Isotope Dilution Gas Chromatography-mass Spectrometry Research Articles

Hormonal diagnosis of 21-hydroxylase deficiency in plasma and urine of neonates using benchtop gas chromatography-mass spectrometry.

We aimed at measuring the first plasma concentrations of 17-hydroxyprogesterone (17OH-P) determined by benchtop isotope dilution/gas chromatography-mass spectrometry (ID/GC-MS) in term neonates with or without 21-hydroxylase deficiency. Plasma samples from normal cord blood specimens (n=30), unaffected neonates (n=38) and neonatal patients with classical 21-hydroxylase deficiency (eight salt-wasters, three simple virilizers) were analyzed. Steroid profiling of random urinary specimens by GC-MS served as a confirmatory test for 21-hydroxylase deficiency. 17OH-P (nmol/l) in cord blood plasma lay between 11.66 and 75.92 (median 24.74). It declined shortly after birth. In the first 8 days of life, the time that screening for 21-hydroxylase deficiency is performed, 17OH-P ranged between undetected levels and an upper limit of 22.87 (median 4.11). Thereafter (days 9-28) its concentrations lay between 2.18 and 20.30 (median 6.22). Except one simple virilizer, all other patients with 21-hydroxylase deficiency had clearly elevated plasma 17OH-P at the time that screening for 21-hydroxylase deficiency would be performed. We suggest ID/GC-MS, which provides the highest specificity in steroid analysis, for checking suspicious concentrations of 17OH-P in neonates and underscore the potential of urinary steroid profiling by GC-MS as a rapid, non-invasive and non-selective confirmatory test for congenital adrenal hyperplasia.

Time-resolved fluoroimmunoassay of plasma daidzein and genistein

We present a method for the determination of the phytoestrogens daidzein and genistein in plasma (serum). These weakly estrogenic isoflavones occur in soybeans and in smaller amounts in some other beans and plants. It has been suggested that they may afford protection against prostate and breast cancer. The method is based on time-resolved fluoroimmunoassay (TR-FIA) using a europium chelate as a label. After synthesis of 4′- O-carboxymethyl-daidzein and 4′- O-carboxymethyl-genistein the compounds are coupled to bovine serum albumin (BSA), then used as antigens to immunize rabbits. The tracers with the europium chelate are synthesized using the same 4′- O-derivative of the isoflavones. After enzymatic hydrolysis and ether extraction the immunoassay is carried out using the VICTOR 1420 multilabel counter (Wallac Oy, Turku, Finland). The antisera cross-reacted to some extent with some isoflavonoids but not with flavonoids. The cross-reactivity seems not to influence the results, which were highly specific for both compounds. The correlation coefficients between the TR-FIA methods and the reference method based on isotope dilution gas chromatography-mass spectrometry were high; r-values were about 0.95–0.99 depending on concentration. The intra-assay coefficients of variation (CV%) for daidzein and genistein at three different concentrations vary 3.2–4.5 and 3.2–4.1, respectively. The inter-assay CVs vary 5.0–6.3 and 4.5–5.3, respectively. The working ranges of the daidzein and genistein assays are 1.0–216 and 1.7–370 nmol/l, respectively. The plasma values ( n = 80) of daidzein and genistein are very low in Finnish subjects (mean for daidzein, 3.8 ± 6.8 and for genistein, 3.2 ± 7.6 nmol/l; median value for daidzein 1.5 and for genistein 1.4 nmol/l).

Improved Stable Isotope Dilution-Gas Chromatography-Mass Spectrometry Method for Serum or Plasma Free 3-Hydroxy-Fatty Acids and Its Utility for the Study of Disorders of Mitochondrial Fatty Acid β-Oxidation

Disorders of fatty acid oxidation (FAO) are difficult to diagnose, primarily because in many of the FAO disorders measurable biochemical intermediates accumulate in body fluids only during acute illness. Increased concentrations of 3-hydroxy-fatty acids (3-OH-FAs) in the blood are indicative of FAO disorders of the long- and short-chain 3-hydroxy-acyl-CoA dehydrogenases, LCHAD and SCHAD. We describe a serum/plasma assay for the measurement of 3-OH-FAs with carbon chain lengths from C(6) to C(16). We used stable isotope dilution gas chromatography-mass spectrometry (GC-MS) with electron impact ionization and selected ion monitoring. Natural and isotope-labeled compounds were synthesized for the assay. The assay was linear from 0.2 to 50 micromol/L for all six 3-OH-FAs. CVs were 5-15% at concentrations near the upper limits seen in healthy subjects. In 43 subjects, the medians (and ranges) in micromol/L were as follows: 3-OH-C(6), 0.8 (0.3-2.2); 3-OH-C(8), 0.4 (0.2-1.0); 3-OH-C(10), 0.3 (0.2-0.6); 3-OH-C(12), 0.3 (0.2-0.6); 3-OH-C(14), 0.2 (0.0-0.4); and 3-OH-C(16), 0.2 (0.0-0.5). 3-OH-FAs were increased in infants receiving formula containing medium chain triglycerides. Two patients diagnosed with LCHAD deficiency showed marked increases in 3-OH-C(14) and 3-OH-C(16) concentrations. Two patients diagnosed with SCHAD deficiency showed increased shorter chain 3-OH-FAs but no increases in 3-OH-C(14) to 3-OH-C(16). Measuring blood concentrations of the 3-OH-FAs with this assay may be a valuable tool for helping to rapidly identify deficiencies in LCHAD and SCHAD and may also provide useful information about the status of the FAO pathway.

Isotope dilution-gas chromatography/mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry for the determination of triiodo-L-thyronine in serum.

Isotope dilution-gas chromatography/mass spectrometry (ID-GC/MS) and isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC/MS/MS) methods have been developed for an determination of triiodothyronine (T3) in serum and their potential as candidate reference methods investigated. In both methods, (13)C(9)-T3 was used as internal standard. Sample pretreatment consisted of deproteinization, extraction and high performance liquid chromatography (HPLC) purification. Conversion of serum thyroxine (T4) to T3 was controlled by adding (13)C(6)-T4. For GC/MS, T3 and (13)C(9)-T3 were converted to the N,O-di-heptafluorobutyryl (HFB) methyl ester derivatives and monitored at m/z 844 and 853. For LC/MS with electrospray ionization, the transitions m/z 652/661 to 606/614 were monitored. For use of the methods as candidate reference methods, special attention was paid to the calibration and the measurement protocol (duplicate analysis of each sample on three occasions). Evaluation of the ID-GC/MS and ID-LC/MS/MS methods showed the absence of interference by reverse T3 and T4, a limit of detection of 100 pg (GC/MS) and 18 pg (LC/MS), a recovery of 100 +/- 1.5% (95% confidence interval) and good precision (the total coefficient of variation, n = 6, was typically 1.5%). In addition to the recovery study, the accuracy of the methods was proven by method comparison (ID-GC/MS vs. LC/MS/MS) on three sera, showing a maximum deviation of 1.1%. Finally, the ID-GC/MS method was applied for measurement of 10 different human sera with a T3 concentration range from 0.6 to 7.3 ng/mL.

Excision of oxidatively damaged DNA bases by the human alpha-hOgg1 protein and the polymorphic alpha-hOgg1(Ser326Cys) protein which is frequently found in human populations.

We have investigated the substrate specificity of the major nuclear form of the human Ogg1 protein, referred as alpha-hOgg1, for excision of damaged bases from DNA exposed to gamma-irradiation. Excision products were identified and quantified using gas chromatography/isotope dilution mass spectrometry (GC/IDMS). The GST-alpha-hOgg1 protein used in this study is a fusion of alpha-hOgg1 to the C-terminus of the GST protein. The results show that GST-alpha-hOgg1 protein excises 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA exposed to gamma-irradiation in a solution saturated with N(2)O or air. Fourteen other lesions, including oxidised purines and pyrimidines, were not excised from these substrates. Catalytic constants were measured for the excision of 8-OH-Gua and FapyGua from DNA gamma-irradiated under N(2)O. The k (cat)/ K (m)values for excision of 8-OH-Gua and FapyGua were 4.47 x 10(-5)and 8.97 x 10(-5)(min(-1)nM(-1)), respectively. The substrate specificity and the catalytic parameters of the wild-type GST-alpha-hOgg1 protein were compared to that of a polymorphic form of alpha-hOgg1 harbouring a Ser-->Cys mutation at codon 326. In the Japanese population, 47.6% of individuals possess both alleles coding for the wild-type alpha-hOgg1-Ser(326)and mutant alpha-hOgg1-Cys(326)proteins. The GST-alpha-hOgg1-Cys(326)protein was purified and its substrate specificity was determined by GC/IDMS analysis. The results show that the GST-alpha-hOgg1-Cys(326)protein efficiently excises 8-OH-Gua and FapyGua from gamma-irradiated DNA. The k (cat)/ K (m)values for excision of 8-OH-Gua and FapyGua were 2. 82 x 10(-5)and 4.43 x 10(-5)(min(-1)nM(-1)), respectively. Furthermore, we compared the capacity of these two forms of alpha-hOgg1 to act on substrates containing 2,6-diamino-4-hydroxy-5- N -methylformamidopyrimidine (Me-FapyGua). The k (cat)/ K (m)values for excision of Me-FapyGua were 278 x 10(-5)and 319 x 10(-5)(min(-1)nM(-1)), respectively. Cleavage of 34mer oligodeoxyribonucleotides containing 8-OH-Gua, 8-hydroxyadenine or an apurinic/apyrimidinic site paired with a cytosine was also investigated. The results show that both GST-alpha-hOgg1-Ser(326)and GST-alpha-hOgg1-Cys(326)catalyse the various cleavage reactions at very similar rates. Furthermore, both proteins efficiently complement the mutator phenotype of the fpg mutY mutant of Escherichia coli.

Phytanic acid α-oxidation: identification of 2-hydroxyphytanoyl-CoA lyase in rat liver and its localisation in peroxisomes

Phytanic acid is broken down by α-oxidation in three steps finally yielding pristanic acid. The first step occurs in peroxisomes and is catalysed by phytanoyl-CoA hydroxylase. We have studied the second step in the α-oxidation pathway, which involves conversion of 2-hydroxyphytanoyl-CoA to pristanal catalysed by 2-hydroxyphytanoyl-CoA lyase. To this end, we have developed a stable isotope dilution gas chromatography-mass spectrometry assay allowing activity measurements in rat liver homogenates. Cell fractionation studies demonstrate that in rat liver 2-hydroxyphytanoyl-CoA lyase is localised in peroxisomes. This finding may have important implications for inherited diseases in man characterised by impaired phytanic acid α-oxidation.

Brain Regional Distribution of Endocannabinoids: Implications for Their Biosynthesis and Biological Function

The amounts, in nine different rat brain regions, of the two endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoylglycerol (2-AG), and of the putative AEA precursorN-arachidonoyl-phosphatidylethanolamine (NArPE), were determined by isotope-dilution gas chromatography-mass spectrometry and compared to the number of cannabinoid binding sites in each region. The distribution of NArPE, reported here for the first time, exhibited a good correlation with that of AEA, the former metabolite being 3–13 times more abundant than the endocannabinoid in all regions. The highest amounts of both metabolites (up to 358.5 and 87 pmol/g wet weight tissue, respectively) were found in the brainstem and striatum, and the lowest in the diencephalon, cortex, and cerebellum. These data support the hypothesis that, in the brain, AEA is a metabolic product of NArPE and may reach levels compatible with its proposed neuromodulatory function. The brain distribution of 2-AG, also described in this study for the first time, was found to correlate with that of AEA with levels ranging from 2.0 to 14.0 nmol/g (in the diencephalon and brainstem, respectively). The distribution of the endocannabinoids did not match exactly with that of cannabinoid binding sites, suggesting either that these compounds are not necessarily produced near their molecular targets, or that they play functional roles additional to the activation of cannabinoid receptors. Regional differences in the ligand/receptor ratios may also lead to predict corresponding differences in the efficiency of receptor activation, as shown by previous studies.

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.

Oxidized amino acids in the urine of aging rats: potential markers for assessing oxidative stress in vivo.

Oxidative damage of proteins has been implicated in disease and aging. In vitro studies demonstrate that two unnatural amino acids, o,o'-dityrosine and o-tyrosine, are stable markers of protein oxidation. We have investigated the possibility that assaying these compounds in urine could provide a noninvasive way to determine levels of protein oxidation in vivo. Isotope dilution gas chromatography-mass spectrometry was used to quantify levels of o,o'-dityrosine and o-tyrosine in skeletal muscle and urine of aging rats subjected to two interventions: 1) dietary antioxidant supplementation and 2) exercise training. In both sedentary rats and exercise-trained rats, antioxidant therapy reduced levels of protein-bound o,o'-dityrosine in skeletal muscle. In contrast, antioxidant therapy or exercise training minimally affected o-tyrosine levels in this tissue. Levels of the oxidized amino acids in urine samples mirrored those of skeletal muscle proteins. Quantification of the levels of oxidized amino acids in urine may thus serve as a noninvasive measure of oxidative stress in vivo because they change in parallel with levels of protein-bound oxidized amino acids in skeletal muscle.

Determination of benzene in aqueous samples by membrane inlet, solid phase microextraction and purge and trap extraction with isotope dilution gas chromatography-mass spectrometry

The determination of benzene in aqueous samples is reported using membrane inlet, solid phase microextraction and purge and trap extraction techniques combined with gas chromatography-mass spectrometry. The membrane inlet and solid phase microextraction techniques have been applied to the analysis of soft drink samples and the performance characteristics of these methods compared to the established purge and trap method. Isotope dilution quantitative procedures with hexadeuterobenzene as internal standard were used in combination with all three sampling inlets. Detection limits were at, or below the parts per billion (µg l–1) level with analytical precision (%RSD) in the range 3–8%.

Influence of soybean processing, habitual diet, and soy dose on urinary isoflavonoid excretion

In an attempt to explain the wide individual variation seen in urinary isoflavonoid phytoestrogen excretion, we conducted a series of 3 human feeding studies: a large crosssectional study of equol production in humans with a soy challenge, a comparison of phytoestrogen metabolism when subjects consumed fermented and unfermented soy products, and a dose-response study of urinary isoflavonoid excretion at the low end of soy consumption. All studies were conducted in young, healthy humans. Urinary isoflavonoids were measured by isotope-dilution gas chromatography–mass spectrometry. Similar to results from other studies, 35% of screened subjects (30 men and 30 women) excreted equol (> 2000 nmol/d). In women, equol excretion was associated with higher intake of dietary fiber and carbohydrate. Fermentation of soy decreased the isoflavone content of the product fed but increased the urinary isoflavonoid recovery, suggesting that fermentation increases availability of isoflavones in soy. When soy-protein powder was fed at 0, 5, 10, and 20 g/d (0–36 mg isoflavones), there was a linear dose response of urinary isoflavonoid excretion to soy consumption that did not differ between subjects with high and low equol excretion. These results suggest that equol excretion may be related to the fermentable carbohydrate content of the diet; additional study is needed. Processing of soy affects isoflavone metabolism and must be considered in recommending exposure to isoflavones from soyfoods. Although optimal isoflavone exposure for disease protection has not been determined, urinary isoflavonoid excretion appears linear at low-to-moderate soy consumption.

Gas Chromatograph/Isotope Dilution Mass Spectrometric Analysis of Airborne Benzo[a]pyrene Using Deuterium and 13C-Labeled Benzo[a]pyrene: A Critical Evaluation

ABSTRACT A critical evaluation has been made of the gas chroma-tography/isotope dilution mass spectrometry (GC/IDMS) analysis of airborne benzo[a]pyrene (B[a]p) using 2H- (deuterium, D) and 13C-labeled benzo[a]pyrene and a low resolution mass spectrometer. Laboratory quality control and field samples were used to determine the suitability of these two isotopically labeled B[a]p as surrogates for GC/ IDMS B[a]p analysis. With method spike QC samples, recovery of the B[a]p was compared with the recoveries of D- and 13C-labeled B[a]p to validate the assumption that these two isotopically labeled B[a]p analogs could be used as method surrogates for IDMS B[a]p analyses. Two collocated Anderson Hi-Vol samplers were used to carry out a field study in which total B[a]p loadings from one sampler were correlated to the second sampler, before and after correcting the total B[a]p loadings against the recovery of the D- and 13C-labeled B[a]p. It is shown that a low-resolution mass spectrometer can be used in GC/ IDMS B[a]p analysis to achieve results of high precision and accuracy. Depending on data quality objectives, D-labeled B[a]p could be used for IDMS B[a]p analysis, but is not recommended.

EXERCISE INDUCES OXIDATIVE DAMAGE OF MITOCHONDRIAL PROTEINS

1835 The goal of this project was to use exercise as a physiological source of oxidative stress. We determined the levels of oxidized amino acids in the mitochondria, cytosol, and urine using stable isotope dilution gas chromatography-mass spectrometry. FigureFigureIn vitro studies show that the oxidized amino acid o,o'-dityrosine is a stable marker for protein oxidation by tyrosyl radicals and o-tyrosine and m-tyrosine are stable markers of protein oxidation by hydroxyl radical. Rats were subjected to a single bout of sustained exercise. Control and exercised animals were sacrificed before and after exercise, mitochondria were isolated from cardiac muscle of the animals, and the levels of oxidation products in mitochondrial and cytosolic protein as well as in urine were determined. Exercise led to a 2-fold increase in oxidation as monitored by the levels of o-tyrosine, m-tyrosine and o,o'-dityrosine in mitochondrial proteins, but not in cytosolic protein. Moreover, there was a similar increase in the level of o,o'-dityrosine in the urine of the exercised animals. These results indicate that exercise is a physiologically relevant oxidative strees, and raise the exciting possibility that this model will be useful to non-invasively study the effects of antioxidant interventions in vivo.

Isoflavonoids and Lignans in Legumes: Nutritional and Health Aspects in Humans

Dietary factors are considered important environmental risk determinants for Western diseases. Studies have revealed beneficial or protective effects of the consumption of legumes with regard to hypercholesterolaemia and coronary heart disease, obesity, diabetes mellitus, and menopause. During the last decade attention has been focused on soy and soybean products. Several constituents have been isolated: isoflavones, phytosterols, protease inhibitors, inositol hexaphosphate, and saponins. Our interest concentrates on hormone-like bisphenolic phytoestrogens of dietary origin, the lignans and isoflavonoids. Their glycosides, converted by gut bacteria to mammalian derivatives with weak estrogenic and antioxidative activity, originate in leguminous seeds. We developed an isotope dilution gas chromatography-mass spectrometry method for quantitative determination of the isoflavones, formononetin, biochanin A, daidzein, genistein and coumestrol, and the lignans secoisolariciresinol and matairesinol, in food samples. We measured the four isoflavonoids and coumestrol, and, for the first time, the two lignans in 52 leguminous seeds and found high concentrations of isoflavonoids (0–1853.35 mg/kg; 0–7.3 mmol/kg dw) but lower amount of lignans (0–15.85 mg/kg; 0.05 mmol/kg dw). The highest plasma levels of their metabolites are found in individuals living in countries or regions with low cancer and cardiovascular disease incidence and these are probably sufficient to influence intracellular enzymes, protein synthesis, growth factor action, malignant cell proliferation, differentiation, and angiogenesis. Leguminous seeds, therefore, in respect to their abundant concentrations of phytoestrogens, are strong candidates for a role as natural cancer-protective food.

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.

Markers of protein oxidation by hydroxyl radical and reactive nitrogen species in tissues of aging rats.

Many lines of evidence implicate oxidative damage in aging. Possible pathways include reactions that modify aromatic amino acid residues on proteins. o-Tyrosine is a stable marker for oxidation of protein-bound phenylalanine by hydroxyl radical, whereas 3-nitrotyrosine is a marker for oxidation of protein-bound tyrosine by reactive nitrogen species. To test the hypothesis that proteins damaged by hydroxyl radical and reactive nitrogen accumulate with aging, we used isotope dilution gas chromatography-mass spectrometry to measure levels of o-tyrosine and 3-nitrotyrosine in heart, skeletal muscle, and liver from young adult (9 mo) and old (24 mo) female Long-Evans/Wistar hybrid rats. We also measured these markers in young adult and old rats that received antioxidant supplements (alpha-tocopherol, beta-carotene, butylated hydroxytoluene, and ascorbic acid) from the age of 5 mo. We found that aging did not significantly increase levels of protein-bound o-tyrosine or 3-nitrotyrosine in any of the tissues. Antioxidant supplementation had no effect on the levels of protein-bound o-tyrosine and 3-nitrotyrosine in either young or old animals. These observations indicate that the o-tyrosine and 3-nitrotyrosine do not increase significantly in heart, skeletal muscle, and liver in old rats, suggesting that proteins damaged by hydroxyl radical and reactive nitrogen species do not accumulate in these tissues with advancing age.

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%.

Assessment of the state-of-the-art trueness and precision of serum total-calcium and glucose measurements in Finnish laboratories - the QSL-Finland study

The purpose of the QSL-Finland study was to assess the state-of-the-art trueness and precision of serum total-calcium and glucose measurements in Finnish clinical laboratories. For this purpose, 21 hospitals and clinical institutes were selected. They measured six single donation sera, the total-calcium (t-calcium) and glucose content of which had been determined by ion chromatography and isotope dilution-gas chromatography-mass spectrometry (ID-GC-MS) reference methods. The results were interpreted in light of specifications for imprecision, bias and total error of routine methods that have been proposed in the past. The data revealed that the performance of t-calcium and glucose methods is generally acceptable in Finnish clinical laboratories. This study did not lead to a separation of laboratories according to accreditation. In consequence, it seems that accreditation, in its present form, cannot substitute dedicated quality assurance practices.

Substrate specificity of Schizosaccharomyces pombe Nth protein for products of oxidative DNA damage.

A gene from Schizosaccharomyces pombe, which encodes a protein with a strong sequence similarity to the Nth protein of Escherichia coli, has recently been identified [Roldán-Arjona, T., Anselmino, C., and Lindahl, T. (1996) Nucleic Acids Res. 24, 3307-3312]. The functional analysis of this eukaryotic enzyme indicated that it is a homologue of E. coli Nth protein. The gene has been subcloned and the protein (Nth-Spo) purified to apparent homogeneity. We investigated the substrate specificity of this eukaryotic enzyme for modified bases in oxidatively damaged DNA, using the technique of gas chromatography/isotope-dilution mass spectrometry (GC/IDMS). DNA substrates containing up to 17 types of modified bases were prepared by gamma-irradiation or by treatment with H2O2 in the presence of Fe(III)-EDTA or Cu(II). The results revealed an efficient excision of five pyrimidine-derived lesions, 5-hydroxycytosine, thymine glycol, 5-hydroxy-6-hydrothymine, 5,6-dihydroxycytosine, and 5-hydroxyuracil. None of the other pyrimidine or purine lesions was excised. Excision was measured as a function of enzyme concentration, time, substrate concentration, and temperature. Kinetic constants were determined. Although some DNA base lesions removed by Nth-Spo protein were similar to those previously described for E. coli Nth protein, differences between substrate specificities of these two enzymes were noted.

Oxidative DNA base damage and antioxidant enzyme levels in childhood acute lymphoblastic leukemia

We have investigated the levels of several antioxidant enzymes and the level of oxidative DNA base damage in lymphocytes of children with acute lymphoblastic leukemia (ALL) and in disease-free children. Children with ALL had just been diagnosed with the disease and had received no therapy prior to obtaining blood samples. A multitude of typical hydroxyl radical-induced base lesions in lymphocyte DNA of children were identified and quantified by gas chromatography-isotope dilution mass spectrometry. Higher levels of DNA base lesions were observed in patients with ALL than in children without the disease. The levels of the antioxidant enzymes glutathione peroxidase, catalase and superoxide dismutase in lymphocytes of ALL patients were lower than in lymphocytes of controls. These findings are in agreement with earlier observations in various types of adulthood cancer. Some of the identified DNA base lesions are known to possess premutagenic properties and may play a role in carcinogenesis. The results may indicate a possible link between decreased activities of antioxidant enzymes and increased levels of DNA base lesions due to oxidative damage, and support the notion that free radical reactions may be increased in malignant cells.