Dilution Gas Chromatography-mass Spectrometry Research Articles

Creatine and guanidinoacetate: diagnostic markers for inborn errors in creatine biosynthesis and transport

In this study, measurements of guanidinoacetate (GAA) and creatine (Cr) in urine, plasma, and cerebrospinal fluid (CSF) were performed using stable isotope dilution gas chromatography–mass spectrometry. Both compounds were analyzed in a single analysis. Reference values were established for GAA and Cr. These values were age dependent. No differences with gender were observed. Eight guanidinoacetate methyltransferase (GAMT) deficient patients and eight creatine transporter SLC6A8 deficient patients were investigated. In urine, plasma, and CSF of GAMT deficient patients increased levels of GAA are present. The SLC6A8 deficient patients all show increased creatine/creatinine (Cr/Crn) ratio in urine demonstrating the importance of the Cr/Crn ratio as a pathognomonic marker of the SLC6A8 deficiency.

Simultaneous determination of mono-, di- and tributyltin in environmental samples using isotope dilution gas chromatography mass spectrometry.

The development of a rapid, precise and accurate speciation method for the simultaneous determination of mono-, di- and tributyltin in environmental samples is described. The method is based on using isotope dilution gas chromatography/mass spectrometry (GC/MS) with electron ionization, a widely used technique in routine testing laboratories. A mixed spike containing (119)Sn-enriched monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT) was used for the isotope dilution of the samples. Five molecular ions were monitored for each analyte, corresponding to the (116)Sn, (117)Sn, (118)Sn, (119)Sn and (120)Sn isotopes. The detection at masses corresponding to (116)Sn and (117)Sn were used to correct for m + 1 and m + 2 contributions of (13)C from the organic groups attached to the tin atom on the (118)Sn, (119)Sn and (120)Sn masses with simple mathematical equations and the concentrations of the butyltin compounds were calculated based on the corrected (118)Sn/(119)Sn and (120)Sn/(119)Sn isotope ratios. The (119)Sn-enriched multispecies spike was applied with satisfactory results to the simultaneous determination of MBT, DBT and TBT in three certified reference materials: two sediments, PACS-2 and BCR 646, and the mussel tissue CRM 477. The method was compared with a previously published GC/inductively coupled plasma MS isotope dilution procedure, developed in our laboratory, by injecting the same samples into both instruments. Comparable analytical results in terms of precision and accuracy are demonstrated for both atomic and molecular mass spectrometric detectors. Thus, reliable quantitative organotin speciation analysis can be achieved using the more widespread and inexpensive GC/MS instrument.

CCQM K27 (a, b): Determination of ethanol in aqueous matrix

A key comparison on the determination of ethanol in aqueous matrix has been successfully completed. Nine national metrology institutes (NMIs) participated in this key comparison and used the techniques of gas chromatography with flame ionization detection (GC-FID), isotope dilution gas chromatography–mass spectrometry (GC-IDMS), gas chromatography–combustion–isotope ratio mass spectrometry (ID-GC-C-IRMS) and headspace-GC-FID for the determinations. Three samples were distributed to participants. Two of these samples (Samples A and B) were aqueous solutions that had been spiked gravimetrically with ethanol (at mass fractions of ethanol representing forensic standards, CCQM-K27a), the other (Sample C) was a commercial (red) wine (representing a traded commodity, CCQM-K27b). The KCRV for Sample A is 0.8040 ± 0.0080 mg g-1, that of Sample B is 120.90 ± 0.35 mg g-1 and that of Sample C is 81.23 ± 0.24 mg g-1 of ethanol in the appropriate aqueous matrix. These results are an improvement over those of the corresponding pilot study (CCQM-P35). The RSD for Sample A is 1.5% compared to 2.3% for a similar mass fraction for the pilot study. For the wine sample (Sample C) corresponding RSDs are 0.44% for this key comparison and 2.3% for the pilot study. This demonstration of capability will enable forensic ethanol standards to be compared across national boundaries. Likewise the ability to measure the typical mass fraction of ethanol found in wine is important since such commodities are traded worldwide and are liable to varying levels of excise duty. Thus this successful key comparison has demonstrated a broad range of capability by NMIs for the measurement of ethanol for both forensic and excise purposes.Main text.To reach the main text of this paper, click on Final Report.The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the Mutual Recognition Arrangement (MRA).

Galactonate determination in urine by stable isotope dilution gas chromatography–mass spectrometry

A stable isotope dilution assay was developed for the sensitive determination of d-galactonic acid. d-[U- 13 C 6 ]galactono-1,4-lactone was prepared as internal standard. Unlabelled and U- 13 C -labelled d-galactonic acid species were converted to the N-(1-butyl)galactonamide pentaacetate derivatives and assessed by gas chromatography–mass spectrometry (GC–MS). Positive chemical ionisation and monitoring of the [ MH-60] +-ions in the galactonate chromatographic peak at m/ z 402 and m/ z 408 were used for quantification. The procedure was applied to study the variability of d-galactonate excretion in healthy subjects and galactosemic patients and to monitor the d-galactonate– d-galactitol ratio in human urine.

Identification of alpha-chloro fatty aldehydes and unsaturated lysophosphatidylcholine molecular species in human atherosclerotic lesions.

A role for myeloperoxidase (MPO) as a mediator of coronary artery disease and acute coronary syndromes has recently received considerable attention. Although active MPO and hypochlorite-modified proteins and peptides have been detected in human atherosclerotic lesions, detection of novel chlorinated oxidized lipid species with proatherogenic properties in vivo has not yet been reported. In this study we show that MPO-generated reactive chlorinating species promote selective oxidative cleavage of plasmalogens, liberating alpha-chloro fatty aldehydes and unsaturated lysophosphatidylcholine in human atherosclerotic lesions. Stable isotope dilution gas chromatography-mass spectrometry methods were used to identify and quantitate the alpha-chloro fatty aldehyde, 2-chlorohexadecanal, in atherosclerotic versus normal human aorta. Compared with normal aorta, 2-chlorohexadecanal levels were elevated more than 1400-fold in atherosclerotic tissues. Parallel electrospray ionization mass spectrometry studies confirmed 34- and 20-fold increases in the plasmalogen cooxidation products, unsaturated lysophosphatidylcholine molecular species containing linoleic and arachidonic acid, respectively, within atherosclerotic compared with normal aorta. Unsaturated lysophosphatidylcholine containing docosahexaenoic acid was also detected in atherosclerotic but not in normal aorta. Exposure of primary human coronary artery endothelial cells to plasmalogen-derived lysophosphatidylcholine molecular species produced marked increases in P-selectin surface expression. The present studies demonstrate that plasmalogens are attacked by MPO-derived reactive chlorinating species within human atheroma. The resultant species formed, alpha-chloro fatty aldehydes and unsaturated lysophospholipids, possess proatherogenic properties, as shown by induction of P-selectin surface expression in primary human coronary artery endothelial cells.

Quantitative trace analysis of estriol in human plasma by negative ion chemical ionization gas chromatography–mass spectrometry using a deuterated internal standard

A stable isotope dilution gas chromatography–mass spectrometry (GC–MS) assay for the trace level determination of estriol in human plasma is described. Negative ion chemical ionization (NICI) MS is used for highly specific detection. The method involves derivatization of the phenolic hydroxyl to the pentafluorobenzyl ether derivative and subsequent reaction of the remaining hydroxyls with heptafluorobutyric anhydride. This derivative allows detection of the strikingly abundant phenolate ion under NICI conditions. [2,4,17β]- 2H 3-labeled estriol was used as an internal standard. For high-level measurements (>313 ng/l) plasma was directly derivatized by extractive alkylation followed by heptafluorobutylation prior to analysis. A rapid and simple sample work up procedure was elaborated for trace level determinations (>5 ng/l plasma) using solid-phase extraction on C 18 with an absolute recovery of 92.9%. For low-level measurements, the calibration curve was linear in the range of 5 to 625 ng/l ( r=0.99993). Inter-assay analytical precisions (RSDs) were 1.29, 2.30 and 2.89% at 39, 156 and 650 ng/l plasma, respectively. For high-level measurements, calibration curve linearity was observed in the range of 0.313 to 20 μg/l ( r=0.99998). Inter-assay analytical precisions (RSDs) were 5.17, 1.92, 2.57 and 2.74% at 0.313, 0.625, 2.5 and 10 μg/l plasma, respectively. Postmenopausal plasma was used for spiked plasma samples. Sensitivity and specificity of the presented method allows adequate determination of estriol in human plasma samples.

Renal excretion of galactose and galactitol in patients with classical galactosaemia, obligate heterozygous parents and healthy subjects.

The age dependence of galactose and galactitol excretion was assessed in overnight-fasted galactose-1-phosphate uridyltransferase-deficient patients under dietary treatment (ages 4-34 years; n = 51), obligate heterozygous parents (ages 25-71 years; n = 49) and healthy subjects (ages 3-58 years; n = 215). Urine concentrations were analysed by stable-isotope dilution gas chromatography mass spectrometry. There was considerable interindividual variability. The intraindividual variation, however, was not age-dependent and was rather low. Excretion estimates were calculated from the creatinine-related concentrations using weight-, age- and sex-related creatinine excretion rates. Experimental evidence is presented underscoring the problems inherent in random sampling and substantiating the primary endogenous origin of galactose and galactitol in postabsorptive urine samples. Age-dependent excretion estimates were best fitted to a simple growth-related model assuming an exponential decrease with age until adulthood. According to the model, mean postabsorptive galactose and galactitol excretion in healthy subjects was similar and decreased exponentially from about 1.2 micromol/kg body weight per day in infants to about 0.2 micromol/kg body weight per day in adults. Excretion in heterozygotes was normal. In galactosaemic patients, galactose excretion was in the normal range. Galactitol excretion, however, was enhanced over 50-fold and decreased from a mean estimate of about 64 micromol/kg body weight per day in infants to about 23 micromol/kg body weight per day in adults. The results are discussed with respect to the significance of galactose and galactitol excretion for whole-body galactose removal and with respect to the applicability of urinary galactitol analysis for metabolic monitoring in galactosaemia.

CCQM-K21 Key Comparison – Determination of pp'-DDT in fish oil

A key comparison on the determination of (pp'-dichlorodiphenyl) trichloroethane (pp'-DDT) in a fish oil matrix has been successfully completed. Nine NMIs participated in this key comparison and used the technique of isotope dilution gas-chromatography mass spectrometry (ID/GC/MS) for the determinations. Two samples (A and B) of fish oil were distributed to participants, each gravimetrically spiked with pp'-DDT. The KCRV for Sample A is 0.0743 ± 0.0020 µg g-1 and that of Sample B is 0.1655 ± 0.0014 µg g-1 of pp'-DDT in fish oil. The results for Sample A showed a RSD of 3.5%, the RSD for Sample B was within 1%. These results were an improvement over those of the corresponding pilot study (CCQM-P21), where at a mass fraction of pp'-DDT in fish oil of 0.311 µg g-1 the RSD was 2.6%. The compound pp'-DDT is a typical organochlorine pesticide and this key comparison has shown that NMIs have the ability to measure such compounds at levels typically found in the environment. The compound (pp'-dichlorodiphenyl) dichloroethylene (pp'-DDE), a metabolite of pp'-DDT, was the subject of a previous key comparison (CCQM-K5). The compound pp'-DDT is technically more challenging than that of pp'-DDE since it can decompose during the measurement procedure. Consequently the success of this key comparison, combined with that of CCQM-K5 demonstrates a broad measurement capability by NMIs for organochlorine compounds in the environment.Main text.To reach the main text of this paper, click on Final Report.Note that this text is that which appears in Appendix B of the BIPMkey comparison database kcdb.bipm.org/.The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the Mutual Recognition Arrangement (MRA).

Determination of selected monohydroxy metabolites of 2-, 3- and 4-ring polycyclic aromatic hydrocarbons in urine by solid-phase microextraction and isotope dilution gas chromatography–mass spectrometry

Eighteen monohydroxy polycyclic aromatic hydrocarbon metabolites (OH-PAHs) representing polycyclic aromatic hydrocarbons (PAHs) containing up to four rings in human urine have been measured. The method includes the addition of carbon-13 labeled internal standards, enzymatic hydrolysis, and solid-phase microextraction followed by gas chromatography with high-resolution mass spectrometry. By using response factors calculated with the carbon-13 labeled standards, results are presented for calibration, relative standard deviations and analyte levels from an unspiked human urine pool. The method detection limits ranged from 0.78 ng/l for hydroxyphenanthrenes to 15.8 ng/l for 1-hydroxynaphthalene, and the recoveries ranged between 6% for hydroxychrysene and 47% for 1-hydroxypyrene. The relative standard deviation was lowest for 3-hydroxyphenanthrene at 2.4% and went up to 18.7% for 6-hydroxychrysene. The method was calibrated from 10 to 1200 ng/l. Eleven of the 18 metabolites were found in background pooled urine samples. This validated method is a convenient and reliable tool for determining urinary OH-PAHs as biomarkers of exposure to eight PAHs.

Ultrasensitive Semiautomated Chemiluminescent Immunoassay for Estradiol

The availability of rapid, non-extraction-based estradiol-17β immunoassays has been important in clinical settings where prompt turnaround time is required, including in vitro fertilization programs, and in large-scale research programs where large numbers of samples are processed. However, the inaccuracy of estradiol-17β assay results has been recognized for some time, as indicated in the Textbook of Reproductive Medicine by L.R. Boots (1), in which he states: “Everyone measures estradiol levels and it is probably assumed that few if any problems exist with this assay…. [T]he most common use of estradiol levels is in relation to levels of estradiol during ovulation stimulation and every assay provides clinically relevant results. These results are clearly inaccurate but usually precise” (1). In the First and Second E2 International Workshops (2), problems associated with the analysis of estradiol-17β were fully discussed. The First E2 International Workshop focused on the need for more specific, precise, and sensitive estradiol-17β assays, and the Second E2 International Workshop explored various means of increasing accuracy in the measurement of estradiol-17β. By consensus, the use of a panel of 22 samples confirmed by isotope dilution–gas chromatography–mass spectrometry (ID-GC-MS) (2) was made available for use in assay development. Indications of a lack of accuracy are also provided by examining survey programs, such as those offered by the College of American Pathologists, that reveal large differences in estradiol-17β values obtained on the same sample. Modern serum assays for estradiol-17β claim detection limits as low as 40 pmol/L or lower, but they lack sufficient sensitivity for pediatric or postmenopausal specimens and are frequently unable to provide adequate analytical precision and accuracy in samples from human males. In postmenopausal women, circulating estradiol-17β is usually 147 pmol/L are rare. In …

Rapid and sensitive method for prenatal diagnosis of propionic acidemia using stable isotope dilution gas chromatography–mass spectrometry and urease pretreatment

Propionic acidemia is one of the most frequent inborn errors of metabolism caused by a deficiency of propionyl-CoA carboxylase. Methylcitric acid, a key indicator of this disorder, is increased in amniotic fluid when a fetus is affected. Therefore, the direct chemical analysis of cell-free amniotic fluid for methylcitric acid, using stable isotope dilution gas chromatography–mass spectrometry, was carried out for the prenatal diagnosis of propionic acidemia. We developed a simple, highly sensitive, and accurate method for quantitation of this polar methylcitric acid in amniotic fluids by applying a simplified urease pretreatment which we devised earlier for urine. As the recovery of methylcitric acid from amniotic fluid was as high as 91% with a coefficient of variation lower than 3% in this procedure, only 0.02 ml of sample was required for the analysis of the affected fetus. This new procedure takes 1 h for sample pretreatment, including derivatization, and 15 min for GC–MS measurement and provides final results within 1.5 h.

Hydroxyl radical generation during exercise increases mitochondrial protein oxidation and levels of urinary dityrosine

Isolated mitochondria are well-established sources of oxidants in vitro. There is little direct evidence that mitochondria promote oxidative stress in vivo, however . Model system studies demonstrate that ortho-tyrosine, meta-tyrosine, and o,o′-dityrosine increase in proteins oxidized by hydroxyl radical. To determine whether mitochondria generate oxidants in vivo, we used isotope dilution gas chromatography mass spectrometry to quantify levels of these markers in the heart muscle of control and exercised rats. Exercise led to a 50% increase in ortho-tyrosine, meta-tyrosine, and o,o′-dityrosine in the mitochondrial proteins but not cytosolic proteins of heart muscle. This increase was transient, and levels returned to normal when exercised animals were allowed to rest. There also was a transient increase in the level of o,o′-dityrosine in the urine of exercised rats. This relationship between mitochondrial and urine levels of o,o′-dityrosine suggests that urine assays of this oxidized amino acid may serve as noninvasive measures of oxidative stress . These observations also provide direct evidence that heart muscle mitochondria produce an intermediate resembling the hydroxyl radical that promotes protein oxidation in vivo.

Analysis of pristanic acid β-oxidation intermediates in plasma from healthy controls and patients affected with peroxisomal disorders by stable isotope dilution gas chromatography mass spectrometry

In this paper we report the development of highly sensitive, selective, and accurate stable isotope dilution gas chromatography negative chemical ionization mass spectrometry (GC-NCI-MS) methods for quantification of peroxisomal β-oxidation intermediates of pristanic acid in human plasma: 2,3-pristenic acid, 3-hydroxypristanic acid, and 3-ketopristanic acid. The carboxylic groups of the intermediates were converted into pentafluorobenzyl esters, whereas hydroxyl groups were acetylated and ketogroups were methoximized. Hereafter, the samples were subjected to clean-up by high performance liquid chromatography. Analyses were performed by selected monitoring of the carboxylate anions of the derivatives. Control values of all three metabolites were established (2,3-pristenic acid: 2–48 nm, 3-hydroxypristanic acid: 0.02–0.81 nm, 3-ketopristanic acid: 0.07–1.45 nm). A correlation between the concentrations of pristanic acid and its intermediates in plasma was found. The diagnostic value of the methods is illustrated by measurements of the intermediates in plasma from patients with peroxisomal disorders. It is shown that in generalized peroxisomal disorders, the absolute concentrations of 2,3-pristenic acid, 3-hydroxypristanic acid, and 3-ketopristanic acid were comparable to those in the controls, whereas relative to the pristanic acid concentrations these intermediates were significantly decreased. In bifunctional protein deficiency, elevated levels of 2,3-pristenic acid and 3-hydroxypristanic acid were found. 3-Ketopristanic acid, although within the normal range, was relatively low when compared to the high pristanic acid levels in these patients.—Verhoeven, N. M., D. S. M. Schor, E. A. Struys, E. E. W. Jansen, H. J. ten Brink, R. J. A. Wanders, and C. Jakobs. Analysis of pristanic acid β-oxidation intermediates in plasma from healthy controls and patients affected with peroxisomal disorders by stable isotope dilution gas chromatography mass spectrometry. J. Lipid Res. 1999. 40: 260–266.

Quantitative determination of trimethylamine in urine by solid-phase microextraction and gas chromatography–mass spectrometry

Trimethylaminuria (fish odour syndrome) is diagnosed from an increase in urinary excretion of trimethylamine with decreased trimethylamine oxide. We report a new quantitative stable isotope dilution gas chromatography–mass spectrometry procedure for the analysis of these metabolites using solid-phase microextraction (SPME). Both polydimethylsiloxane and mixed Carboxen–polydimethylsiloxane SPME fibres were found to be suitable for the headspace extraction of TMA. This new sampling technique could have wide application for the analysis of volatile and semi-volatile compounds by metabolic screening laboratories.

Pristanic acid β-oxidation in peroxisomal disorders: studies in cultured human fibroblasts

To investigate the individual steps of peroxisomal β-oxidation, human fibroblasts from controls and patients affected by different peroxisomal disorders were incubated for 96 h with pristanic acid. Hereafter, 2,3-pristenic acid and 3-hydroxypristanic acid in the incubation medium were quantified by stable isotope dilution gas chromatography mass spectrometry (GC-MS). In control fibroblasts, both intermediates were formed and excreted into the medium in significant amounts. In cells from patients affected with different types of generalized peroxisomal disorders, the formation of both intermediates was absent or low, depending on the clinical severity of the disorder. In fibroblasts from patients affected with bifunctional protein deficiency, the concentrations of 2,3-pristenic acid and 3-hydroxypristanic acid in the medium were higher than in control cell lines.

Subnanomolar quantification of caffeine's in vitro metabolites by stable isotope dilution gas chromatography–mass spectrometry

A method for the quantification of subnanomolar levels of in vitro metabolites of caffeine by an isotope dilution gas chromatographic–mass spectrometric (GC–MS) assay has been developed and applied. Trideuteromethylated analogs of each primary metabolite were synthesized and added after incubations of caffeine with human liver microsomes high in cytochrome P4501A2. HPLC separation of the metabolites prior to GC–MS quantification allowed the isolation of theobromine and paraxanthine which coeluted by GC and enabled quantification over a larger dynamic range. Quantitative analysis was performed on the n-propylated derivatives by selected-ion monitoring of either the M +⋅ ions for the dimethylxanthines or [M−C 3H 6] +⋅ ions for 1,3,7-trimethyluric acid. For the least abundant metabolite (1,3,7-trimethyluric acid), the detection level on column was 200 pg. Replicate analyses exhibited intra- and inter-day variability of 4.2 and 7.9%, respectively. This assay has been successfully used in the quantification of caffeine's primary metabolites in more than 180 incubations, at varying substrate concentrations and with multiple enzyme sources.

Comparison of Oxidative Base Damage in Mitochondrial and Nuclear DNA

The levels of endogenous pig liver cells mitochondrial DNA oxidative base damage have been investigated using isotope dilution gas chromatography mass spectrometry (GC/MS). Higher levels of five measured bases were found in mtDNA in relation to nuclear DNA. We have also detected large differences in the modified base ratios of mitochondrial versus nuclear DNA. These ratios for the bases with promutagenic properties as 8OHGua and 5OHCyt are much lower then for other bases (5OHHyd, 5OHMeHyd, 5OHMeUra).

Quantitative analysis quantitation of 2- n-nonyl-1,3-dioxolane by stable-isotope dilution gas chromatography-mass spectrometry

We report here a quantitative methodology developed for determination of SEPA (2- n-nonyl-1,3-dioxolane) in human serum. The method employed solid-phase extraction of SEPA and internal standard, [ 13C 2]SEPA, from serum followed by gas chromatography–mass spectrometry analysis using EI monitoring m/ z 73 and 75. We have investigated the utility of stable isotope dilution gas chromatography–mass spectrometry (GC–MS) for the determination of SEPA concentrations in serum using chemical ionization (positive ion, CI) or electron ionization (EI). The comparison of the specificity and sensitivity between EI and CI indicated that monitoring the m/ z 73 ion in EI was superior to monitoring either MH + or m/ z 73 using CI. The method was simple, sensitive and accurate, demonstrating a lower limit of quantitation (LLOQ) of 0.25 ng/ml and intra- and inter-assay accuracy and precision of ≤7.5%.

Phytanic acid α-oxidation in peroxisomal disorders: Studies in cultured human fibroblasts

We studied the α-oxidation of phytanic acid in human fibroblasts of controls and patients affected with classical Refsum disease, rhizomelic chondrodysplasia punctata, generalized peroxisomal disorders and peroxisomal bifunctional protein deficiency. Cultured fibroblasts were incubated with phytanic acid, after which medium and cells were collected separately. 2-Hydroxyphytanic acid and pristanic acid were measured in the medium and cells by stable isotope dilution gas chromatography mass spectrometry. In controls, 2-hydroxyphytanic acid and pristanic acid could be detected in the medium after incubation with phytanic acid, proving that α-oxidation of phytanic acid via 2-hydroxyphytanoyl-CoA to pristanic acid was active and intermediates were excreted into the medium. In cells from patients with a defective α-oxidation (Refsum disease, rhizomelic chondrodysplasia punctata and generalized peroxisomal disorders) 2-hydroxyphytanic acid and pristanic acid were low or not detectable, showing that in these disorders the hydroxylation of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA is deficient. In cells with a peroxisomal β-oxidation defect, 2-hydroxyphytanic acid and pristanic acid were formed in amounts comparable to those in the controls.

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.