Handbook of Vitamin C Research

Human Specific Vitamin C Metabolism… 55Measurement of Reactive Oxygen Metabolites (ROM)In order to measure ROM, the d-ROM test was performed using FRAS4 (Diacron,Grosseto, Italy), according to the manufacturer‘s procedures [9]. Briefly, a 20-μL bloodsample (obtained from a finger tip) and 1 ml of buffered solution (R2 reagent of kit, pH 4.8)were gently mixed in a cuvette, and 10 μL of chromogenic substrate (R1 reagent of kit) wasadded. After mixing, the cuvette was centrifuged for 60 s at 37°C and immediately incubatedin the thermostatic block of this analyzer for 5 min at 37°C. Absorbance at 505 nm was thenrecorded, and the results were expressed in terms of Carr U. It has been established that 1Carr U corresponds to 0.08 mg/dL H 2 O 2 . Reference values indicated by the manufacturer(Diacron) range from 250 to 300 Carr U; values higher than 300 Carr U suggest oxidativestress [9].(4) Single Nucleotide Polymorphism (SNP) AnalysisGeneral gene techniques were as described elsewhere [38, 59, 67]. SNPs in xenobioticenzymes (GSTP1-1, GSTP1-2, SOD2 and SOD3) were analyzed by real-time polymerasechain reaction for GSTP1-1 [38]. The Ile105Val SNP of GSTP1-1 is expressed in terms ofnucleotide number A342G (exon 5), or from the 1st codon AGT of exon 1, equivalent tont.A1375G. After the common reaction, a TaqMan genotyping system with an ABI PRISM7900HT unit (Applied Biosystems, Foster City, California, U.S.A.) [59] was used withspecific TaqMan probes and primers. Four SNPs, SOD2 Val16Ala (rs4880), SOD3Arg231Gly (rs1799895), GSTP1 Ile105Val (rs1695) [GSTP1-1 A342G (exon5)] andAla114Val (rs1138272), were analyzed. The sequences of the TaqMan probes were asfollows and polymorphic nucleotides are underlined:For SOD2 Val16Ala, Fam-AGCAGGCAGCTGGCTCCGACT-TAMRA (Ala) and Vic-TGATCTAGCAGGCAGCTGGCTCCGATT-TAMRA (Val);For SOD3 Arg231Gly, Fam-GATCAGGCACTCAGAGCGCAAGAATGG-TAMRA(Gly) and Vic-GCACTCAGAGCGCAAGAATCG-TAMRA (Arg);For GSTP1 Ile105Val, Fam-CAGATCTTGGTGTAGATGAGGGAGTTG-TAMRA (Ile)and Vic-TTGGTGTAGATGAGGGAGTCG-TAMRA (Val);For GSTP1 Ala114Val, Fam-ACGATCCACATAGTCATCCTTGCCTAC-TAMRA(Val) and Vic-CACATAGTCATCCTTGCCTGC-TAMRA (Ala).(5) Statistical AnalysisAfter analysis of variance using Stat View-J4.02 (Macintosh version), multiplecomparisons were performed by Bonferroni/Dunn test [4]. Correlations between clinical andbiochemical values were estimated as reported previously [60].