PLASMA BIOMARKERS IN ALZHEIMERS DISEASE:

A METABONOMIC APPROACH
Luke Whiley1,2, James Heaton1, Simon Lovestone2, Christina Legido-Quigley1
1 Pharmacy Department, School of Biological and Health Sciences, Kings College London, 2 Institute of Psychiatry, Kings College London
luke.whiley@kcl.ac.uk

AD BIOMARKER
DETECTION

MOUSE
BRAIN
HUMAN
PLASMA
LC-QTOF-MS

STATISTICAL DATA TREATMENT

BACKGROUND
With the worldwide incidence of Alzheimers Disease (AD) rapidly increasing[1], the
search for a reliable biomarker is intensifying. Alongside protein[2,3], and genetic[4]
markers, small molecule biomarkers hold the necessary traits and potential to
present themselves as candidates for the role.
Omic sciences, including genomics, proteomics and more relevantly in this
instance, metabonomics utilise highly sensitive analytical methods, including NMR,
GC-MS and LC-MS to identify biomarkers in a non-targeted manner.

OVERVIEW OF SYSTEMS BIOLOGY

Published evidence suggests abnormal concentrations of many lipids and

their metabolites in various tissues including brain[5], cerebrospinal fluid[6],
and plasma[6,7] suggesting evidence for irregular metabolism. This is not
surprising considering that AD is a disease primarily affecting the brain, the
2nd highest lipid containing tissue, only behind adipose tissue.

LITERATURE LIPID ABNORMALITIES IN AD

AIMS

METHOD AND APPROACH

Using this background information, a study was designed to investigate the

potential of using an LC-MS metabonomic approach to determine potential plasma
based AD biomarker candidates. Also investigated was plasma from patients
suffering from mild cognitive impairment (MCI), a condition thought linked to AD.

An initial LC-MS lipidomic method was obtained from the literature[8] and adapted.
The method consisted of a protein precipitation extraction by adding methanol to
plasma (3:1) followed by reversed phase LC analysis (Waters Aquity UPLC) coupled
to a mass spectrometer (Waters Premier QTOF-MS) in ve mode. The elution
method employed 50mM ammonia acetate in both water (A) and acetonitrile (B),
and used a linear gradient (35%-95% B over 9min at 0.6ml/min). The column was a
C18 (Waters Aquity 100mm x 2.1mm x 2.7m) and was kept at 60C.

A preliminary sample set consisting of 12 controls, 10 MCI and 9 AD samples. QCs

were interlaced in the randomised run to ensure method reproducibility.

RESULTS
FEATURE COMPARISONS
A simple Venn feature comparison yielded a large number of molecular features present solely in
each individual group, hence presenting themselves as potential markers for each demographic.
For the creation of the Venn 7 samples were randomly selected from each group to maintain a
similar number of total reproducible features for each group. Only features reproducible in each
of the 7 samples were used.

REPRODUCIBLE FEATURES IDENTIFIED IN ALL QC SAMPLES

0.4
0.2
p(corr)[1]

Features were compared using S-plot analysis, again with a

number of metabolites being highly correlated to a specific
study group. Most promisingly when comparing controls to
MCI a fatty acid showed separation between the groups
(P<0.01), however when comparing controls to AD the
results for the same fatty acid was non-significant
(p=<0.08).

0.6

-0.0
-0.2
-0.4
-0.6
-0.8

-0.3

-0.2

-0.1

-0.0

0.1

0.2

0.3

5.12_282.2496
4.38_253.2160
4.89_255.2292

4.69_279.2312

5.12_281.2431

INTERNAL STANDARD C17:0 FATTY ACID

STANDARD DEVIATION IN QC 13.6%
STANDARD DEVIATION ACROSS ALL SAMPLES 22.9%
14
12

0.4

0.5

0.6

10

0.7

p[1]
SIMCA-P+ 11.5 - 05/10/2010 14:52:22

VIP[5]

FEATURE COMPARISON BETWEEN GROUPS

4.68_559.4665
4.68_715.4922
5.11_649.4342
5.11_661.4907
8.95_116.9294
5.06_263.1267
5.34_308.2673
5.12_282.8362
5.76_409.2076
5.11_646.4302
5.10_790.6243
5.11_564.5106
4.27_205.1909
5.11_290.9113
5.11_563.5055
4.68_280.7272
5.34_307.2622
5.61_291.4775
5.12_789.6256
5.11_648.4394
5.11_647.4334
4.68_281.2405
5.12_644.4349
4.68_285.6137
5.11_287.5841
5.35_309.2710
5.11_885.7148
0.66_632.4942
4.37_101.9356
5.11_883.7116
5.12_613.5264
5.16_99.9277
5.11_662.4893
5.11_614.5299
7.52_747.5657
5.12_284.6959
4.67_774.5615
5.12_291.9267
7.32_393.3747
5.61_590.5199
5.12_283.2580 4.69_280.2355
7.47_710.4815
5.10_828.6165
8.10_223.1619
5.11_400.1540
5.61_383.1917
4.45_116.9293
5.29_113.0426
4.29_796.5432
5.62_115.9222
4.67_775.5686
4.85_258.9642
4.28_208.1492
4.27_280.7228
5.76_310.2822
4.27_275.2014
5.11_884.7120
5.11_287.1360
5.12_166.9835
4.37_235.2064
5.11_347.2667
5.12_586.4919
5.76_309.2762
5.61_290.3972
4.91_356.1699
5.71_397.3491
5.60_333.2834
5.11_696.4439
4.84_801.5847
4.31_265.2197
5.13_585.4766
5.11_177.9047
8.77_862.6752
5.16_116.9298
5.11_381.1789
4.68_282.3837
5.10_529.3146
5.61_288.7243
0.65_447.9799
4.31_279.2317
5.11_176.8957
5.11_601.4598
5.74_381.3139
8.20_777.2425
4.37_257.7400
8.52_894.6575
5.11_382.1838
5.62_591.5246
5.11_117.9311
4.37_255.9889
4.27_277.2154
5.11_295.1923
4.49_415.2596
7.53_146.0976
8.49_908.6625
4.90_255.6037
5.11_290.3819
5.10_587.5049
4.90_610.4514
4.39_341.2298
4.27_577.4313
4.38_411.2112
7.55_395.3909
5.61_589.5185
5.11_285.9892
4.89_256.2374
5.11_137.3121
4.90_806.6675
4.67_288.3585
4.68_438.2300
5.61_384.1935
4.37_353.1454
5.12_283.9838
5.48_557.4756
4.67_848.5928
5.54_334.2875
4.27_281.2123
4.91_355.1623
0.68_884.3846
7.77_198.7103
5.10_671.4143
4.90_237.2225
4.73_244.9765
8.09_219.0235
5.11_379.2057
5.11_383.1861
4.90_607.4709
6.35_437.3693
4.91_247.3003
5.11_113.8588
4.37_259.6730
8.91_660.6265
5.60_287.1128
7.48_748.5692
4.88_776.5791
4.38_254.2215
8.02_304.2357
5.12_565.5189
4.90_805.6648
4.89_533.4486
5.11_281.5627
5.52_555.4458
3.55_249.1878
7.95_116.9307
5.15_159.9779
5.61_284.7627
4.76_268.2374
4.90_330.2549
4.90_246.6179
5.12_402.1497
4.90_608.4730
5.11_615.5223
4.67_437.2343
4.31_259.0693
5.62_212.0756
4.77_267.2315
5.11_136.5873
5.08_265.1836
6.90_101.9366
4.51_243.2222
4.37_335.2253
4.36_414.2980
8.14_283.2632
4.68_285.0581
4.86_259.9902
4.37_254.7195
5.61_285.3789
4.37_255.2222
5.37_577.4166
4.89_717.5152
6.36_438.3783
5.11_788.6203
4.87_778.5939
4.59_581.4131
5.11_288.4508
4.68_281.7451
4.11_228.1937
4.27_278.2070
5.61_284.2797
4.34_260.3072
0.67_503.9549
8.52_895.6587
4.27_233.2279
4.77_780.7049
4.90_238.2274
6.95_859.5313
9.40_212.0740
4.44_445.3339
4.90_329.2468
5.61_283.2668
5.66_148.1132
8.76_937.7168
5.11_289.4160
4.27_278.7387
0.66_557.9833
5.09_140.0597
9.42_136.0445
8.10_815.6045
1.04_162.9061
2.54_174.9937
4.58_297.2350
1.03_116.9316
8.06_851.6921
4.89_850.6013
4.68_286.9705
6.36_387.3228
5.11_439.2438
4.68_279.6576
4.67_657.4444
4.97_308.2371
4.68_560.5271
4.82_262.1303
5.27_331.2643
4.67_716.5021
9.87_281.2464
4.90_261.7297
5.45_295.2642
7.10_871.5418
8.51_893.6641
4.54_594.4806
7.34_837.5590
4.68_345.2493
4.90_534.4593
9.86_212.0734
4.27_279.6771
4.37_531.4291
4.11_230.8607
4.27_182.1302
4.87_777.5851
4.81_261.0515
5.36_830.6241
8.26_860.7888
4.91_669.4003
5.10_829.6183
2.61_197.1534
0.82_137.8951
3.21_116.9303
9.93_146.0977
5.28_255.2330
8.35_783.6314
6.83_353.3432
5.61_286.2249
4.27_282.2185
7.77_466.0479
5.71_158.9757
5.45_296.2678
7.22_861.5514
5.09_396.2594
0.67_414.0118
4.37_605.4248
4.94_263.9877
5.03_588.5046
8.66_655.5193
5.07_293.1085
4.53_478.3496
7.97_94.9817
5.12_786.6211
7.98_809.0790
4.89_851.6003
9.47_214.0714
4.91_340.2143
7.75_534.4946
4.60_363.2183
4.90_594.4197
8.51_778.5789
4.37_253.6819
4.90_257.3712
4.27_281.6412
4.67_361.2416
7.50_643.4954
4.27_435.2159
8.15_814.5965
5.16_178.9762
7.57_827.5637
8.50_845.6708
5.37_141.0057
5.12_139.0356
8.23_801.6565
8.83_101.9374
4.44_772.5458
8.62_935.6890
4.91_609.4519
8.85_646.5930
6.90_365.3419
8.01_582.5109
5.61_289.6714
7.20_786.5311
4.32_179.0540
4.91_414.2289
5.16_98.9407
4.46_446.3362
3.55_226.1910
6.82_339.3283
6.36_337.3118
5.10_285.0505
4.68_777.1802
4.68_375.2250
5.11_142.9364
0.66_800.3773
5.11_395.2541
8.00_826.5685
7.78_208.2636
5.13_965.7246
4.91_333.1982
8.09_519.3855
0.72_517.0281
7.47_932.5772
4.68_287.5526
9.94_214.0726
4.92_596.4925
4.11_227.1986
7.77_194.1804
4.90_248.2062
4.53_996.6300
5.62_141.0058
4.59_163.1148
8.50_869.6279
4.90_413.2255
4.47_213.0787
5.61_286.7142
4.17_477.3749
7.33_382.3789
9.37_158.9763
6.89_834.5252
5.89_557.4579
0.67_936.8979
9.98_121.0310
4.90_130.2282
8.50_972.6532
0.69_924.7234
8.42_925.6445
6.78_354.3448
8.22_700.5345
7.97_753.2470
9.95_154.9466
8.37_859.6420
5.39_450.3018
5.10_111.0296
7.77_204.8680
4.89_249.0332
4.90_311.2490
5.05_415.2180
8.24_702.5213
4.68_159.9785
0.63_517.9961
4.89_321.2478
0.66_624.4529
8.52_808.6041
8.30_777.5621
4.58_593.4794
5.27_332.2692
4.68_286.2728
0.66_436.2949
9.49_116.9301
4.93_307.2684
7.45_116.9300
7.49_687.5433
4.44_773.5535
5.05_475.3115
4.27_469.2827
5.11_528.3093
8.16_754.5831
7.47_127.0040
7.24_886.5521
0.63_878.2993
9.97_256.2374
4.48_354.3011
5.62_368.2381
8.25_138.9741
7.58_843.5515
8.43_784.6274
8.56_331.2690
5.76_297.2800
1.06_397.2042
3.54_225.1856
4.91_765.5838
5.12_140.6666
9.95_158.9766
5.11_890.7502
4.12_309.2113
8.19_778.2046
8.99_148.1144
9.90_284.2678
7.53_659.5276
8.51_868.6353
4.11_230.2051
4.71_291.2115
7.39_101.9343
3.84_354.1536
4.12_231.6893
9.87_134.0460
4.82_136.0479
4.32_258.2049
5.22_356.2689
4.90_535.4666
8.49_663.5175
4.48_242.2178
4.90_267.6984
4.27_127.0785
0.65_798.2906
7.97_824.0441
7.53_719.4794
4.57_502.2828
1.20_369.1722
8.07_245.4067
8.24_920.6198
4.67_177.8640
5.61_289.1172
4.87_505.9849
4.87_513.6825
7.24_811.5325
0.68_420.0174
8.77_922.7154
4.47_241.2171
0.70_495.8560
8.14_712.5148
9.25_96.9626
5.31_146.0989
8.18_835.4952
5.16_213.0805
5.12_365.2362
8.17_502.2893
4.82_584.3853
4.78_583.4051
4.12_239.2030
4.59_149.1340
5.28_463.2952
0.71_132.9733
7.11_835.5355
8.26_752.5601
8.55_158.9753
6.92_857.5159
0.83_134.9176
9.07_154.9473
5.02_367.2511
8.94_648.6005
8.48_760.6239
8.48_160.9770
4.32_413.2952
4.59_428.1654
4.89_593.4773
4.90_764.5831
8.51_909.6669
3.57_313.1729
8.25_756.5810
7.77_915.5970
0.68_635.0099
7.84_821.1921
4.25_425.2745
6.36_338.3162
9.39_141.0084
4.97_306.2662
0.65_587.9085
8.08_761.5636
0.61_471.7560
0.74_464.2483
3.98_251.2019
7.86_446.3534
8.07_725.5259
8.03_100.9352
7.31_158.9761
7.80_796.2796
5.28_623.4805
7.07_701.5174
7.89_774.2479
3.07_223.1683
0.64_477.8794
0.69_556.0572
0.86_513.2711
7.51_84.9926
3.10_158.9768
4.26_340.2072
8.15_802.6079
4.11_229.0826
4.68_168.0466
4.68_122.9876
8.03_142.0174
5.29_156.0122
7.05_913.5807
8.51_809.5991
7.33_832.5702
8.30_923.6428
0.70_366.0052
8.11_99.9284
8.64_154.9480
1.55_231.9206
5.66_331.2691
6.76_212.0779
0.85_512.2712
1.54_229.9219
7.34_381.3714
7.46_445.3568
8.41_858.6546
4.11_229.6725
4.90_332.4509
7.18_331.2748
7.77_671.4605
7.61_452.3315
8.12_704.5659
7.29_712.5011
7.02_141.0033
8.26_729.5544
8.60_212.0762
4.56_214.0561
0.60_134.8873
8.43_798.6448
4.53_507.3706
9.91_94.9808
9.95_759.6258
5.12_149.1356
8.20_922.6392
5.13_168.9788
5.11_194.9129
9.62_283.2634
9.98_309.1727
9.96_445.3490
8.58_115.9232
4.85_779.6220
8.04_901.5988
8.50_918.6653
8.29_757.6084
4.75_269.2550
8.78_923.7071
5.22_355.2651
0.71_363.9898
0.73_412.9442
8.61_281.2481
3.54_397.2266
8.18_762.5265
7.24_858.6071
4.89_266.4103
0.71_978.7088
5.13_366.2231
4.30_388.2532
8.02_537.5063
0.59_594.1504
0.75_276.0069
3.93_271.2284
4.93_473.3024
0.69_956.7331
0.64_509.9350
4.68_611.5049
8.51_748.5471
0.70_750.8022
7.77_898.5763
5.61_265.2536
5.61_351.2543
6.79_550.4355
5.39_244.9770
3.93_326.1876
2.07_496.3053
6.93_100.9348
0.69_459.1369
0.71_988.7211
8.65_146.0984
8.23_816.6256
1.54_227.9237
2.74_450.2553
6.89_833.5211
0.70_278.0433
2.66_297.2448
4.58_173.1345
0.67_707.9652
4.68_779.1420
3.21_201.1833
5.11_889.7285
1.54_307.8785
8.63_618.5840
4.31_506.3309
4.89_256.7694
7.24_885.5464
3.93_252.2066
1.56_116.9304
0.74_401.0691
4.33_412.2766
0.70_680.3399
0.75_161.0067
0.80_136.9525
5.26_158.9766
7.77_816.5637
8.52_754.6117
0.65_806.4087
8.10_873.1558
0.73_918.6693
0.67_378.0018
4.59_427.1580
3.22_277.1893
0.71_445.9955
8.79_682.5792
9.92_873.7016
4.75_217.0089
4.45_511.6765
0.63_625.9054
7.75_794.3689
8.49_941.6968
7.99_391.2393
4.36_397.2026
8.29_464.3149
0.65_656.4815
8.51_939.6797
4.90_331.2775
7.97_750.5591
0.70_420.9442
8.72_645.5287
0.70_364.9800
5.12_141.6671
0.65_804.3967
7.76_758.4911
5.11_114.9101
4.59_219.1380
7.48_931.5936
3.49_100.9339
8.06_148.1150
7.22_910.5547
1.54_388.1508
4.60_283.2031
4.95_305.2396
5.61_365.2747
3.84_373.2769
0.70_598.1077
9.48_134.0454
0.65_864.3755
1.55_390.1445
9.35_445.3541
4.47_457.2351
4.16_475.2618
4.87_514.0606
7.17_713.4891
8.34_116.9299
8.21_817.6411
7.58_842.5919
9.54_255.2333
4.05_441.2026
3.56_441.2517
4.27_339.2010
4.89_258.1995
4.27_359.2189
5.12_401.1833
7.65_415.3623
0.66_649.9931
9.42_154.9520
8.10_230.5320
8.62_934.6956
0.70_281.9913
4.69_436.2555
8.48_445.3548
4.27_450.2641
4.07_576.3307
7.60_112.9865
4.11_337.2052
1.54_387.1505
0.70_340.9358
2.01_392.2903
0.66_611.9736
0.64_886.3328
5.22_256.2367
0.70_480.0155
0.82_528.2527
0.65_543.9420
4.53_574.2447
0.65_730.4447
4.78_800.5777
7.89_305.2492
7.50_431.3549
8.79_527.4440
8.76_632.5948
8.25_843.6576
0.64_354.9516
8.21_142.9926
4.44_221.1310
5.76_311.9483
7.55_396.3925
0.72_423.9565
4.46_461.2645
4.89_549.4372
4.45_580.8984
0.68_579.9993
8.62_656.5271
7.82_769.2035
7.66_119.0358
0.62_207.8850
7.56_690.5030
4.25_158.9758
3.22_200.1743
4.59_229.1969
1.54_389.1514
0.70_422.9379
8.25_769.6116
0.70_378.9554
0.70_362.9810
3.22_199.1695
4.30_141.0040
4.60_201.1650
0.62_231.9623
7.50_378.2772
3.56_398.2302
5.27_413.2337
0.65_449.9784
7.08_671.4551
8.36_800.6031
7.43_888.5777
4.09_621.3046
0.63_91.9801
8.31_776.5593
0.61_326.0933
3.37_309.1729
0.63_742.3416
7.46_749.5637
0.65_784.3930
7.03_148.1143
8.20_156.9860
8.53_874.6604
4.91_251.2015
4.68_305.7222
4.59_329.7083
4.37_352.2858
4.45_581.9276
4.45_582.6593
7.50_645.5321
7.33_675.5321
0.65_699.8802
7.02_884.5373
8.51_953.6988
8.85_647.6041
4.92_595.4927
3.84_226.2243
8.51_802.5797
7.99_825.2050
5.63_281.2501
3.94_305.2153
7.43_656.4239
8.24_709.5018
9.28_717.5724
0.70_280.9780
7.92_822.1663
4.69_561.2514
0.68_161.9654
8.25_174.9674
8.51_835.6343
7.30_912.5660
1.55_309.8822
0.70_339.9603
7.99_419.2561
0.67_528.0104
8.29_564.5312
5.31_595.4980
0.62_100.9697
6.27_311.2950
0.69_438.9245
8.28_832.6029
0.61_193.9825
5.12_138.3029
7.89_831.1890
4.59_195.1424
7.99_195.7062
7.80_850.2221
0.76_561.1154
8.83_621.6022
7.01_116.9315
1.33_136.8930
0.68_218.0346
8.50_926.6591
0.61_407.9869
7.51_474.3655
5.02_496.3339
8.34_726.5520
0.66_744.6828
0.66_742.8373
1.42_99.9271
4.09_149.0075
0.65_822.3839
4.58_187.1507
4.33_203.1802
4.53_973.6342
5.61_366.2833
1.10_398.2037
0.67_609.9785
0.64_640.4750
0.65_676.4968
0.66_93.9699
0.78_135.9570
3.37_310.1767
1.21_370.1773
7.91_719.5467
3.93_325.1854
0.69_338.9371
0.62_89.9761
4.59_121.1029
4.59_175.1406
7.41_848.5471
7.82_846.1505
9.88_213.0778
7.76_869.1704
1.99_393.2951
4.86_461.2691
0.70_494.8538
0.69_496.8697
4.51_503.3114
0.69_591.9078
4.20_598.3144
6.89_645.5028
5.61_665.5165
7.42_702.5044
7.77_817.2418
8.25_829.6455
5.62_606.4091
3.84_619.2861
0.64_258.9746
9.98_107.0405
8.29_787.5796
7.52_800.5485
4.59_181.1229
4.11_209.1914
3.07_267.1474
4.90_339.2220
7.50_432.3563
7.42_442.4079
0.59_453.9494
4.88_489.3968
0.70_512.8152
4.72_606.3874
7.43_655.4221
0.69_198.9826
5.60_465.3037
7.93_786.5693
1.53_146.0989
7.42_799.5336
9.97_156.0127
8.38_159.9776
0.62_171.9851
7.49_213.0782
8.24_898.6280
8.41_255.2327
8.13_936.6042
4.28_283.0610
0.61_425.9540
5.61_442.2642
5.62_605.4070
8.12_705.5696
8.71_98.9412
5.62_116.9297
4.68_778.2012
0.65_790.4330
7.85_146.0980
8.45_799.6630
0.64_816.6265
4.27_159.9766
0.68_200.0246
8.38_884.6199
0.85_250.0433
4.59_249.1871
9.89_255.2359
3.85_272.2320
7.50_306.1782
6.26_312.2973
0.70_444.9750
0.67_493.9784
4.87_511.3237
4.53_580.3521
0.67_589.9437
0.67_597.0158
7.49_616.4704
0.65_722.4429
0.69_102.9935
8.50_771.6405
0.60_136.8888
4.75_141.0075
0.68_808.7603
8.07_821.6204
4.94_176.1472
8.26_882.6110
8.61_921.6972
8.29_951.6790
3.22_297.1526
3.56_311.1691
5.37_515.1861
0.70_540.0626
0.60_569.8733
4.90_573.4515
3.84_620.2902
7.94_726.5417
0.65_764.4406
8.10_776.2490
8.50_971.6537
1.18_313.1196
7.45_100.9352
7.88_765.5228
5.16_115.9226
9.84_116.9306
7.90_780.5530
8.52_779.5770
5.11_143.8849
8.20_803.6304
5.60_178.8894
8.51_849.6459
7.76_870.2820
0.70_219.0210
8.49_913.6383
8.29_929.6330
8.20_953.6240
6.89_366.3462
4.27_377.1407
7.54_475.3711
4.45_503.9794
0.65_690.4296
8.06_730.5637
0.68_108.9483
0.80_248.9201
5.62_99.9265
0.70_118.0038
5.22_804.6168
8.04_158.9760
7.99_196.1019
8.09_226.5067
7.24_887.5584
0.65_952.9455
8.00_303.2339
9.52_325.1875
0.64_332.9237
1.00_332.1954
5.61_441.2590
0.65_601.8541
3.07_96.9612
4.62_284.0938
4.75_526.2952
9.27_127.0032
4.87_148.1137
9.46_831.6588
7.79_844.2042
7.77_198.0624
7.79_916.6035
7.86_925.5994
4.90_264.6355
8.74_960.7457
0.70_338.0490
5.11_341.2555
7.12_445.3569
4.16_474.2636
4.83_503.2939
7.46_529.2862
0.68_614.6681
1.18_423.1775
0.64_196.0050
0.70_387.0082
0.61_125.9545
6.92_858.5232
4.33_303.2339
0.62_303.9643
0.70_380.9656
0.98_448.3046
4.74_527.2944
8.22_753.0777
9.31_99.9274
7.55_780.5219
4.58_135.1186
4.23_156.0133
5.12_182.1295
7.95_856.5587
8.23_867.6404
0.66_894.5813
4.69_258.7156
8.29_937.6588
9.04_331.2683
5.60_352.2584
5.11_440.2456
4.46_508.2122
4.87_513.2138
0.68_552.0298
6.78_576.4475
5.25_573.4865
0.65_608.3502
0.63_110.9116
0.69_104.9539
0.65_766.4225
0.64_806.8068
0.65_860.3890
7.89_213.0799
0.65_874.5246
7.78_969.6133
0.69_319.9586
0.67_486.0036
5.36_517.0744
0.66_553.9506
8.10_562.5073
6.77_717.5326
0.70_305.0101
9.47_759.6182
3.07_266.1528
7.49_523.3311
0.82_679.3381
7.79_770.3081
8.13_784.5907
0.56_192.0076
7.26_911.5603
8.11_950.6161
4.90_337.2420
0.67_403.9978
0.63_402.0075
8.07_849.1580
8.29_930.6276
4.28_293.2098
4.90_323.2228
0.79_510.2539
4.47_544.2821
4.67_303.2299
0.70_141.9682
4.87_509.3704
7.50_761.5809
0.67_205.9551
8.09_918.6148
8.28_928.6303
0.70_280.0436
0.62_323.9542
4.62_392.2749
4.16_407.2231
4.69_487.2138
4.69_559.7760
4.73_605.3738
4.59_677.4561
0.66_896.6095
4.52_382.1856
8.27_448.3187
5.37_525.3016
0.63_579.0086
0.64_642.4840
8.28_846.6179
3.07_265.1463
0.68_150.0288
7.55_741.5275
4.32_257.0457
0.68_396.9078
7.97_782.5582
4.91_146.0967
5.34_148.1151
8.51_932.6660
0.67_944.6796
8.51_954.6969
3.56_312.1690
0.81_310.0828
4.11_385.1956
0.76_438.0410
5.37_464.2834
4.91_520.3434
8.07_731.5688
8.28_890.6694
5.88_311.2933
0.68_145.9950
0.70_388.0241
0.72_436.9516
1.07_223.1178
0.64_360.7067
8.71_529.4644
0.65_534.6177
9.30_94.9814
7.79_845.2120
0.60_878.7862
0.70_888.8358
0.66_251.9971
0.66_559.8857
7.49_592.3960
0.69_668.8211
0.62_664.2745
0.68_728.6019
0.68_316.8982
4.94_212.0754
4.45_506.7510
8.28_780.5892
0.62_302.8475
0.69_306.0180
0.68_446.9347
4.87_507.7615
0.76_80.9661
8.51_940.6854
4.92_474.3127
7.40_775.5329
7.85_163.0755
0.68_962.6572
0.82_272.1301
0.68_349.9051
0.61_477.0032
4.69_479.9764
0.68_577.0312
8.94_634.6085
0.63_789.9829
0.70_141.0144
4.53_477.2800
5.02_495.3278
5.88_312.3064
7.31_674.5287
7.91_718.5393
4.67_799.5724
7.48_156.0142
1.79_171.1404
4.60_177.1606
0.69_478.8558
8.01_536.5030
0.64_648.4470
5.13_137.9093
4.21_148.1144
8.47_156.0159
7.41_159.9769
8.27_823.6325
0.67_852.6979
0.65_872.3155
8.51_875.6590
0.67_886.8465
0.73_970.7283
0.61_381.9656
4.45_512.6470
4.59_678.4647
0.69_734.7655
0.66_734.4792
0.70_223.0123
7.76_802.5558
4.09_502.2927
9.43_760.6233
7.76_803.5636
0.69_318.8991
0.69_398.8937
8.47_279.2329
1.22_371.1832
7.01_99.9260
5.61_101.9338
0.65_828.3729
8.84_620.5995
7.93_727.5513
7.11_735.5550
5.61_552.3631
8.84_683.5968
4.59_327.2293
1.98_391.2819
4.59_328.2341
8.03_820.5933
4.08_503.2949
7.23_810.5313
8.24_332.2681
7.35_367.3582
7.51_103.0421
0.65_846.3415
7.89_910.5709
8.62_920.6705
0.68_278.8453
5.48_357.2799
4.45_434.2361
0.66_616.7259
4.09_622.3058
0.65_626.4881
7.36_822.5245
0.64_898.7980
8.11_683.5189
0.70_126.9981
4.54_196.0381
3.84_595.2877
7.33_836.5434
0.76_385.2004
7.83_757.5297
7.47_148.1144
4.64_153.0138
8.67_644.6017
8.68_102.9497
8.10_156.0130
0.65_844.3469
5.37_242.0794
0.68_328.1357
5.11_425.2356
4.87_462.2755
5.38_516.2201
7.51_591.3907
0.68_844.8679
4.90_338.2424
4.59_534.2556
4.68_561.7741
0.76_576.0204
0.70_118.9744
0.63_256.9779
0.67_198.0247
0.64_226.9422
4.45_564.3320
4.20_597.3029
8.27_840.6041
8.29_848.6331
0.60_203.9809
7.90_756.5565
3.89_158.9773
0.68_550.0808
0.69_260.0023
0.70_618.8341
0.67_652.6167
7.66_755.5429
7.76_767.2328
4.14_119.0338
5.02_118.9865
4.54_197.0406
7.59_866.5855
8.05_974.6194
7.86_430.3760
5.37_515.7390
0.65_563.7526
3.84_643.2875
8.04_854.2146
4.67_232.2260
8.51_899.6548
7.99_805.5750
4.45_565.3351
4.81_582.3739
9.03_760.6215
0.61_166.0400
5.61_213.0799
8.15_279.2324
0.65_382.7062
0.63_507.9843
0.70_570.7827
0.68_602.8758
0.67_636.6240
7.19_747.5066
0.68_866.5009
4.59_230.2008
3.93_276.1799
5.61_287.9756
5.27_369.1806
4.62_471.3089
4.45_510.9294
0.65_714.4607
8.14_732.5773
4.50_176.1479
4.88_322.2477
4.45_578.3408
4.67_304.2371
8.29_772.5826
8.28_847.6188
1.00_368.1633
0.64_288.8411
4.28_302.2217
7.61_713.5595
8.02_753.5635
0.63_138.9700
4.69_180.0192
7.81_255.2333
0.64_358.7350
5.60_466.3096
4.77_494.3210
0.67_504.8194
3.55_569.2699
0.66_786.5035
8.00_886.5811
0.64_910.6108
8.29_915.6116
6.85_340.3343
0.72_385.9992
0.74_611.0065
6.27_629.4596
7.80_697.4930
4.55_500.2754
4.45_579.3490
8.68_100.9343
5.40_212.0751
8.22_896.6316
5.26_269.6814
8.77_936.6996
9.59_331.2687
0.69_632.9752
8.07_717.5421
7.60_136.0440
7.34_785.5830
0.67_802.6163
4.59_191.1811
4.68_306.2063
4.88_351.2234
6.72_655.4243
0.68_736.6829
7.76_226.8973
4.52_476.2729
1.00_367.1564
0.61_284.8992
1.05_179.0251
4.69_224.0692
0.69_250.9418
0.63_348.0352
8.32_727.5485
7.51_305.1818
0.64_390.9068
3.93_275.2047
5.32_179.9951
0.65_858.3243
4.59_309.2236
0.67_654.6149
0.65_768.4235
8.10_913.6038
8.28_946.5797
4.11_362.2362
4.29_378.2155
0.67_434.8952
4.45_513.5218
8.08_830.5826
8.21_855.6309
8.04_716.5191
0.93_210.0208
0.65_596.5524
7.62_728.5271
0.71_162.9911
8.10_799.2262
4.53_972.6159
5.20_275.4004
0.59_390.0456
4.87_417.2431
4.45_460.2583
3.83_596.2924
8.20_645.5509
0.70_686.7270
8.14_737.5403
8.00_818.5766
0.67_232.9127
9.83_833.6580
7.88_788.5443
7.90_445.3534
4.30_280.2254
0.75_152.0643
8.28_834.6018
8.02_850.6181
8.28_897.6439
0.71_112.0473
7.50_128.9817
4.54_331.2684
3.93_454.2871
0.65_464.7161
8.71_530.4603
0.70_538.9044
7.97_751.1541
4.54_140.0120
0.65_830.3682
7.80_220.1520
4.69_905.5695
8.21_970.5843
0.65_289.9589
4.59_330.4384
4.67_629.3776
8.13_800.1828
3.93_363.1951
0.57_572.0425
8.53_680.5358
4.86_581.3660
0.69_402.9707
4.45_519.2770
0.68_502.8117
4.86_521.2891
4.59_328.6548
0.67_536.7158
4.65_301.1775
0.69_552.8957
8.19_755.5894
0.65_670.4731
0.68_950.6867
0.71_954.6769
0.76_605.9198
4.44_505.3315
0.69_116.9719
0.89_246.9363
5.37_510.4218
4.27_301.2175
8.09_831.5927
0.66_460.7651
0.58_265.9956
0.65_418.8301
7.86_429.3744
4.69_486.2448
4.47_627.3532
8.51_753.6083
8.09_785.5400
8.20_146.0981
0.65_842.3503
0.65_370.0056
4.45_459.2502
4.16_481.3103
7.51_646.5340
0.65_756.4463
0.65_782.4058
0.66_820.8107
0.64_818.6792
4.45_952.5878
0.64_427.9647
0.84_567.3192
0.68_678.7435
7.11_720.5398
7.18_916.6044
1.02_344.0745
0.70_647.9247
0.95_244.9151
8.00_778.5749
0.64_324.8035
7.98_698.5094
0.65_476.6721
0.71_376.0883
9.34_100.9348
4.87_510.1950
4.47_588.3309
7.99_199.9454
0.81_220.9898
0.68_444.0865
0.67_674.8517
0.65_754.4382
4.59_167.1085
8.28_916.6304
7.78_970.6178
5.61_468.3157
4.69_490.5118
0.66_716.8178
9.93_137.0344
0.67_926.8711
7.76_742.5236
1.07_194.0168
1.01_146.0538
1.53_212.0751
8.28_773.5874
0.88_158.0379
0.56_220.0157
7.82_714.4954
7.85_818.2072
0.67_296.9791
8.10_217.0108
7.00_883.5371
0.65_520.6772
0.70_134.0399
7.46_391.2398
0.68_456.8217
8.12_850.0319
0.67_360.0732
5.74_493.3432
4.87_510.6944
5.22_520.3429
8.04_807.5914
5.89_313.3053
4.06_442.2074
0.65_740.4335
8.68_886.6238
0.65_922.8347
0.73_693.8689
0.77_108.0440
9.83_832.6655
0.68_234.8873
4.55_501.2817
0.65_154.8878
7.99_806.5831
8.01_724.5216
7.22_909.5513
7.52_377.2738
0.65_384.7213
0.65_610.5837
8.32_136.0446
2.43_237.1505
0.65_812.3983
6.73_634.4478
0.63_668.4212
8.41_860.5684
0.68_732.8644
8.09_770.5539
4.86_580.3583
4.95_478.2913
0.71_306.9512
4.67_798.5696
0.67_148.0019
0.65_578.5970
8.43_101.9351
7.50_801.5569
8.42_156.9917
6.90_881.5247
0.96_247.9215
0.67_608.8632
0.70_119.9691
8.26_841.6056
8.09_926.6052
0.71_458.0948
7.18_787.5175
0.65_810.4524
4.69_487.9444
5.09_606.3845
0.64_140.0274
7.78_917.6003
0.63_700.3341
0.85_170.0702
0.85_169.0443
0.63_286.8159
7.77_826.5615
7.55_740.5234
0.65_96.0855
5.38_216.9991
7.92_800.5819
8.11_331.2691
0.65_594.5500
0.65_500.7511
4.96_574.4267
0.64_590.6834
0.69_252.9575
0.68_376.8621
0.61_462.0753
7.97_491.3547
2.57_500.9259
9.92_141.0069
0.67_470.0078
0.66_522.6718
7.78_820.1630
4.59_193.1222
7.85_847.0149
8.28_904.6297
0.68_317.9242
0.62_475.9639
4.69_558.7892
4.47_589.3480
7.10_588.4457
0.65_692.4274
7.73_868.2113
9.91_265.1513
4.63_127.0878
5.25_279.2348
0.67_529.9329
4.85_506.3281
8.09_854.5875
8.09_845.6003
8.16_767.5386
0.68_224.0233
0.66_374.8993
0.62_379.9632
0.65_462.7382
0.67_384.2050
2.53_212.0739
4.47_543.2768
9.37_146.0982
7.78_205.7597
7.99_862.6060
5.50_358.2905
8.02_473.4005
7.64_508.4669
4.46_512.1357
4.87_511.7515
7.47_525.3224
0.62_525.9515
0.61_511.9061
0.61_583.8420
4.87_508.3999
0.66_96.9366
0.67_292.8611
4.69_483.3889
0.69_217.0271
8.31_751.5477
0.63_212.8976
0.75_308.9629
9.28_718.5795
4.40_212.0750
0.65_592.5548
1.01_211.0189
9.98_874.6957
0.59_263.9877
7.92_801.5943
4.53_971.6124
4.59_147.1186
0.66_814.4756
0.65_850.6398
3.55_233.1558
4.69_557.7896
0.63_638.2279
7.85_768.2392
0.64_848.5817
8.19_852.1901
0.64_908.6235
8.50_927.6880
7.91_796.8999
3.85_355.1544
0.70_519.9097
0.68_902.8318
0.76_187.0056
0.63_143.0033
7.77_817.5729
8.00_819.5824
4.46_529.3165
8.00_804.5693
0.80_195.0957
4.47_602.3462
0.57_262.9978
4.94_479.2962
0.71_320.9965
7.89_741.5407
0.66_95.6925
7.50_141.0018
4.49_99.9281
7.89_789.5735
4.67_599.3227
8.27_447.2951
4.41_509.3956
0.65_440.6975
2.05_370.8909
7.98_436.2816
7.48_450.3634
7.68_704.5169
0.66_772.4145
4.24_146.0969
4.90_394.3330
4.88_297.0524
4.44_504.3130
0.77_107.0450
8.31_750.5390
7.69_791.5406
7.91_764.5208
0.72_480.9781
4.80_158.9755
4.86_567.3502
7.13_663.4766
0.65_840.3511
8.10_874.6944
0.66_638.6481
0.65_696.4790
3.84_319.2188
4.16_481.9260
4.69_488.6499
0.67_498.0943
0.65_565.8594
0.65_708.4608
0.58_763.9712
0.66_932.6861
5.45_653.4071
5.27_271.3641
5.18_100.9345
8.17_743.5563
4.44_100.9346
0.67_174.9510
4.55_525.2821
6.77_158.9761
5.21_349.2562
4.45_433.2353
0.67_624.9178
7.40_774.5346
0.71_672.8749
6.90_882.5221
0.68_238.0235
4.59_133.1029
4.59_161.1322
0.62_892.5650
8.10_927.6135
1.03_427.1999
7.46_530.2877
6.27_548.4132
0.64_862.3216
0.68_976.8753
8.09_771.5651
4.55_524.2757
0.73_261.0058
7.96_838.5896
0.65_518.6589
7.45_99.9283
7.99_699.5039
0.63_348.9113
7.61_787.5967
0.67_404.8297
5.60_467.3068
4.45_508.7019
8.19_701.5185
9.27_832.6615
8.22_873.6324
7.53_889.5679
3.86_295.1812
0.66_486.9205
4.16_497.3041
4.47_531.7816
4.48_566.3357
0.65_698.8762
8.51_99.9282
0.61_784.8896
5.04_405.2441
0.72_421.9799
0.60_451.9562
7.77_480.3152
0.80_613.1904
5.38_94.9838
0.69_109.7047
7.07_136.0446
7.99_159.9805
3.92_411.2453
0.67_634.0577
0.69_705.7521
0.68_150.9493
7.65_717.5278
7.83_827.5727
4.86_566.3466
4.47_626.3475
0.71_176.9399
2.07_368.9568
2.55_499.9316
0.59_178.0087
8.01_281.2487
0.64_400.7077
0.65_580.5765
0.63_600.8661
4.54_995.6115
0.68_454.9179
4.45_520.2771
8.09_233.6800
4.69_497.2792
0.68_749.9872
0.71_171.0278
4.46_528.3033
7.99_745.5459
0.68_551.0004
4.68_975.6457
0.99_511.2927
8.09_758.5633
0.66_544.8965
0.65_558.6712
0.70_554.8307
0.67_514.8885
0.65_758.7300
7.48_617.4821
8.27_685.5005
7.78_708.4971
0.72_120.2729
0.65_900.3083
5.12_384.1845
7.67_696.4777
0.60_88.0020
7.76_743.5337
8.15_742.5373
4.47_553.3159
4.47_603.3490
0.98_144.0469
5.03_556.3410
8.01_779.5804
0.80_121.0199
6.77_807.5040
8.30_883.6191
7.47_446.3580
7.69_754.5429
0.65_838.6065
8.23_213.0774
4.59_315.2318
0.66_498.6646
4.87_522.2971
0.65_770.4171
6.77_136.0437
8.10_798.3066
8.09_844.5915
8.28_833.6082
0.57_264.9994
1.00_331.1911
7.61_815.5696
1.01_465.2642
5.48_535.3601
0.57_506.0593
7.34_711.5454
9.92_99.9290
7.40_798.5269
0.65_824.3760
7.63_179.0554
3.92_327.1844
0.64_428.9212
7.45_566.3543
8.11_851.1520
5.26_353.2387
0.68_531.9872
8.14_535.5060
5.37_570.3676
0.66_92.9336
8.09_794.5601
0.65_210.8771
3.85_293.1787
8.23_212.0754
7.77_279.2343
2.05_366.9008
0.65_458.6602
8.23_128.9810
0.68_274.9204
0.65_516.6354
4.68_976.6471
0.67_634.7157
7.95_734.5023
8.20_938.6140
6.77_732.5373
4.59_107.0872
3.85_367.2165
6.88_646.4898
0.67_714.9813
0.83_308.0965
5.12_364.2618
4.90_399.2132
5.37_513.9868
5.37_585.9095
4.68_120.5720
4.87_507.3295
0.57_242.0002
8.10_795.5710
0.64_208.8883
1.48_164.0046
7.58_814.5593
7.86_715.5454
0.66_484.9685
8.22_94.9821
0.57_221.0297
4.69_485.5616
8.21_188.9744
5.19_101.9348
0.68_362.0589
4.42_510.2752
0.68_556.7483
0.65_650.4852
1.49_165.0093
8.11_872.2303
0.81_583.2603
0.66_738.4627
6.75_141.0086
4.09_541.2670
0.72_561.8785
2.53_158.9680
7.05_213.0792
5.37_419.2628
0.65_533.9412
4.16_479.2968
1.50_498.2934
4.84_502.2915
2.04_475.3283
0.62_298.9632
0.78_383.1724
0.71_99.9699
8.27_822.6081
0.65_204.9810
7.46_94.9828
7.27_762.5330
0.85_242.0000
0.69_628.7497
4.49_612.3514
0.57_741.9774
7.94_750.2445
7.18_915.6000
7.25_578.4600
8.50_653.4904
0.65_710.8635
0.57_783.9583
0.64_974.5681
0.66_468.9960
8.36_872.6628
7.41_688.5223
0.64_321.9787
8.23_378.2713
7.97_877.6161
5.18_438.2864
0.85_159.0566
7.47_429.3528
5.71_119.0018
0.65_716.1509
4.59_189.1647
4.97_468.3345
0.70_966.6100
4.46_552.3069
8.26_774.5420
1.33_391.2840
7.48_524.3349
0.67_121.9925
8.06_155.0022
7.98_855.5489
0.56_331.9603
7.58_752.5288
4.69_231.2122
0.70_690.9497
7.77_212.6353
4.88_524.3343
8.10_226.1360
3.93_455.2812
4.69_636.3582
0.67_691.9963
7.99_744.5377
0.63_268.8423
0.62_270.8666
0.64_326.8010
7.99_723.5139
7.81_739.5085
8.27_775.5460
4.63_205.1763
8.55_127.0031
7.18_737.5054
0.63_586.8643
0.65_750.4546
8.06_900.5966
7.51_476.3614
5.37_512.6990
0.66_557.0278
0.63_792.6147
0.72_591.0283
7.58_753.5292
7.46_331.2678
0.76_192.9861
7.82_890.5897
0.56_169.9765
0.66_560.6911
0.61_584.8792
5.60_551.3553
9.86_148.1143
0.62_155.9882
1.23_254.1109
7.77_205.2884
4.16_538.3253
0.67_612.5946
4.67_628.3657
0.58_678.1570
7.98_813.5869
0.68_830.7928
0.68_772.7666
3.93_381.1987
0.61_483.9085
5.12_491.3275
7.65_731.4886
5.11_139.6909
7.83_479.3886
0.68_928.8954
0.58_976.1309
0.65_94.9302
7.65_716.5199
8.15_766.5279
7.81_738.5028
7.45_564.3429
0.62_236.9562
0.65_576.5848
1.54_507.2274
8.07_760.5575
8.14_736.5216
8.27_786.6102
4.17_389.2114
4.69_410.2412
0.63_598.7566
0.65_826.3715
5.11_350.2504
3.07_424.2489
0.61_535.0837
4.49_567.2920
5.61_655.4240
6.76_659.5064
0.66_746.4601
4.87_514.7841
4.74_545.2944
8.27_661.4887
5.11_294.0075
5.37_509.3619
7.33_673.5276
4.16_480.3019
0.65_442.6812
0.64_684.9496
7.72_689.5091
0.70_748.8849
0.65_103.9984
0.60_840.8805
4.68_904.5651
0.67_600.1422
0.65_706.4508
0.79_110.0602
0.64_788.4004
0.65_808.4080
5.37_516.7213
0.74_83.0159
4.16_478.2888
7.76_767.5396
0.68_89.0252
0.65_152.8891
1.05_464.3028
7.85_327.2335
0.63_542.9029
7.48_451.3590
7.13_662.4945
8.03_839.6064
9.43_148.1122
8.24_559.4090
8.29_866.6229
0.65_862.8571
0.58_686.1434
0.65_794.6148
0.60_826.8758
4.68_347.2240
0.67_694.5861
0.58_720.0360
0.65_752.4435
7.91_301.2167
1.54_508.2332
6.78_577.4533
7.23_614.4518
0.65_704.7468
0.60_623.8000
7.52_714.5066
2.08_168.9888
0.67_322.9051
0.66_574.6347
3.85_294.1835
0.70_262.0178
0.66_568.9062
0.67_764.8542
7.58_768.4560
7.85_783.5288
5.28_351.2574
4.17_493.2658
8.16_875.1586
5.62_564.3429
7.18_732.5244
8.15_962.6020
7.48_477.3708
5.49_608.3934
7.76_766.5305
8.09_869.6067
0.69_148.9924
0.63_182.9996
4.92_568.3623
8.29_914.6127
0.60_748.1479
4.16_196.0367
8.24_906.6018
7.46_138.9755
5.27_562.4931
0.67_712.5973
7.75_762.5048
4.74_530.3275
4.69_482.7686
7.84_740.5217
7.69_776.5464
0.77_123.0470
8.16_880.6072
7.91_751.5488
8.26_728.5512
4.74_532.3339
7.63_729.5305
4.76_454.2857
5.37_377.2683
8.12_874.1208
0.66_870.8604
0.58_900.8932
0.66_920.8168
5.22_273.6398
0.61_667.7877
8.15_894.6203
5.09_532.3433
4.73_557.3555
8.23_84.9922
7.61_737.4960
0.65_226.0340
0.65_562.6666
7.89_832.6349
0.64_528.9248
0.67_406.7828
6.78_808.5135
0.65_411.9871
4.87_512.4949
0.66_880.7719
7.75_841.5748
4.69_495.2769
7.41_738.5057
0.62_128.0056
4.68_485.0004
4.52_321.2116
7.81_891.5991
0.60_105.9796
7.99_812.5784
1.51_213.0784
0.71_294.0467
0.66_692.8867
0.69_397.9137
5.27_427.2436
7.41_699.5438
4.69_482.3158
8.03_748.5047
0.66_129.0306
7.95_852.5938
0.66_172.9565
0.66_222.0082
0.62_416.0108
0.61_370.9739
5.59_492.3454
0.67_516.0158
8.20_939.6146
5.12_363.2603
7.35_368.3620
0.64_732.1122
0.63_834.6455
8.30_487.2973
0.61_641.7938
7.74_840.5664
7.74_763.5103
0.58_230.0238
5.26_272.2369
7.98_752.5430
7.45_565.3492
0.65_334.0300
0.63_640.8664
0.67_630.7788
0.64_724.4377
7.46_734.4883
7.41_849.5378
4.11_768.5183
4.74_390.2666
2.56_498.9264
7.99_722.5072
4.74_531.3273
8.03_879.5947
0.70_113.0034
4.73_604.3612
0.67_180.0464
7.11_719.5340
0.77_81.9575
6.73_336.3251
7.58_779.4891
0.60_842.8896
4.69_486.7430
4.74_590.3434
8.09_237.9721
0.68_906.6700
0.69_914.6806
0.68_573.9906
8.50_790.5902
7.46_528.2924
7.58_769.8077
4.69_481.3274
6.77_657.4984
7.87_829.5777
8.04_749.5118
0.61_344.9236
4.69_409.2361
0.62_426.9709
5.13_493.3520
8.27_781.5972
0.72_190.0414
8.30_486.2960
0.66_470.9540
8.00_759.5421
0.68_828.9181
4.45_518.3307
0.67_537.0153
4.69_489.0976
0.68_655.0036
4.69_555.3486
7.86_843.5852
7.61_736.4928
4.68_555.7812
0.57_684.0309
3.85_225.2231
5.26_561.4791
8.99_156.0133
0.66_822.7736
7.46_255.2314
8.17_480.3091
4.92_299.2656
4.67_423.2158
5.62_100.9344
0.63_182.0114
0.60_167.9966
1.55_141.0025
0.61_230.9830
0.61_102.0569
4.69_391.2281
7.48_507.3323
4.69_480.3103
4.69_554.3451
8.08_868.5959
0.62_314.9773
7.11_659.5091
8.30_871.6431
7.21_686.5310
5.37_583.3799
0.58_242.9990
8.12_141.0070
6.77_658.5005
9.36_761.6202
0.59_368.9784
0.57_353.9973
7.11_661.5211
4.74_225.0582
0.61_418.0209
0.64_510.8909
4.97_538.3548
0.67_738.8214
7.74_795.2294
4.70_540.3334
9.83_831.6613
8.05_893.6095
0.62_294.9370
0.60_330.9411
7.22_760.5657
7.89_848.6028
5.37_512.2129
4.10_601.3380
7.21_700.4975
0.63_858.7526
0.76_482.1051
0.58_991.0928
8.05_892.6023
5.03_494.3236
4.97_464.3122
8.04_878.5917
5.13_492.3464
4.17_498.2644
0.57_286.0064
4.97_465.3183
0.57_240.0190
7.29_689.4981
0.56_244.0434
7.77_328.2334
7.92_377.2725
0.64_615.1201
4.17_483.2747
5.27_273.0029
0.68_656.8727
8.16_976.6266
5.61_656.4265
7.21_759.5650
0.71_116.0711
5.37_511.3757
7.89_824.5788
0.59_316.0186
1.27_408.2846
4.52_299.2582
4.17_499.2672
0.68_236.0003
0.59_288.0171
0.71_754.8108
0.69_234.0061
5.27_337.2383
7.50_642.4795
0.62_607.8452
8.24_112.9861
0.60_342.9533
7.97_747.5034
8.09_127.0020
7.44_715.5061
0.67_125.0210
4.97_467.3332
7.95_853.5988
7.99_773.4738
7.57_691.5152
0.71_770.7981
7.07_159.9779
4.53_300.2627
0.65_796.6089
8.04_214.0741
8.21_870.6154
0.63_123.9956
7.41_788.5373
0.57_284.0240
0.82_212.0544
0.58_302.0178
4.77_453.2822
0.63_526.9341
4.52_492.3095
7.18_687.5357
1.55_148.1140
4.09_600.3326
7.18_736.5026
0.63_564.7645
7.97_836.5915
0.64_572.8561
0.65_824.8156
0.62_521.0192
5.24_792.6129
1.55_236.1071
5.13_490.3323
0.65_688.8652
8.03_848.1391
5.24_274.3938
0.72_639.9650
1.27_407.2772
5.37_568.3623
0.56_682.0380
0.68_176.0142
7.21_746.5584
4.64_206.1770
0.59_431.9533
0.63_646.8809
0.63_854.7721
5.62_495.3626
7.87_768.5448
4.77_452.2763
0.67_131.0489
0.71_179.0323
1.89_100.9365
7.99_876.6147
4.68_120.0161
4.10_297.2435
0.61_672.2577
0.62_798.8235
0.67_499.0346
0.57_935.1745
4.48_532.7641
7.99_673.4831
7.77_484.8971
0.57_311.0043
0.61_185.0125
8.31_797.6081
0.73_164.0686
8.13_791.5438
7.63_212.0812
8.15_881.6117
8.21_377.2731
5.62_494.3609
0.76_575.0045
6.77_731.5357
0.63_532.7647
1.46_450.3133
8.20_952.6235
5.37_513.3264
0.64_790.7775
0.62_776.6997
8.07_924.5930
5.26_270.2855
7.86_842.5793
4.69_541.3370
4.69_484.1581
8.14_202.9895
0.63_506.8484
0.57_662.0716
4.31_468.3033
0.71_85.0315
7.81_781.5176
0.65_756.7715
7.99_775.4227
7.46_430.3569
2.56_315.2505
8.21_796.5777
8.21_856.6028
7.88_828.5731
7.70_850.5592
7.11_734.5543
0.65_530.8601
0.62_409.9931
0.64_804.9176
0.63_622.8104
0.63_774.7182
8.03_474.3969
0.62_882.8203
1.78_398.9342
4.09_527.2968
7.88_793.5512
0.61_228.9741
4.16_496.3054
8.03_810.5795
8.10_809.5466
5.37_582.3719
0.68_146.9895
0.56_214.0009
8.14_790.5427
0.56_312.0190
3.93_453.2829
7.11_660.5164
7.99_772.5052
1.78_399.9409
7.99_700.4967
7.18_761.5714
7.39_750.5052
5.37_224.0697
7.76_219.6147
7.79_771.1806
5.47_536.3657
0.63_720.8034
0.62_997.0107
5.61_427.2484
4.97_466.3279
7.40_789.5404
1.33_134.8960
5.54_391.1708
0.62_540.9048
0.61_452.9498
4.69_542.3411
8.24_864.6015
0.57_705.0300
0.67_868.7347
4.53_322.2114
0.72_559.0288
7.69_790.5335
0.73_130.0857
7.87_769.5534
7.11_733.5493
8.15_895.6255
0.69_174.0253
7.21_745.5501
0.60_258.0217
0.63_606.2952
0.62_206.9948
0.58_355.9896
0.64_780.7252
5.50_197.0700
5.37_524.3121
0.63_872.7793
4.69_977.6459
0.58_282.9810
0.60_266.9228
7.72_864.5670
7.72_865.5761
4.32_467.2971
7.73_777.5493
7.40_739.5091
7.40_813.5502
0.59_704.0387
0.60_202.0193
5.37_569.3673
0.65_788.9552
0.65_700.8599
0.67_752.8535
0.65_846.8709
8.22_635.4856
0.67_371.9546
4.09_526.2909
3.93_452.2753
7.97_746.5193
7.41_862.5578
0.65_246.0068
7.35_403.3058
0.66_359.0911
4.67_600.3289
0.57_952.1435
5.38_508.3452
4.69_556.3908
0.66_188.0155
0.67_566.9753
4.53_493.3193
8.11_808.5813
0.64_726.8777
5.37_127.0022
5.62_505.3294
0.59_737.0540
5.24_793.6129
4.69_562.3282
1.50_448.3053
7.41_812.5419
5.37_584.3783
0.61_682.9706
0.60_395.9955
0.65_492.0028
0.57_958.1657
0.57_300.9993
7.85_792.5503
0.67_190.9607
0.67_812.9627
7.38_751.5168
0.63_768.8908
0.60_489.9113
7.92_822.9886
7.21_685.5238
4.32_466.2921
0.62_145.0528
7.85_867.5912
0.63_474.8280
5.37_523.3082
0.59_482.8978
0.59_373.9997
2.09_218.9900
0.77_400.0650
5.37_514.7404
0.66_392.0388
0.61_215.0255
0.59_424.9544
0.57_272.0130
0.59_616.0589
0.64_766.9148
7.78_720.4974
0.60_490.9300
0.69_492.9453
0.58_367.9927
0.69_706.9222
0.61_782.9205
0.63_864.8237
5.27_251.2379
0.59_239.0270
0.60_662.9363
0.65_508.8984
0.63_666.9499
0.62_710.2797
0.63_856.5512
0.62_585.9667
0.57_996.0908
0.61_432.9819
0.74_180.9982
0.62_604.8527
0.61_718.9303
0.60_345.9923
0.64_762.8208
0.58_563.1085
7.85_866.5849
8.30_857.6297
0.64_588.8281
4.68_496.2862
0.64_836.7239
0.60_726.1580
0.66_876.5770
0.57_995.1057
5.48_534.3567
7.58_825.5497
0.58_254.0037
0.58_630.1174
0.60_467.9396
4.69_558.3092
0.66_800.7582
5.50_392.1511
7.76_732.5124
7.58_765.5254
0.57_255.0307
0.58_696.9644
7.78_721.4987
0.57_957.1490
0.57_185.9787
0.58_628.1374
7.88_825.5840
1.50_449.3110
7.58_824.5443
5.23_462.2983
5.21_437.2794
0.62_582.9129
0.57_210.0354
0.58_658.0608
0.57_990.1090
0.58_466.9273
1.79_400.9344
5.08_436.2836
0.59_346.9248
7.70_851.5653
8.01_811.5696
0.64_473.9780
0.58_959.1400
0.66_388.9869
0.57_951.1552
0.60_248.1136
0.71_87.0105
0.64_832.5608
7.58_764.5184
0.58_212.0358
0.60_622.1846
0.64_670.9064
7.33_758.5370
0.73_631.9328
7.58_838.5553
0.60_621.0287
0.59_524.8890
7.41_863.5639
0.59_688.0698
0.65_448.8953
0.57_975.1305
0.62_201.0352
0.61_203.0667
0.66_410.9820
0.61_548.8847
0.59_393.9987
0.66_581.0496
5.45_480.3079
0.57_292.0806
0.61_114.0560
0.57_352.9952
0.58_270.0274
0.60_450.9415
0.56_698.0322
0.60_463.0412
0.67_567.9918
5.45_481.3125
0.73_165.0578
0.66_730.9880
0.65_154.0460
0.63_651.0430
0.63_760.7096
0.57_998.0767
0.58_350.9972
0.65_648.9973
0.59_115.0107
0.62_778.7062
0.64_890.6285
0.61_357.0050
0.60_272.9398
0.58_408.9703
7.60_839.5635
0.62_372.9744
0.61_416.9625
0.57_183.9964
0.63_546.8272
0.57_974.1352
5.45_482.3193
0.57_268.0412
0.57_629.1105
5.50_393.1467
0.57_300.0041
0.57_227.9886
0.62_225.0006
0.60_676.9803
5.45_196.0378
8.10_912.6016
0.60_542.0397
0.57_659.0481
0.64_167.0051
0.61_414.9665
0.56_660.0700
5.27_269.2489
0.65_189.0452
0.61_430.9372
0.62_472.9202
0.56_86.0265
0.57_674.0288
0.60_660.9838
0.69_98.9900
0.57_256.0198
0.58_352.0097
0.58_646.1049
0.59_157.0806
0.57_330.0189
0.58_366.9880
0.60_665.0149
0.57_274.0778
0.59_296.0354
0.58_298.0077
0.62_606.8153
0.59_341.9977
0.57_679.0384
0.60_143.9911
4.10_437.2055
0.58_329.0089
0.57_159.1055
0.57_247.0925
0.60_132.0228
0.60_627.0202
0.57_197.0463
0.58_158.0915
3.93_467.2401
0.59_291.0214
0.57_241.0528
0.56_160.0985
0.60_358.0211
0.57_973.1284
0.62_488.8824
0.57_313.0437
0.57_314.0450
0.58_680.9963
0.59_645.0181
0.59_216.0360
0.58_687.0563
0.59_702.9756
0.59_740.9639
0.65_722.8259
0.58_643.0589
0.63_746.9750
5.74_464.3141
0.60_724.9701
0.62_708.9820
0.57_644.0889
0.59_343.9946
0.60_392.9929
0.63_434.0112
0.57_989.1079
0.60_394.9873
0.61_336.9986
0.61_277.0076
0.58_666.0997
0.59_335.02240.58_336.0428

0.8

8
6
4

4.46_552.3

CONCLUSIONS

4.67_303.2

0
4.44_504.3

P<0.01

Var ID (Primary)
SIMCA-P+ 11.5 - 05/10/2010 15:01:30

This preliminary study demonstrates the successful implementation of a non-targeted LC-MS metabonomics approach in AD biomarker discovery. A number of molecules have been
identified as candidate markers, which now require confirmation using an external standard approach. Furthermore through this study the clear potential of a metabonomic approach in
the discovery of AD biomarkers has been demonstrated.

OTHER PROJECTS
AD small molecule biomarker candidate identification in
human plasma
AD small molecule biomarker candidate identification in
mouse brain
Targeted AD biomarker discovery LC-MS
Optimisation of LC-QTOF-MS lipidomics (see right)
Systems biology modelling

POSITIVE
MODE

NEGATIVE
MODE

IN TEXT REFERENCES - [1] C. Ferri et al, Lancet. 366, 2112-2117 (2005). [2] A. Hye et al, Brain. 129, 3042-3050 (2006). [3] M. Thambisetty et al, Arch. Gen. Psychiatry. 67, 739-748 (2010). [4] C. Reitz, R. Mayeux, Ann. N Y. Acad. Sci. 1180, 75-96 (2009).
Neurochem. Int. 48, 679-686 (2006). [6] D. Pratico et al, Ann. Neurol. 48, 809-812 (2000). [7] Y. Yoshida et al, Neurobiol. Aging. 30, 174-185 (2009). [8] P. Rainville et al, J. Proteome. Res. 6, 552-558 (2007).

[5] W.

Vlkel et al,

Acknowledgements Many thanks to the National Institute for Health Research (NIHR) Biomedical Research Centre for Mental Health based at the Institute of Psychiatry for the funding of the PhD program.
Further thanks must go to the Chromatographic Society for the provision of the John Dolphin Fellowship providing the funding to attend ICAS2011
For completion of the Venn diagram thanks to - Oliveros, J.C. (2007) VENNY. An interactive tool for comparing lists with Venn Diagrams http://bioinfogp.cnb.csic.es/tools/venny/index.html