NORMAL GENOMIC

• Evans MD, Singh R, Mistry V, Farmer PB, Cooke MS
• Analysis of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine by liquidchromatography-tandem mass spectrometry.
• Methods Mol Biol. 2010; 610: 341-51
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• The ability to non-invasively monitor DNA oxidation and its repair hassignificant utility in large-scale, population-based studies. Such studiescould include assessments of the efficacy of antioxidant interventionstrategies, pathological roles of DNA oxidation in various disease statesand population or inter-individual differences in antioxidant defence andDNA repair. The analysis of urine, or indeed any extracellular matrix, for8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), using chromatographic orimmunoassay procedures, is by far the most popular method tonon-invasively assess oxidative insult to the genome. The actualbiological significance of the presence of extracellular 8-oxodG is stilla subject for debate however. Studies are slowly ruling out confoundingfactors such as diet and cell turnover, which would leave endogenousprocesses, notably repair, as the sole source of extracellular 8-oxodG.The method described herein exploits the non-invasive properties of urinesampling, coupled with efficient extraction of 8-oxodG by a validatedsolid-phase extraction procedure. Subsequent analysis by liquidchromatography-tandem mass spectrometry has the advantages of sensitivity,internal standardisation and robust peak identification.

• Liu MY, Meng SN, Wu HZ, Wang S, Wei MJ
• Pharmacokinetics of single-dose and multiple-dose memantine in healthychinese volunteers using an analytic method of liquidchromatography-tandem mass spectrometry.
• Clin Ther. 2008; 30: 641-53
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• OBJECTIVE: This study consisted of 2 phases: development of a liquidchromatography-tandem mass spectrometry (LC/MS) method for determinationof memantine in human plasma and characterization of single-dose andmultiple-dose pharmacokinetic profiles of memantine in healthy Chinesevolunteers using the LC/MS method. METHODS: An analytic method of LC/MSfor determination of memantine in human plasma was developed and validatedand was applied to this single-center, open-label, single-dose andmultiple-dose pharmacokinetic study conducted in healthy native Chinesevolunteers. Subjects were randomized to receive a single dose of 5, 10, or20 mg of memantine to study the linear characteristics ofpharmacokinetics, or a multiple dose of 5 mg once daily for 14 days tostudy the drug accumulation. The pharmacokinetic parameters calculatedincluded C(max), T(max), AUC, t(1/2),mean residence time (MRT), maximumsteady-state plasma concentration (C(ssmax)), minimum steady-state plasmaconcentration ((ssmin)), average steady-state plasma concentration(C(ssav)), and fluctuation percentage (DF). All values were expressed asmean (SD). Sequential blood samples were collected from 0 to 360 hours forsingle-dose pharmacokinetic determinations after the dose on day 1; in themultiple-dose pharmacokinetic arm, the sequential blood samples were alsoobtained from 0 to 360 hours on day 14 after collecting the predosesamples at 0 hour on days 11, 12, and 13. Memantine concentrations inplasma were determined by LC/MS method. A calibration curve wasconstructed by 7 memantine concentrations and processed by least-squareslinear regression analysis (w=1/x(2)). Safety assessments, includingadverse events (AEs), were performed at all study visits. RESULTS: TheLC/MS method for determination of memantine in human plasma was developedand validated. The standard calibration curve for spiked human plasmacontaining memantine was linear in the concentration range of 0.2 to 200.0ng/mL. The correlation coefficient was greater than 0.9960 (n = 6). Thelower limit of quantification for memantine in human plasma was 0.2 ng/mL,and the intraday and interday coefficients of variation were all lowerthan 15%. The mean recoveries of the 0.4, 20.0, and 180.0 ng/mL levelswere 78.87%, 81.55%, and 81.98%, respectively. The coefficients ofvariation were all lower than 15% after being treated at room temperaturefor 24 hours, for 45 days at -40 degrees , and within 3 freeze-and-thawcycles in plasma samples. Forty native Chinese subjects (10 [5 men, 5women] subjects per group; mean [SD] age, 21.6 [1.6] [range, 19-27] years;weight, 63.0 [7.7] [range, 52-82] kg; height, 170.0 [7.0] [range, 155-185]cm) were enrolled in the study. After single-dose oral administration, themain pharmacokinetic parameters found for memantine at doses of 5, 10, and20 mg were as follows: C(max), 6.20 (0.75), 11.60 (1.95), and 25.34 (8.34)ng/mL, respectively; T(max), 5.70 (1.64), 6.00 (1.33), and 6.89 (1.41) h;AUC(0-t), 486.19 (80.00), 889.32 (239.49), and 1772.91 (784.07) ng x h/mL;AUC(0-infinity), 540.05 (89.68), 932.07 (230.82), and 1853.29 (776.85) ngx h/mL; t(1/2), 66.86 (11.75), 63.57 (12.58), and 62.06 (9.26) h; and MRT,99.37 (16.96), 91.73 (18.16), and 89.56 (13.77) h. The mainpharmacokinetic parameters found for memantine at doses of 5 mg once dailyfor 14 days were as follows: T(max), 6.80 (2.46) h; C(ssmax), 19.69 (2.00)ng/mL; C(ssmin), 12.76 (2.80) ng/mL; C(ssav), 16.10 (2.46) ng/mL; t(1/2),64.57 (15.78) h; MRT, 93.17 (23.38) h; AUC(ss),386.37 (59.00) ng x h/mL;and DF, 44.47% (15.27%). One female subject withdrew from the study aftera single 20-mg dose due to an AE (dizziness and vomiting); no othersubjects experienced an AE. CONCLUSIONS: In these healthy Chinesesubjects, the t(1/2) and MRT values were fixed and did not increasefollowing the increased dose, and the AUC(infinity) and C(max) valuesincreased following the increasing dosage of memantine. Linearpharmacokinetics was found at doses from 5 to 20 mg. The multiple-dosepharmacokinetic parameters (other than C(max)) were nearly similarcompared with the single-dose administration. The maximum plasmaconcentration of memantine after multiple-dose administration was greaterthan that after a single-dose administration, suggesting memantineaccumulation with multiple-dose administration of 5 mg and requiringfurther confirmation in larger studies.

• Al-Dirbashi OY, Rashed MS, Al-Qahtani K, Al-Mokhadab MA, Kurdi W, Al-Sayed MA
• Quantification of N-acetylaspartic acid in urine by LC-MS/MS for thediagnosis of Canavan disease.
• J Inherit Metab Dis. 2007; 30: 612-612
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• Canavan disease is an autosomal recessive leukodystrophy characterized byexcessive excretion of N-acetylaspartic acid (NAA) in urine. The diseaseis caused by deficiency of aspartoacylase, the enzyme responsible for thehydrolysis of NAA into acetate and l-aspartate. Patients, who are oftenasymptomatic in their early months, show a wide spectrum of clinicalpresentation thereafter that includes macrocephaly, poor head control,seizures, abnormal muscle tone, optic atrophy, significant developmentaldelay and death. In this work, we describe a simple liquidchromatography-tandem mass spectrometry (LC-MS/MS) method for thedetermination of NAA in urine. The internal standard d3-NAA was added tountreated urine and the mixture was injected into the LC-MS/MS systemoperated in the negative ion mode. Detection was achieved in multiplereaction monitoring (MRM) mode by monitoring m/z 174 --> 88, 174 --> 130and 174 --> 58 for NAA and 177 --> 89 for the internal standard.Separation was carried out on a C8 column (2.1 x 150 mm) using a mixtureof acetonitrile and water (1:1 v/v) containing 0.05% formic acid at a flowrate of 0.25 ml/min. NAA was eluted at 1.6 min and the run time wasapproximately 2 min. Using spiked urine, the assay was linear up to 2mmol/L with limit of quantification at 1 micromol/L (S/N = 12). NAA inpatients' urine (n = 17) ranged between 366 and 21,235 mmol/mol creatininecompared to controls of <39 mmol/mol creatinine (n = 159). This LC-MS/MSmethod for NAA as described involved no extraction and no derivatization,showed no interference, and gave excellent recovery with low variabilityand short analytical time.

• Malayappan B, Garrett TJ, Segal M, Leeuwenburgh C
• Urinary analysis of 8-oxoguanine, 8-oxoguanosine, fapy-guanine and8-oxo-2'-deoxyguanosine by high-performance liquidchromatography-electrospray tandem mass spectrometry as a measure ofoxidative stress.
• J Chromatogr A. 2007; 1167: 54-62
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• A sensitive and specific assay aimed at measuring the oxidized nucleicacids, 8-oxoguanine (8-oxoGua), fapy-guanine (Fapy-Gua), 8-oxoguanosine(8-oxoGuo), 8-oxo-2'-deoxyguanosine (8-oxodG) has been developed bycoupling reversed phase liquid chromatography (HPLC) with electrospraytandem mass spectrometry detection (MS/MS) and isotope dilution. TheHPLC-MS/MS approach with multiple reaction monitoring (MRM) allowed forthe sensitive determination of 8-oxoGua, Fapy-Gua, 8-oxoGuo, and 8-oxodGin human urine samples. There is no sample preparation needed except forthe addition of buffer and (13)C- and (15)N-labeled internal standards tothe urine prior to sample injection into the HPLC-MS/MS system. Thismethod was tested in urine samples from non-smokers, smokers, non-smokerswith chronic kidney disease (CKD) and smokers with CKD, to assess thelevel of oxidative damage to nucleic acids. Markers of both RNA and DNAdamage were significantly increased in the smokers with and without CKDcompared to their respective control subjects. These findings suggest thata highly specific and sensitive analytical method such as isotope dilutionHPLC-MS/MS may represent a valuable tool for the measurement of oxidativestress in human subjects.

• Prieme H, Loft S, Klarlund M, Gronbaek K, Tonnesen P, Poulsen HE
• Effect of smoking cessation on oxidative DNA modification estimated by8-oxo-7,8-dihydro-2'-deoxyguanosine excretion.
• Carcinogenesis. 1998; 19: 347-51
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• BACKGROUND: Reactive oxygen species from, e.g. tobacco smoke are suggestedto be involved in carcinogenesis by oxidative modification of DNA. Theurinary excretion rate of the oxidized nucleoside8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) has been validated as abiomarker of the rate of oxidative DNA modification with mechanisticrelation to carcinogenesis. In cross-sectional studies, the urinaryexcretion rate of 8-oxodG has been shown to be elevated in smokerscompared with non-smokers. PURPOSE: In this randomised, controlled smokingcessation study, we investigated whether cigarette smoking per se causesoxidative DNA modification. METHODS: Of the 182 healthy smokers included,100 were randomized to quit smoking after baseline samples had been taken,and 82 were randomized to continue usual smoking. Before the start of thestudy and after 4 weeks, the subjects collected 24-h urine samples thatwere analysed for 8-oxodG content by high-pressure liquid chromatographywith electrochemical detection. The subjects randomized to smokingcessation were followed up after 26 weeks. RESULTS: Four weeks of smokingcessation resulted in a 21% decrease in 8-oxodG excretion rate (from mean+/- SD, 30.5 +/- 13.9 to 24.1 +/- 10.5 nmol/24 h, P < 0.001) in 58quitters included in per-protocol data analysis. Sixty-five continuedsmokers included in per-protocol analysis showed a 9% decrease in 8-oxodGexcretion rate (from 31.6 +/- 13.2 to 28.7 +/- 12.6 nmol/24 h, P = 0.026).After 4 weeks, the 8-oxodG excretion rate was 16% (95% confidence interval4 to 28%) higher in the continued smokers than in the quitters (P =0.0085, ANCOVA), demonstrating the effect of smoking per se. A 23% (P <0.005) decrease in 8-oxodG excretion rate was sustained for 26 weeks in 27quitters who completed the study. CONCLUSION: Smoking cessationsignificantly reduces the urinary excretion rate of 8-oxodG, giving directand controlled evidence that cigarette smoking causes an increased rate ofoxidative DNA modification. This could represent a mechanism by whichtobacco smoke is carcinogenic.