Abstract
(Englisch)
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THERE EXISTS IN THE SCIENTIFIC LITERATURE SIGNIFICANT DISCREPANCY BETWEEN THE ENDOGENOUS LEVELS OF THE BIOMARKER 8- OXO-7,8-DIHYDROGUANINE (8-0XO-GUA) OR 8-0XO-7,8-DIHYDRO-2'-DEOXYGUANOSINE (8-0XO-DG) IN DNA. THIS SUGGESTS VARIABILITY IN MEASUREMENTS DEPENDING ON THE LABORATORY AND/OR THE MEASUREMENT TECHNIQUE. THE ESCODD WAS ESTABLISHED TO RESOLVE PROBLEMS ASSOSCIATED WITH THE MEASUREMENT OF OXIDATIVE DAMAGE TO DNA, VII. 8-0XO-GUA AND 8-0XO-DG, IN VARIOUS BIOLOGICAL TISSUES. IN TOTAL 28 LABORATORIES -MAINLY IN EUROPE -ARE PARTICIPATING, AND THE TASKS ARE DESIGNED IN THREE 1-YEAR STAGES (EACH COVERED BY ONE WORKPACKAGE).
OBJECTIVE OF THE FIRST STAGE
THE MAIN OBJECTIVE OF PHASE 1 WAS TO ASSESS THE ABILITY OF THE DIFFERENT LABORATORIES AND METHODS TO MEASURE OXIDATIVE DNA DAMAGE, USING VARIOUS CONTEMPORARY ANALYTICAL TECHNIQUES SUCH AS HPLC-ECD, GC-MS AND LC-MS/MS. THIS WAS ACHIEVED BY A SERIES OF DISTRIBUTIONS OF REFERENCE STANDARD SAMPLES OF THE FOLLOWING:
BATCH 1) 8-0XO-GUA/8-0XO-DG PROVIDED IN SOLUTION AT DIFFERENT CONCENTRATIONS; BATCH 2) 8-0XO-GUA/8-0XO-DG BUILT INTO A SYNTHETIC OLIGONUCLEOTIDE, AND BASELINE LEVELS OF 8-0XO-GUA/8-0XO-DG IN CALF-THYMUS DNA (3 SAMPLES); BATCH 3) A SERIES OF CONCENTRATIONS OF 8-0XO-GUA/8-0XO-DG INDUCED EXPERIMENTALLY (DIFFERENT DOSES OF LIGHT AND PHOTO-SENSITIZER) IN CALF THYMUS DNA. RESULTS WERE COLLATED AND THE PERFORMANCE WAS ASSESSED BY THE PROJECT CO-ORDINATOR.
RESULTS AND MILESTONES
DEVELOPMENT OF THE LC-MSIMS METHOD DURING THIS PERIOD, WE HAVE DEVELOPED AN ISOTOPE-DILUTION LC-MS/MS METHOD IN ORDER TO ANALYSE 8-0XO-GUA/8-0XO-DG BY THIS MORE SUPERIOR TECHNIQUE. THE ISOTOPE LABELLED COGNATES WERE SYNTHESIZED IN HOUSE BY A METHOD DEVELOPED IN OUR LABORATORY (STADLER ET AL., CHEM. RES. TOXICOL. 7, 784-791, 1994). IN ESSENCE, THE METHOD USES REVERSED PHASE LC COUPLED TO ELECTROSPRAY IONIZATION MS/MS, AND ENTAILS PURIFICATION OF DNA OVER QIAGEN ION EXCHANGE COLUMNS FOLLOWED BY ENZYMATIC HYDROLYSIS WITH NUCLEASE P1 AND ALKALINE PHOSPHATASE. ELECTROSPRAY IONIZATION IN THE POSITIVE MODE IS USED FOR MS MEASUREMENTS, AND A (M-+4) INTERNAL STANDARD FOR QUANTIFICATION BY ISOTOPE DILUTION MS. THE METHOD WAS ABLE TO DETECT SUB-PICOGRAM LEVELS OF 8-0XODG ON-COLUMN. SIMULTANEOUS DETECTION OF DEOXYGUANOSINE IS DONE, AND EXHIBITED A LINEAR RESPONSE FROM 0.1 -1 MICROGRAM FOR DEOXYGUANOSINE (DG), BUT NOT OVER THE COMPLETE RANGE UP TO 4 MICROGRAM. IN THIS CASE, TWO CALIBRATION CURVES MUST BE CONBSTRUCTED TO ACHIEVE GOOD LINEARITY FOR DG.
ANAL YS/S OF THE SAMPLES OF BATCH 1,2 AND 3
THREE BATCH OF SAMPLES WERE TO BE ANAL YSED:
.BATCH ONE: FOUR DIFFERENT 8-0XO-DG. STANDARD SAMPLES AND ONE DG STANDARD SAMPLE, TO BE ANALYSED IN TRIPLICATE ON THREE OCCASIONS, AT LEAST TWO DAYS APART. RECEIVED IN APRIL 2000.
.BATCH TWO : FOUR DIFFERENT 8-0XO-DG. STANDARD SAMPLES AND THREE CALF THYMUS DNA SAMPLE (THREE TUBES OF EACH), TO BE ANAL YSED IN TRIPLICATE ON THREE OCCASIONS. RECEIVED IN JUNE 2000.
.BATCH THREE: FOUR SAMPLES OF CALF THYMUS DNA WITH DIFFERENT DOSES OF LIGHT AND PHOTO-SENSITIZER TO INDUCE 8- OXO-DG. ANALYSIS SHOULD BE ON 3 OCCASSIONS AT LEAST 2 DAYS APART.
USING THE LC-MS/MS BASED METHOD, WE CONDUCTED THE RELEVANT ANALYSES ON ALL SAMPLES FOR BATCHES 1 AND 2, AND THE RESULTS WERE PROVIDED TIMELY TO THE ESCODD CO-ORDINATOR FOR COLLATION AND STATISTICAL TREATMENT.
HOWEVER, BATCH 3 COULD NOT BE ANALYZED WITHIN THE GIVEN TIME-FRAME DUE TO AN UNFORESEEN LONGER PERIOD OF ILLNESS OF THE TECHNICIAN PERFORMING THE ANALYSES IN OUR LABORATORY (MR. E. GREMAUD). A CHANGE TO ANOTHER TECHNICIAN WAS NOT RECOMMENDED, DUE TO THE INTRODUCTION OF AN ADDITIONAL POTENTIAL VARIABLE.
ESCODD SUMMARY AND CONCLUSION OF THE PROGRESS ACHIEVED IN PHASE 1
AS MENTIONED ABOVE, THE MAIN OBJECTIVE IN THE FIRST YEAR WAS TO IMPROVE THE PERFORMANCE OF THE ANALYTICAL
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APPROACHES TO THE MEASUREMENT OF OXIDATIVE DNA DAMAGE. THE OVERALL RESULTS OF THE 8-OXO-DG, OLIGONUCLEOTIDE, AND CALF THYMUS SAMPLES CAN BE SUMMARIZED AS FOLLOWS:
8-OXO-DG: INTRA-TUBE VARIABILITY WAS ASSESSED AS THE CV OF N = 3 DETERMINATIONS FROM ONE SAMPLE TUBE. FOR SAMPLE 0, 2/3 OF RESPONSES HAS A CV < 5%; FOR SAMPLE A (LOWEST CONCENTRATION OF 8-0XO-DG), 1/3 OF THE RESPONSES HAD A CV < 5%. INTER- TUBE VARIABILITY WAS ASSESSED ON THE 3 DIFFERENT TUBES TO BE ANALYZED ON DIFFERENT DAYS. A CV < 10% WAS ACHIEVED BY MORE THAN % OF THE LABORATORIES. THE BATCH WITH THE HIGHEST 8-0XO-DG LEVEL SHOWED GREAT VARIABILITY, WITH CLUSTERING AROUND THE MEDIAN VALUES AS WELL AS CONSISTENT UNDER/OVER ESTIMATION FOUND IN CERTAIN LABORATORIES. VARIABILITY WAS WORSE IN BATCH 2 RESULTS, ATTRIBUTED PERHAPS TO THE EXCESSIVE TEMPERATURES EXPERIENCED IN MANY PLACES DURING THE SUMMER.
DG SAMPLE: THIS ENTAILS STRAIGHTWORWARD ANALYSIS OF THE UNALTERED NUCLEOSIDE/BASE, AND 4/5 OF RESULTS WERE WITHIN 20% OF THE MEDIAN, AND INTRA AND INTER-TUBE CVS WERE < 5% FOR MOST LABORATORIES.
OLIGONUCLEOTIDES: THESE WERE SYNTHESIZED WITH NO DG OR CONTAINING 0,1, OR 5 8-0XO-DGS. MOST HPLC METHODS PRODUCED VALUES WITHIN A FACTOR OF 2 OF THE TARGET. GC-MS TENDED TO OVERESTIMATE THE RATIO, AND LC-MS/MS VALUES WERE VERY CLOSE TO TARGET LEVELS. THIS EXERCISE ALSO ENABLED A CHECK OF EFFICIENCY OF HYDROLYSIS OF THE DNA.
CALF THYMUS DNA WITH ADDITIONAL DAMAGE INDUCED BY LIGHT AND RO: THE ABILITY TO DETECT A DOSE-RESPONSE IN THE 4 SAMPLES WAS THE MAIN PERFORMANCE CRITERION. 7 OF 24 LABORATORIES DETECTED THE DOSE RESPONSE OVER THE WHOLE RANGE OF CONCENTRATIONS. ALL BUT ONE LABORATORY DETECTED THE DOSE RESPONSE OVER 3 OF THE 4 SAMPLES.
IN CONCLUSION, CONSIDERABLE PROGRESS WAS MADE DURING THE FIRST YEAR OF ESCODD. MOST LABORATORIES WERE ABLE TO MEASURE REASONABLY ACCURATELY THE SAMPLES OF 8-0XO-DG THAT WERE PROVIDED. ALMOST ALL LABORATORIES DETECTED THE DOSE RESPONSE IN THE SAMPLES OF DNA THAT HAD BEEN TREATED TO INDUCE OXIDATIVE DAMAGE. PROBLEMS OF INDIVIDUAL LABORATORIES WERE IDENTIFIED AND SOLUTIONS SUGGESTED.
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