper is described. The method involves stable isotope dilution gas chromatography-mass spectrometry with [methyl-2H3]-MMA as the internal standard. MMA is stable in dry paper samples stored at room temperature for at least 2 weeks. The extraction efficiency of MMA from paper was 56-58%. The concentration of urinary MMA in dried filter paper specimens from 190 normal controls was 1.21 +/- 1.34 (mean +/- SD) mmol/mol of urinary creatinine. Age-related reference values are also reported. The concentrations, normalized to the urinary creatinine concentration, decrease with age. The applicability of this method to rapid screening for cobalamin (vitamin B12)-related disorders and methylmalonic aciduria is demonstrated.

Four frequently used test systems for serum cholesterol evaluated by isotope dilution gas chromatography-mass spectrometry candidate reference method

We evaluated the performance of four frequently used cholesterol test systems, using split-sample measurements with a panel of 79 patients' specimens and isotope dilution gas chromatography-mass spectrometry (ID GS-MS) as a comparison method. The test systems were from Beckman, a Boehringer Mannheim, Merck, and Johnson & Johnson Clinical Diagnostics, performed on the Synchron CX7, Hitachi 717, Mega, and Ektachem 250 analyzers, respectively. The liner regression data for the method comparison [ID GS-MS as independent variable (x)] were for Beckman: slope = 1.012, intercept = 0.0243 mmol/L, dispersion (S(y/x)) = 0.1303 mmol/l, and correlation coefficient (r) = 0.9867; for Boehringer Mannheim: slope = 1.002, intercept = 0.114 mmol/L, Sy/x = 0.0759 mmol/L, r = 0.9954; for Merck: slope = 1.034, intercept = -0.0613 mmol/L, Sy/x = 0.0886 mmol/L, r = 0.9941; and for Johnson & Johnson Clinical Diagnostics: slope = 1.007, intercept = 0.01 mmol/L, Sy/x = 0.15 mmol/L, and r = 0.9811. These data demonstrate excellent state-of-the-art cholesterol measurement for some of the most widely used test systems.

Quality control in the determination of cortisol in plasma/serum by using, on every sample, two different three-step separation methods including ultrafiltration, restricted-access high-performance liquid chromatography and reversed-phase high-performance liquid chromatography, and contrasting results to immunoassays

Tests of HPLC columns with restricted access, polymer covered alumina, polymer, and different ODS phases showed that base-acid compatible ODS columns gave the best peak shapes of cortisol, internal standard, as well as of plasma/serum (P/S) matrix components. Further trials with cortisol in P/S showed that three separation steps were essential in order to obtain chromatographic data which were superior to immunoassay data. Also, sufficient confidence in results required determination of each sample with two newly developed separation methods: (a) pre-separation with a restricted access column, concentration of the desired cut with a 20 mm base-acid compatible ODS column, and analysis with a 250 mm column filled with the same ODS; (b) pre-separation with an ultrafilter followed by the last two steps in (a). For detection UV was preferred over fluorescence. This twin multistep chromatography showed that immunoassays were very treacherous in that they produced a spectrum of results ranging from good to untenable without any warning whatever about functionality. The measurement of official controls, with reference values derived via gas chromatography-isotope dilution mass spectrometry, also demonstrated the superiority of the double HPLC method.

Isotope Dilution Gas Chromatographic–Mass Spectrometric Method for the Determination of Isoflavonoids, Coumestrol, and Lignans in Food Samples

We present a method for the quantitative determination of the phytoestrogens formononetin, biochanin A, daidzein, genistein, and coumestrol and simultaneously the lignans secoisolariciresinol (SECO) and matairesinol in plant-derived foods. These compounds are measured by isotope dilution gas chromatography–mass spectrometry in the selected ion monitoring mode (ID/GC/MS/SIM) using synthesized deuterated internal standards for the correction of losses during the procedure. A three-step hydrolysis—a rehydration with distilled H2O, followed by enzymatic and acid hydrolysis—has been applied in order to convert the diphenolic glycosides into their respective aglycones. Purification and separation are carried out in two ion-exchange chromatographic steps followed by derivatization and GC–MS. The within-assay imprecision values vary 3.1–9.6% and the between-assay imprecision 7.0–21.2%. The mean recovery of authentic standards processed through the whole procedure varied from 95.5 to 105.5%. Values for some different food samples are presented. The simultaneous determination of the biologically most interesting phytoestrogens and lignans in foods has not been carried out previously and the method will be useful for screening of important foods in populations with different risk of cancer and coronary heart disease, and for metabolic studies.

Three routine methods for serum cortisol evaluated by comparison with an isotope dilution gas chromatography-mass spectrometry method

Three routine methods for serum cortisol evaluated by comparison with an isotope dilution gas chromatography-mass spectrometry method

Synthesis of 5,6,6-[ 2H 3]finasteride and quantitative determination of finasteride in human plasma at picogram level by an isotope-dilution mass spectrometric method

Finasteride is a potent inhibitor of the enzyme steroid 5α-reductase now approved as a drug for the treatment of benign prostatic hyperplasia. We describe an original method for the quantitative determination of finasteride at picogram level in human plasma by isotope-dilution gas chromatography mass spectrometry. 5,6,6-[ 2H 3]Finasteride was synthetized with an high ratio of trideuteration ( finasteride/[ 2 H 3] finasteride = 0.007) allowing its optimal use as internal standard. Plasma samples were purified in a single-step procedure on solid-phase extraction C 18 columns with a recovery ⩾90%. Samples were injected in the GC-MS instrument without any derivatization and the minimum detection level of finasteride was 50 pg with a singal-to-noise ratio of 6:1. The coefficients of variation for the 5 and 10 ng/ml (plasma) concentrations were 5.8% and 4%, respectively. the method has been applied to the determination of the plasma pharmacokinetic of finasteride in five male volunteers treated with a single 5-mg dose of the drug, affording kinetic parameters which are in good agreement with the values previously reported with a different methodology. The present method results accurate, specific, sensible and reliable for a routinely determination of finasteride at picogram levels.

Regulation of sex hormone-binding globulin production by isoflavonoids and patterns of isoflavonoid conjugation in HepG2 cell cultures

The effect of the isoflavonoid phytoestrogens daidzein, equol, and genistein on sex hormone-binding globulin (SHBG) levels, SHBG mRNA transcript levels, and SHBG gene methylation was studied in HepG2 cell cultures by fluoroimmunometric SHBG assay and Northern and Southern hybridizations, respectively. The effect of 17β-estradiol on these parameters was studied as a control. The metabolism of isoflavonoids in HepG2 cells was determined by isotope dilution gas chromatography-mass spectrometry, after ion-exchange chromatography. Daidzein and equol increased SHBG levels in parallel intracellularly and extracellularly, whereas genistein increased SHBG levels only within the cells, resembling thus the effect of 17β-estradiol. The difference may originate from the fact that genistein has more hydroxyl groups than daidzein and equol. The regulation of SHBG production by phytoestrogens appears to occur at the post-transcriptional level. Firstly, daidzein, equol, or genistein did not have a clear effect on the steady-state SHBG mRNA levels. Secondly, no effect on SHBG gene methylation was observed by genistein. The findings applied also to 17β-estradiol. However, as the SHBG gene was more methylated in SHBG-negative MCF-7 cells than in SHBG-positive HepG2 cells, DNA methylation may play a role in the tissue-specific activation of this gene. The metabolism of isoflavonoids in HepG2 cells yielded mainly unconjugated and sulfated compounds. Similar metabolism in hepatocytes in vivo might retain their biological activity in tissues responsive to estrogens.