Urinary biomarkers of nucleic acid oxidation and methylation in workers exposed to low concentrations of benzene.

The study aims to investigate the influence of exposure to low concentrations of benzene on urinary biomarkers of nucleic acid oxidative damage and methylation. Benzene exposure was characterized for 93 coke production workers by measuring both airborne benzene and S-phenylmercapturic acid (SPMA) and unmodified benzene (U-B) in urine samples, collected at the end of the shift (ES) and at the next morning before shift (next BS). In the same urinary samples, biomarkers of nucleic acid oxidative damage and methylation were determined. Urinary concentrations of cotinine and creatinine were also determined to evaluate the smoking effect and to normalize urinary concentrations of analytes, respectively. The biomarkers of benzene internal dose, of oxidative damage (8-hydroxyy-7,8-dihydroguanine, 8-hydroxy-7,8-dihydroguanosine and 8-hydroxy-7,8-2'deoxyguanosine) and some of the biomarkers of nucleic acid methylation (5-Methyl-Cytosine, 1-Methyl-Guanine and 7-Methyl-Guanine) were higher in the ES than the next BS samples. Positive associations between ES 5-Methyl-Cytosine and both SPMA and U-B were found. In conclusion, occupational exposure to low levels of benzene seems to be related to urinary ES 5-Methyl-Cytosine that could be a possible biomarker to evaluate the changes of the nucleic acid methylation status.

Environmental cadmium exposure induces alterations in the urinary metabolic profile of pregnant women.

Cadmium (Cd) is a well-recognized, hazardous toxic heavy metal, and the adverse effects of high-level Cd exposure on human health have been well documented. However, little is known about the health effects of low-level environmental Cd exposure on pregnant women. The objective of this study was to assess urinary metabolic alterations in pregnant women exposed to environmental Cd, and to identify informative biomarkers. Urine samples from 246 pregnant women in the first trimester of pregnancy were collected, and urinary Cd concentrations were quantified using inductively coupled plasma mass spectrometry (ICP-MS). Urinary metabolomics was analyzed by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Cd-related metabolic biomarkers were examined by comparing the samples of the first and third tertiles of Cd exposure classifications, using a partial least-squares discriminant (PLS-DA) model. Five putative biomarkers were identified, including L-cystine, L-tyrosine, dityrosine, histamine, and uric acid, all of which were related to oxidative stress and nephrotoxic effects induced by Cd exposure. The results show that low-level environmental Cd exposure could induce metabolite profile alterations in pregnant women, which might be associated with adverse health effects. Our findings provide new insights into the early molecular events following Cd exposure, and may be valuable for the health risk assessment of Cd exposure during pregnancy.

Potential biomarkers for monitoring the toxicity of long-term exposure to atrazine in rat by metabonomic analysis.

1. Herbicide atrazine (ATR) poses harmful effects on human health. The purpose of this study is to study potential biomarkers used for monitoring the toxic effects after chronic exposure to ATR by studying urine metabolites. 2. Rats were assigned into clinical chemistry and metabonomics arms, and each arm was divided into low-dose, high-dose and control groups. ATR was administered to rats along with their feed. At the end of 16, 20 and 24 weeks, clinical parameters and histopathologic changes was assessed to monitor the toxic effects. Twenty-four hour urine samples was analyzed by UPLC-MS, to find the significant alterations in metabolic profiling. 3. The body weight of rats in ATR group was lower than that of control starting from 12th week; abnormal levels of serum biochemistry and histopathologic alterations of organs were found initially from 16th and 20th week, respectively. Five exogenous and five endogenous metabolites were found which showed significant differences between ATR groups and control group at above-mentioned time points. 4. These metabolites may be used as potential indicators to monitor ATR toxicity, and also may provide some clues for understanding the mechanism of toxicity of ATR. The exact relationship between endogenous metabolites and ATR toxicity needs further investigation.

Detection of human urinary 5-hydroxymethylcytosine by stable isotope dilution HPLC-MS/MS analysis.

The sixth DNA base 5-hydroxymethylcytosine (5hmC) is the major oxidation product of the epigenetic modification 5-methylcytosine (5mC), mediating DNA demethylation in mammals. Reduced 5hmC levels are found to be linked with various tumors and neurological diseases; therefore, 5hmC is an emerging biomarker for disease diagnosis, treatment, and prognosis. Due to its advantages of being sterile, easily accessible in large volumes, and noninvasive to patients, urine is a favored diagnostic biofluid for 5hmC analysis. Here we developed an accurate, sensitive, and specific assay for quantification of 5mC, 5hmC, and other DNA demethylation intermediates in human urine. The urinary samples were desalted and enriched using off-line solid-phase extraction, followed by stable isotope dilution HPLC-MS/MS analysis for 5hmC and 5mC. By the use of ammonium bicarbonate (NH4HCO3) as an additive to the mobile phase, we improved the online-coupled MS/MS detection of 5mC, 5hmC, and 5-formylcytosine (5fC) by 1.8-14.3 times. The recovery of the method is approximately 100% for 5hmC, and 70-90% for 5mC. The relative standard deviation (RSD) of the interday precision is about 2.9-10.6%, and that of the intraday precision is about 1.4-7.7%. By the analysis of 13 volunteers using the developed method, we for the first time demonstrate the presence of 5hmC in human urine. Unexpectedly, we observed that the level of 5hmC (22.6 ± 13.7 nmol/L) is comparable to that of its precursor 5mC (52.4 ± 50.2 nmol/L) in human urine. Since the abundance of 5hmC (as a rare DNA base) is 1 or 2 orders of magnitude lower than 5mC in genomic DNA, our finding probably implicates a much higher turnover of 5hmC than 5mC in mammalian genomic DNA and underscores the importance of DNA demethylation in daily life.

Selective, sensitive and fluorometric determination of urinary cytosine with 4-trifluoromethylbenzamidoxime and N,N-dimethylformamide.

BACKGROUND: Cytosine in urine is one of the biomarkers for the diagnosis of metabolic immunodeficiency. It has been mentioned that a high level of cytosine is found in urine of children having immunodeficiency. In this study, we have developed a fluorescence (fluorescence) derivatization reaction of cytosine using 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent. METHODS: In this reaction, cytosine was mixed with 4-TFMBAO, K3[Fe(CN)6], N,N-dimethylformamide (DMF) and KOH in an aqueous solution. The mixture was heated at 100°C for 20 min. The fluorescence intensity of the mixture was measured with a spectrofluorometer. RESULTS: Under the optimized reaction conditions, a strong fluorescence was produced only from cytosine amongst 62 compounds including structurally related bio-substances. The selectivity and sensitivity of this method were compared with a conventional fluorescence one using 2-bromoacetophenone that reacts with cytosine, adenine and their related substances. The present method was sufficiently selective toward cytosine, and approximately 50 times more sensitive than the conventional one. CONCLUSIONS: Our method permitted the quantitative determination of cytosine in human urines without any pretreatment for a primary screening test of inborn disorder in pyrimidine metabolism with immunodeficiency, and indicated the lower detection limit of 0.1 µmol/l cytosine which gave 3 times greater fluorescence intensity than that observed for the reagent blank.

First pharmacokinetic and safety study in humans of the novel lipid antiviral conjugate CMX001, a broad-spectrum oral drug active against double-stranded DNA viruses.

CMX001 is a novel, broad-spectrum lipid antiviral conjugate (LAC) that produces high intracellular levels of the active antiviral agent cidofovir diphosphate (CDV-PP). Study CMX001-102 was a randomized, double-blind, placebo-controlled, parallel group, dose-escalating study in healthy volunteers. The objectives of the study were to evaluate the safety and pharmacokinetic parameters of CMX001 after single and multiple doses. Single doses ranging from 0.25 to 2.0 mg/kg of body weight and multiple doses ranging from 0.1 to 1.0 mg/kg (3 total doses, administered every 6 days) were given orally. Safety was assessed using comprehensive clinical and laboratory evaluations, including enhanced monitoring for potential gastrointestinal (GI) effects using wireless capsule endoscopy (WCE). Serial plasma and pooled urine samples were collected to estimate pharmacokinetic parameters for both CMX001 and cidofovir (CDV). No adverse events occurred that prevented dose escalation. No clinically significant drug-related changes in blood chemistry, hematology, renal function, or intraocular pressure were observed. No CMX001-related gastrointestinal mucosal changes were observed by WCE. CMX001 was absorbed rapidly, with maximum plasma concentrations observed 2 to 3 h postdose. Maximum plasma drug concentration and systemic exposure of CMX001 increased approximately in proportion to dose following single and multiple doses; no significant accumulation of CMX001 or CDV was observed following multiple doses. We conclude that CMX001 is orally bioavailable and well tolerated in healthy volunteers at doses up to 2 mg/kg, approximately 140 mg in a typical adult. This is the first demonstration of the use of phospholipid conjugation technology to achieve plasma drug exposures that are expected to result in activity against multiple double-stranded DNA viruses.

¹H nuclear magnetic resonance based metabolic urinary profiling of patients with ischemic heart failure.

OBJECTIVES: We sought to identify metabolic pathways characterizing human heart failure (HF) using ¹NMR based urinary metabolomic analysis in conjunction with multivariate statistics. DESIGN AND METHODS: Patients with systolic HF of ischemic origin (n=15) and healthy controls (n=20) participated in this study. Patients with type 2 diabetes mellitus were excluded. RESULTS: The results showed that the urine of the HF patients had higher levels of metabolites for acetate (p<0.05) and acetone (p<0.01) compared to the healthy controls. In addition, there was a perturbation in methylmalonate metabolism as shown by increased urinary levels of methylmalonic acid (p<0.001) in the HF patients. HF patients also had increased urinary levels of cytosine (p<0.01) and phenylacetylglycine (p<0.01) and decreased 1-methylnicotinamide (p<0.05) compared to healthy controls. CONCLUSIONS: TCA cycle metabolites and fatty acid metabolism were modified in the HF patients, indicating altered energy metabolism. Moreover, perturbations of metabolism in nucleotide and methylmalonate were observed.

Effect of gender and cigarette smoking on urinary excretion of etheno DNA adducts in humans measured by isotope dilution gas chromatography/mass spectrometry.

Endogenous formation of the promutagenic DNA adducts 1,N(6)-ethenoadenine (epsilon Ade) and 3,N(4)-ethenocytosine (epsilon Cyt) has been considered as biomarkers originated from lipid peroxidation. Elevated levels of epsilon Ade and epsilon Cyt were observed in cancer-prone tissues, suggesting the validity of these adducts in cancer risk assessment. The presence of DNA base adducts in biological fluids is considered to derive primarily from base excision repair (BER) systems. In this study, a modified gas chromatography/mass spectrometry (GC/MS) method is developed for simultaneous analysis of epsilon Ade and epsilon Cyt in human urine. After adjusting for creatinine concentration, urinary excretion of epsilon Ade, as well as epsilon Cyt, is much higher in 18 male smokers than in 10 male nonsmokers (p=0.003 for epsilon Ade and p=0.04 for epsilon Cyt). Furthermore, excretion of epsilon Ade and epsilon Cyt in 14 female nonsmokers is much higher than in 10 male nonsmokers (p=0.002 for epsilon Ade and p=0.005 for epsilon Cyt). These results suggest a statistically significant association between gender, as well as smoking, and excretion of epsilon Ade and epsilon Cyt. Moreover, urinary excretion of epsilon Ade in these 42 subjects correlates with that of epsilon Cyt (R(2)=0.6846, p<0.0001). Measurement of urinary epsilon Ade and epsilon Cyt excretion should provide valid noninvasive biomarkers for carcinogenesis and chemoprevention studies.

Determination of DMF modified DNA base N4-methylcarbamoylcytosine in human urine using off-line sample clean-up, two-dimensional LC and ESI-MS/MS detection.

A sensitive internal standard method for the analysis of a DNA-adduct of N,N-dimethylformamide (N4-methylcarbamoylcytosine, NMC-C) in human urine has been developed. A sample pre-treatment involving an acidic hydrolysis is followed by the sample clean-up performed with solid-phase extraction (SPE) technique using a cation-exchange resin. A two-dimensional liquid chromatography is used to separate the target analyte from the matrix using first a C18 reversed phase column with incorporated hydrophilic moieties and then a C8 bonded reversed phase column for the final separation. Quantification is carried out by positive electrospray ionisation and mass spectrometry detection of the transitions from molecule ions to product ions (169-->112 and 172-->115) for the analyte and the labelled internal standard, respectively. The detection limit in urine reaches down to 8 ng/L (48 pmol/L). In the general population NMC-C could not be detected. In 10 out of 32 urine samples of occupationally to DMF exposed subjects NMC-C could be detected. The concentrations ranged up to 172 ng/L (1023 pmol/L) with a 95th percentile of 121 ng/L (720 pmol/L).

Urinary excretion of 3,N4-etheno-2'-deoxycytidine in humans as a biomarker of oxidative stress: association with cigarette smoking.

Smokers are known to have elevated levels of lipid peroxidation, a form of oxidative stress. Etheno DNA adduct formation can originate from endogenous lipid peroxidation or from exogenous exposure of carcinogens. Using a modified stable isotope dilution GC/negative ion chemical ionization/MS assay originally developed for urinary 3,N(4)-ethenocytosine (epsilonCyt), the nucleoside 3,N(4)-etheno-2'-deoxycytidine (epsilondCyd) was detected for the first time in human urine. The presence of epsilondCyd in human urine was confirmed by LC/electrospray ionization/tandem MS. Concentrations of epsilondCyd in the 24 h urine samples from healthy individuals not occupationally exposed to industrial chemicals were in the range between 0 and 0.80 nM. A statistically significant correlation was established between cigarette smoking and urinary excretion of epsilondCyd after being adjusted for creatinine (p = 0.004). Furthermore, the urinary total antioxidant capacity was found to correlate inversely with the epsilondCyd levels (r = -0.50, p = 0.02). The results indicate that urinary epsilondCyd may provide a valuable noninvasive biomarker for oxidative DNA damage.

Effect of cigarette smoking on urinary 3,N4-ethenocytosine levels measured by gas chromatography/mass spectrometry.

Etheno DNA adducts are DNA damages derived from exogenous carcinogens as well as endogenous lipid peroxidation and oxidative stress. Elevated levels of etheno DNA adducts were found in cancer-prone tissues and blood samples, suggesting that these promutagenic lesions correlate with risk of cancers. We previously reported the detection of 3,N4-ethenocytosine (epsilon Cyt) in the urine samples of two smokers using the isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) assay (Chen et al., 2001, Chem. Res. Toxicol. 14, 1612-1619). Since smokers are found to have elevated levels of lipid peroxidation and oxidative stress, we examined the association between urinary epsilon Cyt levels with cigarette smoking. Among the 23 samples analyzed, the average concentration of urinary epsilon Cyt in smokers was significantly higher than that of nonsmokers, 2.65 +/- 4.0 versus 0.61 +/- 0.90 ng/kg/g creatinine (p= 0.03). Albeit the number of subjects is limited, the results indicate that the measurement of epsilon Cyt in human urine may provide a useful noninvasive biomarker for oxidative DNA damage and cancer chemoprevention studies.

Antiviral activity and pharmacokinetics of 1-(2,3-dideoxy-2-fluoro-beta-L-glyceropent-2-enofuranosyl)cytosine.

1-(2,3-Dideoxy-2-fluoro-beta-L-glyceropent-2-enofuranosyl)cytosine (L-2'-Fd4C) is an L-nucleoside analogue with both anti-human immunodeficiency virus (HIV) and anti-hepatitis B virus (HBV) activity with median effective concentrations of 0.12 microM in peripheral blood mononuclear cells and 0.002 microM in HepG2-2.2.15 cells, respectively. The purpose of this study was to examine the antihepadnavirus potency and pharmacokinetics of L-2'-Fd4C in vivo. HBV-transgenic mice treated intraperitoneally with L-2'-Fd4C showed a reduction of HBV levels in their blood comparable to that produced by lamivudine. The pharmacokinetics of L-2'-Fd4C in rhesus monkeys was evaluated after intravenous and oral administration. The concentrations in plasma declined in a biexponential manner after intravenous administration, with a long terminal-phase half-life of 5.02 h. The steady-state volumes of distribution and systemic clearance were 1.09 liter x kg(-1) and 0.25 liter x h(-1) x kg(-1), respectively, with a renal clearance of 0.16 liter x h(-1) x kg(-1). The oral bioavailability was approximately 44%. About 53% of the compound administered intravenously and 19% of that administered orally were recovered unchanged in the urine within the 24-h urine collection period, and no other metabolite was detected. The compound penetrated the central nervous system at concentrations that exceeded the median effective antiviral concentration against HIV in cell cultures. Based upon these observations, further testing to develop this agent for treatment of HIV and HBV infections is warranted.

Analysis of 3,N(4)-ethenocytosine in DNA and in human urine by isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry.

The promutagenic etheno DNA adducts have been detected in tissue DNA of rodents and humans from various exogenous and endogenous sources. While other etheno DNA adducts have been detected and quantified by isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS), similar analysis for 3,N(4)-ethenocytosine (epsilonCyt) has not been available. In this report, a GC/NICI/MS assay was developed for detection and quantification of epsilonCyt in DNA and in human urine samples. The stable isotope of epsilonCyt with 7 mass units higher than the normal epsilonCyt was synthesized and used as internal standard of the assay. The adduct-enriched fraction of DNA hydrolysate was derivatized with pentafluorobenzyl bromide before GC/NICI/MS analysis with selective ion monitoring at [M - 181](-) fragments of pentafluorobenzylated epsilonCyt and its isotope analogue. One femtogram (S/N > 40) of pentafluorobenzylated epsilonCyt was detected when injected on column with selective ion monitoring mode. The limit of quantification for the entire assay was 7.4 fmol of epsilonCyt, which was approximately one thousand times lower than that of the HPLC/fluorescence assay for the nucleoside 3,N(4)-etheno-2'-deoxycytidine in DNA. Analysis of chloroacetaldehyde-treated calf thymus DNA by both GC/NICI/MS and HPLC/fluorescence methods provided similar adduct levels and thus verified the assay. This GC/NICI/MS method was used for analysis of epsilonCyt in two smokers' urine samples and the average level of epsilonCyt was 101 +/- 17 pg/mL/g of creatinine. Thus, quantification of epsilonCyt in DNA and in urine by this highly specific and ultrasensitive isotope dilution GC/NICI/MS assay may facilitate research on the role of epsilonCyt in carcinogenesis and in cancer development.

Clinical pharmacokinetics of the antiviral nucleotide analogues cidofovir and adefovir.

Cidofovir and adefovir are members of a new class of antiviral compounds. They are acyclic phosphonate analogues of deoxynucleoside monophosphates. Both compounds undergo intracellular activation to form diphosphates that are potent inhibitors of viral DNA polymerases. Cidofovir has broad spectrum antiviral activity against herpesviruses, papillomaviruses and poxviruses, whereas adefovir has potent activity against retroviruses and certain DNA viruses, including herpesviruses and hepadnaviruses. Intravenous cidofovir is approved for treatment of cytomegalovirus retinitis in patients with AIDS. Cidofovir and adefovir are dianionic at physiological pH and have low oral bioavailability in animals and humans. After intravenous administration to HIV-infected patients, the pharmacokinetics of both drugs are independent of dose and are consistent with preclinical data. Systemic exposure is proportional to the intravenous dose and both drugs are cleared by the kidney and excreted extensively as unchanged drug in the urine. Intracellular activation of a small fraction (< 10%) of the dose by cellular kinases leads to prolonged antiviral effects that are not easily predicted from conventional pharmacokinetic studies. The observed rate of elimination of cidofovir and adefovir from serum may not reflect the true duration of action of these drugs, since the antiviral effect is dependent on concentrations of the active phosphorylated metabolites that are present within cells. For both drugs, > 90% of an intravenous dose is recovered unchanged in the urine over 24 hours. Metabolism does not contribute significantly to the total clearance of either drug. Concomitant oral probenecid decreases both the renal clearance of cidofovir and the incidence of nephrotoxicity, presumably by blocking its active tubular secretion. This is the basis of the clinical use of concomitant probenecid as a nephroprotectant during cidofovir therapy. Subcutaneous administration produces exposure equivalent to that following intravenous administration. Drug interaction studies with cidofovir are ongoing, but there is no evidence of an interaction between zidovudine and either cidofovir or adefovir. Clearance of cidofovir in patients with renal impairment showed a linear relationship to creatinine clearance. The low oral bioavailability of adefovir has led to the development of an oral prodrug, adefovir dipivoxil, currently in development for the treatment of HIV and hepatitis B infections.

Bioavailability and metabolism of cidofovir following topical administration to rabbits.

The bioavailability and metabolism of the antiviral nucleotide analog cidofovir (HPMPC) were examined in New Zealand white rabbits following topical administration to normal and abraded skin. Male rabbits (four per group) received 14C-cidofovir (100 microCi/kg) intravenously (1 mg/kg) as a solution or topically (2 mg/animal) as a 1% w/w gel containing hydroxyethylcellulose (HEC) with or without propylene glycol (PG). The same PG/HEC formulation was applied topically to an abraded skin site in a fourth group of animals. All radioactivity detected in plasma and skin was accounted for by cidofovir. Plasma concentrations of radioactivity declined multiexponentially following intravenous administration, with a terminal half-life of 5.4 h. For intact skin, the absolute bioavailabilities of the HEC and PG/HEC formulations were 0.2 and 2.1%, respectively. For abraded skin, the bioavailability for the PG/HEC gel was 41%. Radioactivity in kidneys was attributed to cidofovir ( > 95%) and cyclic HPMPC. Concentrations in kidney following topical administration of cidofovir to normal skin were < 4% of those following intravenous dosing. Topical application of cidofovir to intact skin led to negligible systemic exposure to the drug. The topical bioavailability and hence the flux of cidofovir through intact skin was enhanced by the presence of PG in the formulation. Abrasion of the skin removed the principal barrier to absorption and led to significant systemic exposure to cidofovir.

Effect of probenecid on the distribution, metabolism, and excretion of cidofovir in rabbits.

The effect of concomitant probenecid on the tissue distribution, metabolism, and urinary excretion of cidofovir was examined in New Zealand white rabbits. Two groups of six male rabbits received intravenous [3H]cidofovir (5 mg/kg, 20 mu Ci/kg) alone or with concomitant intravenous probenecid (90 mg/kg). Radioactivity in kidney at 15 min postdose was decreased 70% by probenecid; plasma levels at 15 min postdose were increased 65% by probenecid. These effects were diminished at later time points. The estimated elimination half-life of cidofovir from the kidney was 16 hr in the presence of probenecid and 11 hr without probenecid. Two additional groups of six rabbits received intravenous [14C]cidofovir (15 mg/kg, 100 mu Ci/kg) alone or 1 hr after oral administration of probenecid (90 mg/kg). Radioactivity was highest in the kidney (approximately 700 mu g-eq/g at 30 min postdose). Probenecid did not affect the gross distribution of radioactivity. However, autoradiography of left kidneys revealed localization of the drug in the renal cortex; radioactivity in the cortex at 30 min postdose was decreased 50% by probenecid. These data are consistent with inhibition of tubular secretion of cidofovir by probenecid. More than 73% of the cidofovir dose was recovered in the urine in 24 hr. Urine contained unchanged cidofovir (>97%) and a metabolite coeluting with authentic cidofovir phosphocholine (2%). This metabolite also accounted for approximately 1-4% of the radioactivity in rabbit kidney.

Comparative mutagenicity testing of bropirimine, 3. Bropirimine does not induce cytogenetic damage in vivo in the rat but does produce micronuclei in the mouse.

Bropirimine is a biological response modifier (BRM) with potential antineoplastic and antiviral indications. Recent results have documented the negative findings in the Ames Salmonella assay, the in vitro UDS assay and the mouse lymphoma TK+/- assay as well as positive findings in the in vitro cytogenetic assay in CHO cells. Extensive mechanistic studies failed to establish the reason for positive findings in the in vitro cytogenetic assays. The data reported here cast doubt on the relevance of the in vitro cytogenetic results and suggest limited in vivo genotoxic potential. At doses as high as 150 mg/kg (i.p.) and 6.73 g/kg (p.o.), no evidence of chromosome aberration induction was observed in rat bone marrow cytogenetic assays. Consistent with these data, plasma and bone marrow tissue levels in similarly treated animals were well below those required for activity in the in vitro chromosome aberration assays. Positive results were obtained in the mouse micronucleus assay. However, the significance of these findings may be explained by markedly different pathways of metabolism in that species as compared to the rat. Hence, the findings in the mouse are of questionable relevance to human risk assessment. Exposure of humans to bropirimine, under therapeutically acceptable regimens is unlikely to constitute a genotoxic health hazard.

[Age dependency of normal and modified nucleobases in urine in correlation to growth velocity (author's transl)]. / Die Altersabhängigkeit der normalen und modifizierten Nucleobasen im Urin als Ausdruck der Wachstumsgeschwindigkeit.

Up to now no age-correlated normal values of normal and modified nucleobases in urine have been published. In this paper such normal values are presented, obtained from 116 healthy subjects from 0 to 16 years by high performance liquid chromatography of creatinine-correlated single urine specimens. A narrow correlation between the excretion of nucleobases and growth velocity was found. A polynomial function including the puberal growth spurt could be fitted to the analytical data. It is felt that this method should be suitable to assess growth processes of different kinds, e.g. effects of nutrition or drugs and hormones on growth velocity, furthermore intrauterine and malignant growth.

Cytosine and orotic acid in urine of immunodeficient children.

We describe procedures for determining cytosine and orotic acid in urine. We determine cytosine by cation-exchange analysis with either HCl or pH 5.2 buffer as eluent. Orotic acid is first separated by an anion-exchange separative procedure; after lyophilization, the product is subjected to "high-pressure" liquid chromatography for further separation and detection. We analyzed urine from normal subjects and from immunodeficient children. Three children with severe combined immunodeficiency had increased levels of cytosine in urine (23-160 mmol/mol creatinine); one child with severe combined immunodeficiency and two children with other immunodeficiencies had normal urinary levels (less than 2 mmol/mol creatinine). Orotic acid excretion in urine was normal (1-5 mmol/mol creatinine) in all of th immunodeficient children. We discuss the possible significance of the increased cytosine excretion in the three children with severe combined immunodeficiency.

High-resolution liquid chromatographic analysis of methylated purine and pyrimidine bases in transfer RNA.

Methylated and major purine and pyrimidine bases were separated and quantified by high-resolution liquid chromatography after hydrolyzing transfer ribonucleic acids (tRNAs). Separation was accomplished by eluting the hydrolyzed samples from an anion-exchange column with a concentration gradient of ammonium acetate at pH 9.2. Isolated sample of tRNA were hydrolyzed to the free bases with a trifluoroacetic acid-formic acid mixture of 200 degrees. Detection limits of 100-200 ng/ml were measured for the methylated bases; analytical data are reported for ten methylated bases plus the four major bases of calf liver and rat liver tRNA.