Isotope Dilution Mass Spectrometric Assay Research Articles

Identification and determination of the urinary metabolite of iodotyrosine invivo

BackgroundCongenital hypothyroidism screening traditionally relies on detecting elevated thyroid-stimulating hormone levels, yet this approach may not detect a specific type of congenital hypothyroidism caused by iodotyrosine dehalogenase-1 (Dehal1) deficiency. The deficiency of this enzyme prevents the deiodination of mono-iodotyrosine (MIT) and di-iodotyrosine (DIT) in the process of iodine recycling. This underscores the potential use of iodotyrosine or its metabolites as non-invasive urinary biomarkers for early diagnosis of congenital hypothyroidism. However, the urinary metabolites of MIT/DIT have not yet been discovered. Thus, this study aimed to identify the urinary metabolites of iodotyrosine in experimental models. MethodGas chromatography mass spectrometry was used to identify the urinary metabolites of iodotyrosine following intraperitoneal injection of MIT in rats. An isotope dilution mass spectrometric assay was developed for assessment of identified metabolites. Urine samples from Dehal1 knockout mice were used to confirm the results. ResultsWe identified novel iodotyrosine metabolites, 3-iodo-4-hydroxyphenylacetic acid (IHPA), and 3,5-diiodo-4-hydroxyphenylacetic acid (Di-IHPA) as the primary urinary metabolites of MIT and DIT respectively. The concentrations of urinary IHPA and Di-IHPA were significantly higher in Dehal1 knockout mice. ConclusionOur findings suggest that IHPA is detected in larger quantities and may hold more clinical significance than previously identified biomarkers like MIT and DIT, making it a promising candidate for diagnosing congenital hypothyroidism or other conditions associated with iodine recycling inhibition.

Chronic Cyclic Bladder Over Distention Up-Regulates Hypoxia Dependent Pathways

Bladder over distention secondary to anatomical or functional obstruction can eventually lead to pathological changes, including decreased elasticity and contractile dysfunction. We hypothesized that chronic bladder distention in a murine model would activate hypoxia dependent signaling pathways despite intermittent relief of distention. Female C57Bl/6 mice were oophorectomized at age 5 to 6 weeks and underwent urethral catheterization and 90-minute bladder distention. Acute and chronic time points were evaluated. Bladder tissue was harvested for hematoxylin and eosin, and immunohistochemical staining with the hypoxia markers Glut-1 (EMD Millipore, Merck, Darmstadt, Germany) and Hypoxyprobe™-1. Bladder tissue was also harvested for real-time polymerase chain reaction and oxidative stress measurement. Hypoxia polymerase chain reaction arrays were done to determine changes in gene expression. Oxidative stress was measured using F2-IsoP. Functional bladder changes were evaluated using voided urine blots. After acute distention and 5 consecutive distentions, bladders showed marked inflammatory changes on hematoxylin and eosin staining, and evidence of tissue hypoxia on immunohistochemistry. Quantitative real-time polymerase chain reaction revealed up-regulation of hypoxia and oxidative stress related genes, including Hif1a, Arnt2, Ctgf, Gpx1 and Hmox1. Measurements of oxidative stress with F2-IsoP did not change. Voided urine blots before and after bladder distention showed marked changes with an overactive voiding pattern. Chronic bladder distention is possible in the female mouse. It generates hypoxic injury, as characterized functionally by increased voiding patterns. This bladder injury model might more closely replicate bladder dysfunction in patients with poor bladder emptying due to neurological disease, including those noncompliant with intermittent catheterization.

Oxidative Stress Measured by Urine F2-Isoprostane Level is Associated With Prostate Cancer

Oxidative stress is implicated in prostate cancer by several lines of evidence. We studied the relationship between the level of F2-isoprostanes, a validated biomarker of oxidative stress, and prostate cancer and high grade prostatic intraepithelial neoplasia. This case-control analysis within the Nashville Men's Health Study included men recruited at prostate biopsy. Body morphometrics, health history and urine were collected from more than 2,000 men before biopsy. F2-isoprostanes were measured by gas chromatography/mass spectrometry within an age matched sample of Nashville Men's Health Study participants that included 140 patients with high grade prostatic intraepithelial neoplasia, 160 biopsy negative controls and 200 prostate cancer cases. Multivariable linear and logistic regression was used to determine the associations between F2-isoprostane level, and high grade prostatic intraepithelial neoplasia and prostate cancer. Mean patient age was 66.9 years (SD 7.2) and 10.1% were nonwhite. Adjusted geometric mean F2-isoprostane levels were higher in patients with prostate cancer (1.82, 95% CI 1.66-2.00) or high grade prostatic intraepithelial neoplasia (1.82, 95% CI 1.68-1.96) than in controls (1.63, 95% CI 1.49-1.78, p <0.001), but were similar across Gleason scores (p = 0.511). The adjusted odds of high grade prostatic intraepithelial neoplasia and prostate cancer increased with increasing F2-isoprostane quartile (p-trend = 0.015 and 0.047, respectively) and the highest F2-isoprostane quartile was associated with significantly increased odds of prostate cancer (OR 2.44, 95% CI 1.17-5.09, p = 0.017). Pre-diagnosis urine F2-isoprostane level is increased in men with high grade prostatic intraepithelial neoplasia or prostate cancer, suggesting urinary F2-isoprostane provides a biomarker for the role for oxidative stress in prostate carcinogenesis. F2-isoprostanes may also serve to estimate the efficacy of interventions targeting oxidative stress mechanisms in prostate cancer prevention or treatment.

Quantification of F-ring isoprostane-like compounds (F 4-neuroprostanes) derived from docosahexaenoic acid in vivo in humans by a stable isotope dilution mass spectrometric assay

Lipid peroxidation has been implicated in the pathophysiological sequelae of human neurodegenerative disorders. It is recognized that quantification of lipid peroxidation is best assessed in vivo by measuring a series of prostaglandin (PG) F 2-like compounds termed F 2-isoprostanes (IsoPs) in tissues in which arachidonic acid is abundant. Unlike other organs, the major polyunsaturated fatty acid (PUFA) in the brain is docosahexaenoic acid (DHA, C22:6 ω-6), and this fatty acid is particularly enriched in neurons. We have previously reported that DHA undergoes oxidation in vitro and in vivo resulting in the formation of a series of F 2-IsoP-like compounds termed F 4-neuroprostanes (F 4-NPs). We recently chemically synthesized one F 4-NP, 17-F 4c-NP, converted it to an 18O-labeled derivative, and utilized it as an internal standard to develop an assay to quantify endogenous production of F 4-NPs by gas chromatography (GC)/negative ion chemical ionization (NICI) mass spectrometry (MS). The assay is highly precise and accurate. The lower limit of sensitivity is approximately 10 pg. Levels of F 4-NPs in brain tissue from rodents were 8.7±2.0 ng/g wet weight (mean±S.D.). Levels of the F 4-NPs in brains from normal humans were found to be 4.9±0.6 ng/g (mean±S.D.) and were 2.1-fold higher in affected regions of brains from humans with Alzheimer’s disease ( P=0.02). Thus, this assay provides a sensitive and accurate method to assess oxidation of DHA in animal and human tissues and will allow for the further elucidation of the role of oxidative injury to the central nervous system in association with human neurodegenerative disorders.

Urinary Metabolites of Leukotriene B4 in the Human Subject

Leukotriene B4 (LTB4) is a potent chemoattractant for neutrophils and is thought to play a role in a variety of inflammatory responses in humans. The metabolism of LTB4 in vitro is complex with several competing pathways of biotransformation, but metabolism in vivo, especially for normal human subjects, is poorly understood. As part of a Phase I Clinical Trial of human tolerance to LTB4, four human subjects were injected with 150 nmol/kg LTB4 with one additional subject as placebo control. The urine of the subjects was collected in two separate pools (0-6 and 7-24 h), and aliquots from these urine collections were analyzed using high performance liquid chromatography, UV spectroscopy, and negative ion electrospray ionization tandem mass spectrometry for metabolites of LTB4. In the current investigation, 11 different metabolites of LTB4 were identified in the urine from those subjects injected with LTB4, and none were present in the urine from the placebo-injected subject. The unconjugated LTB4 metabolites found in urine were structurally characterized as 18-carboxy-LTB4, 10,11-dihydro-18-carboxy-LTB4, 20-carboxy-LTB4, and 10,11-dihydro-20-carboxy-LTB4. Several glucuronide-conjugated metabolites of LTB4 were characterized including 17-, 18-, 19-, and 20-hydroxy-LTB4, 10-hydroxy-4,6,12-octadecatrienoic acid, LTB4, and 10,11-dihydro-LTB4. The amount of LTB4 glucuronide (16.7-29.4 pmol/ml) and 20-carboxy-LTB4 (18.9-30.6 pmol/ml) present in the urine of subjects injected with LTB4 was determined using an isotope dilution mass spectrometric assay before and after treatment of the urine samples with beta-glucuronidase. The urinary metabolites of LTB4 identified in this investigation were excreted in low amounts, yet it is possible that one or more of these metabolites could be used to assess LTB4 biosynthesis following activation of the 5-lipoxygenase pathway in vivo.

Improved assay for the quantification of the major urinary metabolite of the isoprostane 15-F 2t-Isoprostane (8-iso-PGF 2α) by a stable isotope dilution mass spectrometric assay

Background: The F 2-isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical catalyzed peroxidation of arachidonic acid. One IsoP, 15-F 2t-IsoP (8-iso-PGF 2α), has been shown to be formed in abundance in vivo and to exert potent biological activity. Methods: As a means to assess the endogenous production of this compound, we previously developed a method to quantify the major urinary metabolite of 15-F 2t-IsoP, 2,3-dinor-5,6-dihydro-15-F 2t-IsoP (2,3-dinor-5,6-dihydro-8-iso-PGF 2α, 15-F 2t-IsoP-M ), by gas chromotography (GC)/negative ion chemical ionization mass spectrometry (MS) employing stable isotope dilution methodology. While useful, we found that the assay occasionally suffered from the presence of impurities that co-elute on GC with 15-F 2t-IsoP-M, making the measurement of this compound difficult. We now report a modified assay for the quantification of 15-F 2t-IsoP-M employing GC/MS that alleviates this problem. Results: Precision of the assay is ±7% and the accuracy is 96%. The lower limit of sensitivity is approximately 8 pg. Normal concentrations of this metabolite in urine were found to be 0.46±0.09 ng/mg creatinine (mean±1 S.D.) Urinary excretion of 15-F 2t-IsoP-M is markedly altered in situations associated with increased or decreased oxidant stress in vivo. Conclusions: This assay provided a sensitive and accurate method to assess endogenous IsoP generation and can be used to further explore the role of oxidant injury in human disease.

Quantification of the Major Urinary Metabolite of 15-F2t-Isoprostane (8-iso-PGF2α) by a Stable Isotope Dilution Mass Spectrometric Assay

The isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical-catalyzed peroxidation of arachidonic acid. The first series of IsoPs characterized contained F-type prostane rings analogous to PGF2α. One F-ring IsoP, 15-F2t-IsoP (8-iso-PGF2α) has been shown to be formed in abundancein vivoand to exert potent biological activity. As a means to assess the endogenous production of this compound, we developed a method to quantify the major urinary metabolite of 15-F2t-IsoP, 2,3-dinor-5,6-dihydro-15-F2t-IsoP (2,3-dinor-5,6-dihydro-8-iso-PGF2α), by gas chromotography/negative ion chemical ionization mass spectrometry. This metabolite was chemically synthesized and converted to an18O2-labeled derivative for use as an internal standard. After purification, the compound was analyzed as a pentafluorobenzyl ester trimethylsilyl ether. Precision of the assay is ±4% and accuracy is 97%. The lower limit of sensitivity is approximately 20 pg. Levels of the urinary excretion of this metabolite in 10 normal adults were found to be 0.39 ± 0.18 ng/mg creatinine (mean ± 2 SD). Substantial elevations in the urinary excretion of the metabolite were found in situations in which IsoP generation is increased and antioxidants effectively suppressed metabolite excretion. Levels of 2,3-dinor-5,6-dihydro-15-F2t-IsoP were not affected by cyclooxygenase inhibitors. Thus, this assay provides a sensitive and accurate method to assess endogenous production of 15-F2t-IsoP as a means to explore the pathophysiological role of this compound in human disease.

Quantification of glutaric acid by isotope dilution mass spectrometry for patients with glutaric acidemia type I: selected ion monitoring vs. selected ion storage

An isotope dilution mass spectrometric assay for the quantification of glutaric acid in urine and serum samples was developed. The performance of a quadrupole mass filter (QMF) gas chromatography–mass spectrometry (GC–MS) instrument, operated in the selected ion monitoring mode, and a quadrupole ion trap (QIT) GC/MS instrument, operated in the selected ion storage mode, was compared. Both instruments gave linear standard curves with glutaric acid concentrations between 0.19 and 3.8 μM. The average coefficients of correlation were 0.9998 and 0.9993 for the QMF and the QIT system, respectively. There was good agreement between the glutaric acid concentrations measured with the two instruments. The run-to-run precision was between 1.2 and 3.7% and between 6.2 and 8.6%, the average recovery of glutaric acid in urine and serum samples was 96 and 103% with the QMF and QIT instrument, respectively. We conclude that although the QMF has a slightly better performance, both instruments can be used to reliably measure glutaric acid concentrations from urine and serum patient samples.

Nonenzymatic Free Radical-catalyzed Generation of Thromboxane-like Compounds (Isothromboxanes) in Vivo

The isoprostanes (IsoPs) are novel bioactive prostaglandin-like compounds produced in vivo by free radical-catalyzed peroxidation of arachidonyl-containing lipids. Previously, we have identified IsoPs containing F-type and D- and E-type prostane rings that are formed by reduction and rearrangement of IsoP endoperoxide intermediates, respectively. We now explore whether thromboxane B2 (TxB2)-like compounds, termed B2-isothromboxanes (B2-IsoTxs), are formed by rearrangement of IsoP endoperoxides. Detection of these compounds was carried out using a stable isotope dilution mass spectrometric assay originally developed for quantification of cyclooxygenase-derived TxB2. Incubations of arachidonic acid with Fe/ADP/ascorbate for 30 min in vitro generated a series of peaks representing putative B2-IsoTx at levels of 62.4 +/- 21.0 ng/mg arachidonate. Using various chemical modification and derivatization approaches, it was determined that these compounds contained hemiacetal ring structures and two double bonds, as would be expected for B2-IsoTx. Analysis of the compounds by electron ionization mass spectrometry yielded multiple mass spectra similar to those of TxB2. B2-IsoTxs are also formed esterified to phospholipids; oxidation of arachidonyl-containing phosphatidylcholine in vitro followed by hydrolysis resulted in the release of large amounts of these compounds. To explore whether B2-IsoTxs are also formed in vivo, a well characterized animal model of lipid peroxidation consisting of orogastric administration of CCl4 to rats was used. Levels of B2-IsoTx esterified in lipids in the liver increased 41-fold from 2.5 +/- 0.5 to 102 +/- 30 ng/g of liver. In addition, circulating levels of free compounds increased from undetectable (<5 pg/ml) to 185 +/- 30 pg/ml after CCl4, a 37-fold increase. Thus, we have provided evidence that IsoTxs constitute another novel class of eicosanoids produced in vivo nonenzymatically by free radical-catalyzed lipid peroxidation. These studies thus expand our understanding of products of lipid peroxidation formed in vivo from the free radical-catalyzed peroxidation of arachidonic acid.

Tiglylglycine excreted in urine in disorders of isoleucine metabolism and the respiratory chain measured by stable isotope dilution GC-MS

Tiglyglycine (TG), an intermediate product of the catabolism of isoleucine, is increased in the urine of patients with beta-ketothiolase deficiency or with disorders of propionate metabolism. It is also implicated as a useful diagnostic marker in disorders of the respiratory chain. We present a method for the synthesis of TG and tiglyl[13C, 15N]glycine and the development of a stable isotope dilution mass spectrometric assay for TG. We compare data from controls with that from subjects with beta-ketothiolase deficiency and propionyl-CoA carboxylase deficiency, and with six patients with enzyme-confirmed disorders of the respiratory chain. TG was increased in the urine from all of the patient groups. The increased TG excretion did not persist in one patient with a respiratory chain defect, which suggests that, in some patients, multiple sample analysis may be necessary to identify a respiratory chain defect. This is the first urinary compound to be implicated as a potential marker of disorders of the respiratory chain.

Plasma cis-dec-4-enoic acid measured by isotope dilution mass spectrometry; an improved assay to diagnose medium-chain acyl-CoA dehydrogenase deficiency.

An isotope dilution mass spectrometric assay for plasma cis-dec-4-enoic acid is described. It is quicker, more reliable and more accurate than previous methods. It confirmed previous findings that cis-dec-4-enoic acid is a reliable indicator for medium-chain acyl-CoA dehydrogenase deficiency (MCAD). The plasma cis-dec-4-enoic acid levels of both asymptomatic and symptomatic MCAD patients (3.5-71 mumol/L) are demonstrably higher than those of normal children (0.2-1.7 mumol/L), MCAD heterozygotes (0.1-1.5 mumol/L), those with other fatty acid oxidation defects (0.2-2.2 mumol/L) or those receiving high doses of valproic acid (0.2-0.4 mumol/L).

Automated Analysis for Free and Short-Chain Acylcarnitine in Plasma with a Centrifugal Analyzer

We describe a fully automated, spectrophotometric assay of free and total carnitine in plasma ultrafiltrates. The method, suitable for routine application in most hospital laboratories, incorporates the hydrolysis of acylcarnitines to free carnitine within the program of a Cobas Fara II centrifugal analyzer. The hydrolysis is monitored and calibrated with standard solutions containing octanoylcarnitine. Results correlated well with those from a reference isotope-dilution mass spectrometric assay. The ability to analyze a batch of samples for both free and total carnitine within 90 min enables analysis of > or = 100 samples per day. Used in conjunction with acylcarnitine species identification by mass spectrometry, the Cobas assay facilitates the diagnosis of carnitine-deficiency syndromes and specific metabolic disorders.

Quantification of the major urinary metabolite of prostaglandin D 2 by a stable isotope dilution mass spectrometric assay

Prostaglandin D 2 (PGD 2) has been found to be an important pathophysiological mediator in a number of human disorders. Thus a means to assess the endogenous production of PGD 2 is of considerable clinical value. To accomplish this goal, we developed a method for the quantification of the major urinary metabolite of PGD 2, 9α, 11β-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid, by gas chromatography/negative ion chemical ionization mass spectrometry. This metabolite was chemically synthesized and converted to an 18O 4-labeled derivative for use as an internal standard. Novel derivatization and purification procedures were incorporated in the assay taking advantage of the ability of the lower side chain of this molecule to undergo cyclization at acidic pH to form a hemiketal, γ-lactone, and uncyclization with methoximation. Precision of the assay is ±7% and accuracy is 96%. The lower limit of sensitivity is approximately 50 pg. Normal levels for the urinary excretion of this metabolite in 18 normal adults was found to be 1.08 ± 0.72 ng/mg creatinine (mean ± 2SD). Substantial elevations in the urinary excretion of the metabolite were found in clinical situations in which prostaglandin D 2 has been shown to be released in increased quantities. Thus, this assay provides a sensitive and accurate method to assess endogenous production of prostaglandin D 2 as a means to explore the pathophysiological role of prostaglandin D 2 in human disease.

Comparison of gas-liquid chromatography, mass fragmentography, and isotope dilution-mass spectrometry for determination of pentobarbitone in human plasma

Comparative studies of gas-liquid Chromatographic, mass fragmentographic methods, and isotope dilution-mass spectrometric assay for the quantitative determination of pentobarbitone in human plasma, are described. Butabarbitone is used as internal standard for the measurement of the drugs by gas-liquid chromatography and mass fragmentography, and deuterated pentobarbitone is used for quantification by the isotope dilution-mass spectrometric method.

Arachidonic acid metabolism in isolated pancreatic islets: IV. Negative ion-mass spectrometric quantitation of monooxygenase product synthesis by liver and islets

Deuterium-labelled standards of four regionally isomeric epoxyeicosatrienoic acids (EETs) and their hydrolysis products, the dihydroxyeicosatrienoic acids (DHETs), have been prepared and analyzed by capillary column gas chromatography (GC)-negative ion (NI)-methane chemical ionization (MCI)-mass spectrometry (MS) as the pentafluorobenzyl esters. As little as 40 pg of these compounds were readily visualized by these methods, and the deuterium-labelled standards were used in a stable isotope dilution mass spectrometric assay which was linear from near the detection limit over several orders of magnitude. NADPH-dependent synthesis of both EETs and DHETs from arachidonate by hepatic microsomal cytochrome P-450-monooxygenase activity was demonstrable with these methods and was significantly suppressed by the compound BW755C (500 μM), but not by eicosa-5,8,11,14-tetraynoic acid (ETYA, 20 μM) or by nordihydroguaiaretic acid (NDGA, 50 μM). All three compounds suppress glucose-induced insulin secretion and 12-hydroxyeicosatetraenoic acid (12-HETE) synthesis by isolated pancreatic islets with similar concentration dependence. Microsomes derived from isolated pancreatic islets synthesized less than 3% of the EET and DHET compounds as a comparable amount of hepatic microsomes. Intact islets synthesized less than 3% by mass of the EET and DHET compounds compared to the mass of 12-HETE produced by the islets. Islets also failed to convert 3H-labelled arachidonate to 3H-labelled EETs or DHETs under conditions where conversion to [ 3H]12-HETE and to [ 3H]prostaglandin E 2 (but not to [ 3H]leukotriene C 4, D 4, or E 4) was clearly demonstrable. Neither exogenous EETs nor leukotriene C 4 stimulated insulin secretion from the isolated islets or reversed the suppression of glucose-induced secretion by the lipoxygenase inhibitor BW755C. The cytochrome P-450-monooxygenase inhibitor, metyrapone (50 μM), did not influence insulin secretion from the isolated islets under conditions where the lipoxygenase inhibitor, NDGA, suppressed glucose-induced secretion. These observations argue against the recently suggested hypothesis that EETs derived from arachidonate by monooxygenase action participate in glucose-induced insulin secretion by isolated pancreatic islets.

66] Quantification of the PGD2 urinary metabolite 9α-hydroxy-11,15-dioxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid by stable isotope dilution mass spectrometric assay

This chapter describes the methods developed for isolation and preparation of a deuterium- and tritium-labeled internal standard and specific assay procedures for quantification of the prostaglandin (PG) D 2 urinary metabolite 9α-hydroxy-11,15-dioxo-2,3,18,19-tetranorprost-5-ene- 1,20- dioic acid (PGD-M). The method chosen for quantification of a PGD 2 urinary metabolite was a stable isotope dilution mass spectrometric assay because of the high degree of specificity, accuracy, and sensitivity associated with this assay methodology. Required for a stable isotope dilution mass spectrometric assay is an internal standard of the compound being assayed that has been labeled with both stable and radioactive isotopes. Infusion of labeled PGD 2 into an experimental animal could provide a means to generate biologically the labeled PGD 2 urinary metabolites. Unlabeled PGD-M is isolated and purified from 5 liters of urine collected from the patients with mastocytosis. In the absence of a radiolabeled metabolite, column fractions containing PGD-M are identified during chromatographic purification procedures by analyzing aliquots of collected fractions with mass spectrometric-selected ion monitoring of major ions in the mass spectrum of PGD-M.