MTBLS1: NMR based metabolomics of Human Type 2 Diabetes urine samples    

Reza Salek , Jules Griffin

Study Submission Date: 14-Feb-2012   Study public release date: 14-Feb-2012

Type 2 diabetes mellitus is the result of a combination of impaired insulin secretion with reduced insulin sensitivity of target tissues. There are an estimated 150 million affected individuals worldwide, of whom a large proportion remains undiagnosed because of a lack of specific symptoms early in this disorder and inadequate diagnostics. In this study, NMR-based metabolomic analysis in conjunction with uni- and multivariate statistics was applied to examine the urinary metabolic changes in Human type 2 diabetes mellitus patients compared to the control group. The human population were un medicated diabetic patients who have good daily dietary control over their blood glucose concentrations by following the guidelines on diet issued by the American Diabetes Association. Note: This is part of a larger study, please refer to the original paper below.

Organism(s):
Homo sapiens (Human)

Study Design Description:
  • type 2 diabetes mellitus
  • Metabolic syndrome
  • Human Study Subject
  • Urine global profiling
  • NMR spectroscopy
  • Metabolomics

Publications
A metabolomic comparison of urinary changes in type 2 diabetes in mouse, rat, and human.

Experimental Factors
  • Gender: [ Male Gender(Male_Gender) , Female Gender(Female_Gender) ]
  • Metabolic syndrome: [ Control Group , type 2 diabetes mellitus ]
Protocol Description
Sample collection For the human studies, midstream urine (~15 ml) samples were collected and frozen from each volunteer. In total, 84 samples were collected from 12 healthy volunteers (7 time points, 8 males and 4 females) and 50 samples from 30 T2DM patients (1-3 time points, 17 males and 13 females) with well-controlled blood glucose maintained at normal concentrations by diet, following the guidelines issued by the American Diabetes Association, rather than medication. T2DM patients agreed to stop treatment with oral anti-diabetic agents during the study. Subjects went through a washout period of 4 wk before sample collection and abstained from alcohol during the study; diet was controlled throughout the study.
NMR sample Aliquots of 400 µl urine samples were made up to 600 µl with phosphate buffer (0.2 M, pH 7.4) and any precipitate removed by centrifugation. In total, 500 µl of supernatant were transferred to 5-mm NMR tubes with 100 µl of sodium 3-trimethylsilyl-(2,2,3,3-2H4)-1-propionate (TSP)/D2O/sodium azide solution (0.05% wt/vol TSP in D2O and 1% wt/vol sodium azide).
NMR spectroscopy The spectra of human urine samples were acquired on a Bruker DRX700 NMR spectrometer using a 5 mm TXI ATMA probe at a proton frequency of 700.1 MHz and ambient temperature of 27℃.
NMR assay A 1D NOESY presaturation pulse sequence was used to analyze the urine samples. For each sample 128 transients were collected into 64k data points using a spectral width of 14.005 kHz (20 ppm) and an acquisition time of 2.34 s per FID.
Data transformation Spectra were processed using ACD/1D NMR Manager 8.0 with Intelligent Bucketing Integration (Advanced Chemistry Development, Toronto, ON, Canada). Spectra were integrated 0.20-9.30 ppm excluding water (4.24-5.04 ppm), glucose (3.19-3.99 ppm, 5.21-5.27 ppm), and urea (5.04-6.00 ppm). Intelligent bucketing ensures that bucket edges do not coincide with peak maxima, preventing resonances from being split across separate integral regions; a 0.04 ppm bucket width and a 50% looseness factor were used. All spectra were normalized to total area excluding the water, urea, and glucose regions
Metabolite identification Assignments were confirmed by two dimensional spectroscopy including homonuclear 1H- 1H Correlation Spectroscopy (COSY), 1H-13C Heteronuclear Signal Quantum Coherence (HSQC) and 1H-13C Heteronuclear Multiple Bond Correlation (HMBC) Spectroscopy.

Technology - Platform: NMR spectroscopy - metabolite profiling - Bruker
Source Metabolic syndrome Gender
ADG19007u_437 Control Group Male Gender(Male_Gender)
ADG19007u_244 Control Group Female Gender(Female_Gender)
ADG19007u_303 Control Group Male Gender(Male_Gender)
ADG10003u_117 type 2 diabetes mellitus Male Gender(Male_Gender)
ADG10003u_053 type 2 diabetes mellitus Male Gender(Male_Gender)
ADG10003u_073 type 2 diabetes mellitus Male Gender(Male_Gender)
ADG19007u_476 Control Group Male Gender(Male_Gender)
ADG19007u_285 Control Group Male Gender(Male_Gender)
ADG19007u_403 Control Group Male Gender(Male_Gender)
ADG19007u_091 Control Group Female Gender(Female_Gender)
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a-Hydroxy-n-butyrate (CHEBI:1148) C4H8O3 [0.85 .. 0.90] t 0.90 CCC(C(=O)O)O InChI=1S/C4H8O3/c1-2-3(5)4(6)7/h3,5H,2H2,1H3,(H,6,7) 0.65738 0.70845 0.79577 0.69651 0.83728 0.78334 0.81323 0.68001 0.70588 0.70414 0.75543 0.8791 0.71739 0.69668 0.84853 1.5065 0.87096 2.0571 0.66625 0.65715 0.69855 1.4837 2.1094 0.60666 1.0473 0.66253 0.7276 0.65311 0.71471 0.95758 0.76342 0.74092 0.69132 0.58798 0.72842 0.53611 0.73655 0.98086 0.85238 0.66343 0.60851 0.64309 0.65494 0.76503 0.64768 0.57672 0.6895 0.64344 0.71845 0.96912 0.85623 0.65403 0.65636 0.59469 1.0331 0.75257 0.74383 0.77416 0.56124 0.69271 0.62447 0.78025 0.58495 0.64518 0.56448 0.64934 0.62079 0.5896 0.63624 0.47653 0.59834 0.57986 0.49678 0.54097 0.55446 0.64562 0.56631 0.56882 0.69186 0.54739 0.49641 0.5453 0.64955 0.63971 0.7333 0.73146 0.53588 0.53305 0.61139 0.62199 0.6476 0.67038 0.69277 0.54995 0.598 0.64563 0.65525 0.54626 0.59861 0.64178 0.51115 0.55647 0.64742 0.82422 0.50621 0.60429 0.67727 0.52806 0.53165 0.64513 0.68975 0.61255 0.62126 0.73655 0.71039 0.7187 0.74581 1.0112 0.69888 0.71576 0.68772 0.70576 0.70815 0.76134 0.75525 0.66553 0.69083 0.54973 0.379 0.46882 0.54912 0.65024
Description Formula Chemical shift Multiplicity Smiles Inchi ADG10003u_007 ADG10003u_008 ADG10003u_009 ADG10003u_010 ADG10003u_015 ADG10003u_016 ADG10003u_017 ADG10003u_021 ADG10003u_022 ADG10003u_023 ADG10003u_051 ADG10003u_052 ADG10003u_053 ADG10003u_066 ADG10003u_067 ADG10003u_071 ADG10003u_072 ADG10003u_073 ADG10003u_087 ADG10003u_088 ADG10003u_089 ADG10003u_097 ADG10003u_098 ADG10003u_112 ADG10003u_113 ADG10003u_114 ADG10003u_115 ADG10003u_116 ADG10003u_117 ADG10003u_118 ADG10003u_120 ADG10003u_151 ADG10003u_152 ADG10003u_153 ADG10003u_155 ADG10003u_156 ADG10003u_160 ADG10003u_161 ADG10003u_162 ADG10003u_166 ADG10003u_167 ADG10003u_168 ADG10003u_177 ADG10003u_178 ADG10003u_179 ADG10003u_185 ADG10003u_186 ADG10003u_187 ADG19007u_011 ADG19007u_012 ADG19007u_013 ADG19007u_014 ADG19007u_015 ADG19007u_016 ADG19007u_017 ADG19007u_071 ADG19007u_072 ADG19007u_073 ADG19007u_074 ADG19007u_075 ADG19007u_076 ADG19007u_077 ADG19007u_091 ADG19007u_092 ADG19007u_093 ADG19007u_094 ADG19007u_095 ADG19007u_096 ADG19007u_097 ADG19007u_161 ADG19007u_162 ADG19007u_163 ADG19007u_164 ADG19007u_165 ADG19007u_166 ADG19007u_167 ADG19007u_191 ADG19007u_192 ADG19007u_193 ADG19007u_194 ADG19007u_195 ADG19007u_196 ADG19007u_197 ADG19007u_241 ADG19007u_242 ADG19007u_243 ADG19007u_244 ADG19007u_245 ADG19007u_246 ADG19007u_247 ADG19007u_281 ADG19007u_282 ADG19007u_283 ADG19007u_284 ADG19007u_285 ADG19007u_286 ADG19007u_287 ADG19007u_301 ADG19007u_302 ADG19007u_303 ADG19007u_304 ADG19007u_305 ADG19007u_306 ADG19007u_307 ADG19007u_351 ADG19007u_352 ADG19007u_353 ADG19007u_354 ADG19007u_355 ADG19007u_356 ADG19007u_357 ADG19007u_401 ADG19007u_402 ADG19007u_403 ADG19007u_404 ADG19007u_405 ADG19007u_406 ADG19007u_407 ADG19007u_431 ADG19007u_432 ADG19007u_433 ADG19007u_434 ADG19007u_435 ADG19007u_436 ADG19007u_437 ADG19007u_471 ADG19007u_472 ADG19007u_473 ADG19007u_474 ADG19007u_475 ADG19007u_476 ADG19007u_477
a-Hydroxy-n-valerate (CHEBI:60630) C5H9O3- [0.90 .. 0.96] t 0.92 CCCC(C(=O)[O-])O InChI=1S/C5H10O3/c1-2-3-4(6)5(7)8/h4,6H,2-3H2,1H3,(H,7,8)/p-1 0.68444 0.87504 0.86846 0.72335 0.8903 0.77572 0.85805 0.71397 0.754 0.66498 0.78102 0.90594 0.97188 0.76008 0.81961 0.54415 0.56562 0.65923 0.78541 0.81781 0.88794 0.63269 0.61958 0.80315 1.3539 0.78665 0.88788 0.82273 0.91465 0.98002 0.87736 0.83098 0.76854 0.7815 0.7132 0.59186 0.79994 0.88219 0.82289 0.90304 0.73611 0.7657 0.79092 0.89067 0.71189 0.65316 0.76553 0.69558 0.87757 1.2164 1.0134 0.86012 0.8596 0.73116 1.2607 0.81059 0.84141 0.80987 0.66816 0.83571 0.75889 0.7153 0.65423 0.72458 0.61757 0.68796 0.74608 0.6885 0.64606 0.55485 0.76198 0.68323 0.58231 0.7145 0.67372 0.81372 0.7007 0.74417 0.81172 0.64973 0.65548 0.72301 0.75334 0.78129 0.93391 0.854 0.71371 0.75581 0.8193 0.84106 0.79292 0.88612 0.85387 0.69374 0.79796 0.77927 0.84874 0.64768 0.80716 0.75362 0.65727 0.75505 0.85099 0.92216 0.64059 0.79668 0.82092 0.64689 0.68947 0.73516 0.75129 0.68679 0.74811 0.79083 0.75579 0.80464 0.81561 0.80154 0.85079 0.86539 0.80282 0.81271 0.87079 0.89467 0.84166 0.80101 0.88645 0.66394 0.48274 0.58727 0.66892 0.69982
isoleucine (CHEBI:24898) C6H13NO2 [0.90 .. 0.96] t 0.94 C(CCC(=O)O)CCN InChI=1S/C6H13NO2/c7-5-3-1-2-4-6(8)9/h1-5,7H2,(H,8,9) 0.68444 0.87504 0.86846 0.72335 0.8903 0.77572 0.85805 0.71397 0.754 0.66498 0.78102 0.90594 0.97188 0.76008 0.81961 0.54415 0.56562 0.65923 0.78541 0.81781 0.88794 0.63269 0.61958 0.80315 1.3539 0.78665 0.88788 0.82273 0.91465 0.98002 0.87736 0.83098 0.76854 0.7815 0.7132 0.59186 0.79994 0.88219 0.82289 0.90304 0.73611 0.7657 0.79092 0.89067 0.71189 0.65316 0.76553 0.69558 0.87757 1.2164 1.0134 0.86012 0.8596 0.73116 1.2607 0.81059 0.84141 0.80987 0.66816 0.83571 0.75889 0.7153 0.65423 0.72458 0.61757 0.68796 0.74608 0.6885 0.64606 0.55485 0.76198 0.68323 0.58231 0.7145 0.67372 0.81372 0.7007 0.74417 0.81172 0.64973 0.65548 0.72301 0.75334 0.78129 0.93391 0.854 0.71371 0.75581 0.8193 0.84106 0.79292 0.88612 0.85387 0.69374 0.79796 0.77927 0.84874 0.64768 0.80716 0.75362 0.65727 0.75505 0.85099 0.92216 0.64059 0.79668 0.82092 0.64689 0.68947 0.73516 0.75129 0.68679 0.74811 0.79083 0.75579 0.80464 0.81561 0.80154 0.85079 0.86539 0.80282 0.81271 0.87079 0.89467 0.84166 0.80101 0.88645 0.66394 0.48274 0.58727 0.66892 0.69982
leucine (CHEBI:25017) C6H13NO2 [0.96 .. 1.01] t 0.96 CC(C)CC(C(=O)O)N InChI=1S/C6H13NO2/c1-4(2)3-5(7)6(8)9/h4-5H,3,7H2,1-2H3,(H,8,9) 0.35269 0.65685 0.56701 0.55969 0.70704 0.56233 0.59148 0.43643 0.43411 0.38359 0.41119 0.48236 0.49428 0.37887 0.47025 0.46733 0.41556 0.58016 0.46125 0.49644 0.48319 0.4786 0.50844 0.36367 0.50486 0.4387 0.4514 0.47117 0.50721 0.53038 0.52719 0.44654 0.40624 0.37848 0.4496 0.36044 0.44046 0.52666 0.53928 0.48674 0.4185 0.43427 0.49579 0.48465 0.42499 0.3898 0.42821 0.43806 0.53421 0.60256 0.53589 0.53175 0.53744 0.47391 0.57243 0.48085 0.49349 0.4627 0.42645 0.46757 0.43733 0.41334 0.4038 0.44164 0.3714 0.42741 0.47986 0.40233 0.3944 0.35307 0.5072 0.4375 0.36965 0.46183 0.43365 0.53016 0.42671 0.45577 0.47145 0.39208 0.41044 0.44816 0.43696 0.47182 0.50767 0.47953 0.39712 0.47912 0.4202 0.48087 0.42228 0.45638 0.4399 0.37815 0.41946 0.41161 0.45601 0.40584 0.50272 0.47967 0.40539 0.50637 0.51424 0.57612 0.35498 0.40714 0.39801 0.36173 0.3847 0.40746 0.41622 0.39238 0.45525 0.46644 0.44949 0.4697 0.45176 0.47759 0.47181 0.51111 0.45613 0.47636 0.49081 0.47143 0.4477 0.40924 0.46544 0.36197 0.29492 0.33756 0.36308 0.39778
valine (CHEBI:57762) C5H11NO2 [0.96 .. 1.01] t 0.97 CC(C)C(C(=O)O)N InChI=1S/C5H11NO2/c1-3(2)4(6)5(7)8/h3-4H,6H2,1-2H3,(H,7,8)/t4-/m1/s1 0.35269 0.65685 0.56701 0.55969 0.70704 0.56233 0.59148 0.43643 0.43411 0.38359 0.41119 0.48236 0.49428 0.37887 0.47025 0.46733 0.41556 0.58016 0.46125 0.49644 0.48319 0.4786 0.50844 0.36367 0.50486 0.4387 0.4514 0.47117 0.50721 0.53038 0.52719 0.44654 0.40624 0.37848 0.4496 0.36044 0.44046 0.52666 0.53928 0.48674 0.4185 0.43427 0.49579 0.48465 0.42499 0.3898 0.42821 0.43806 0.53421 0.60256 0.53589 0.53175 0.53744 0.47391 0.57243 0.48085 0.49349 0.4627 0.42645 0.46757 0.43733 0.41334 0.4038 0.44164 0.3714 0.42741 0.47986 0.40233 0.3944 0.35307 0.5072 0.4375 0.36965 0.46183 0.43365 0.53016 0.42671 0.45577 0.47145 0.39208 0.41044 0.44816 0.43696 0.47182 0.50767 0.47953 0.39712 0.47912 0.4202 0.48087 0.42228 0.45638 0.4399 0.37815 0.41946 0.41161 0.45601 0.40584 0.50272 0.47967 0.40539 0.50637 0.51424 0.57612 0.35498 0.40714 0.39801 0.36173 0.3847 0.40746 0.41622 0.39238 0.45525 0.46644 0.44949 0.4697 0.45176 0.47759 0.47181 0.51111 0.45613 0.47636 0.49081 0.47143 0.4477 0.40924 0.46544 0.36197 0.29492 0.33756 0.36308 0.39778
a-Hydroxyisovalerate (CHEBI:64669) C5H10O3 [0.96 .. 1.01] CC(C)(C(=O)OC)O InChI=1S/C5H10O3/c1-5(2,7)4(6)8-3/h7H,1-3H3 0.35269 0.65685 0.56701 0.55969 0.70704 0.56233 0.59148 0.43643 0.43411 0.38359 0.41119 0.48236 0.49428 0.37887 0.47025 0.46733 0.41556 0.58016 0.46125 0.49644 0.48319 0.4786 0.50844 0.36367 0.50486 0.4387 0.4514 0.47117 0.50721 0.53038 0.52719 0.44654 0.40624 0.37848 0.4496 0.36044 0.44046 0.52666 0.53928 0.48674 0.4185 0.43427 0.49579 0.48465 0.42499 0.3898 0.42821 0.43806 0.53421 0.60256 0.53589 0.53175 0.53744 0.47391 0.57243 0.48085 0.49349 0.4627 0.42645 0.46757 0.43733 0.41334 0.4038 0.44164 0.3714 0.42741 0.47986 0.40233 0.3944 0.35307 0.5072 0.4375 0.36965 0.46183 0.43365 0.53016 0.42671 0.45577 0.47145 0.39208 0.41044 0.44816 0.43696 0.47182 0.50767 0.47953 0.39712 0.47912 0.4202 0.48087 0.42228 0.45638 0.4399 0.37815 0.41946 0.41161 0.45601 0.40584 0.50272 0.47967 0.40539 0.50637 0.51424 0.57612 0.35498 0.40714 0.39801 0.36173 0.3847 0.40746 0.41622 0.39238 0.45525 0.46644 0.44949 0.4697 0.45176 0.47759 0.47181 0.51111 0.45613 0.47636 0.49081 0.47143 0.4477 0.40924 0.46544 0.36197 0.29492 0.33756 0.36308 0.39778
isoleucine (CHEBI:24898) C6H13NO2 [1.01 .. 1.03] d 1.01 C(CCC(=O)O)CCN InChI=1S/C6H13NO2/c7-5-3-1-2-4-6(8)9/h1-5,7H2,(H,8,9) 0.083834 0.097845 0.10966 0.085085 0.12014 0.12089 0.13525 0.10463 0.086128 0.084556 0.10075 0.10798 0.090919 0.087954 0.091142 0.070861 0.077182 0.089031 0.08994 0.098865 0.10037 0.07503 0.081867 0.084151 0.11539 0.1071 0.10401 0.10466 0.1166 0.12654 0.17752 0.09427 0.089507 0.063785 0.10211 0.090369 0.10262 0.13131 0.12615 0.086988 0.08446 0.092822 0.090644 0.092411 0.094961 0.084025 0.077102 0.096917 0.11881 0.15248 0.14016 0.12743 0.099417 0.099762 0.15871 0.10848 0.090415 0.097562 0.083399 0.093592 0.098237 0.095621 0.079372 0.076605 0.072264 0.083938 0.088211 0.087611 0.092622 0.072568 0.08354 0.099456 0.085639 0.081462 0.094033 0.12236 0.097777 0.091287 0.10169 0.096857 0.087768 0.088842 0.11309 0.10017 0.10051 0.096334 0.083007 0.075535 0.087165 0.10294 0.10639 0.11523 0.112 0.087655 0.091376 0.10232 0.1424 0.090734 0.11246 0.098962 0.089334 0.093113 0.11364 0.13374 0.086659 0.079254 0.080767 0.089472 0.078522 0.092927 0.095108 0.088751 0.078369 0.092502 0.098709 0.08753 0.085616 0.089237 0.12617 0.10691 0.10543 0.12958 0.10225 0.10702 0.11653 0.094023 0.10129 0.088191 0.07973 0.080197 0.098218 0.10527
a-keto-b-methyl-N valerate (CHEBI:35932) C6H10O3 [1.03 .. 1.09] d 1.075 CCCC(=O)C(=O)OC InChI=1S/C6H10O3/c1-3-4-5(7)6(8)9-2/h3-4H2,1-2H3 0.74011 0.76453 0.70767 0.60742 0.74635 0.57071 0.58937 0.51939 0.47797 0.47593 0.47386 0.55347 0.63418 0.45194 0.53713 2.4813 1.9415 4.3365 0.53125 0.57248 0.56731 2.8198 3.4271 0.42391 0.6227 0.52028 0.54073 0.55055 0.61958 0.62556 0.5917 0.53169 0.53598 0.44656 0.41502 0.4271 0.54765 0.59639 0.60975 0.57121 0.54929 0.49119 0.80981 0.70669 0.6402 0.44665 0.51737 0.55718 0.59369 0.63409 0.57349 0.53842 0.61879 0.52781 0.57255 0.59266 0.66089 0.57377 0.57172 0.60901 0.55015 0.49593 0.50895 0.60001 0.44267 0.51862 0.62489 0.48401 0.50071 0.38375 0.63113 0.49642 0.44811 0.56508 0.49478 0.55666 0.48178 0.57074 0.53803 0.48714 0.53989 0.55598 0.46318 0.51613 0.60122 0.5222 0.48277 0.66691 0.54256 0.60632 0.55921 0.68972 0.57757 0.56785 0.61751 0.53364 0.54816 0.46264 0.67376 0.64501 0.48123 0.71097 0.57284 0.68914 0.4548 0.58272 0.54281 0.4961 0.59469 0.54982 0.51516 0.43941 0.58451 0.58919 0.55739 0.68482 0.70074 1.3155 0.55628 0.6841 0.55909 0.5796 0.65601 0.58987 0.44458 0.53596 0.63667 0.44595 0.39559 0.51223 0.52112 0.53707
2-oxoisovalerate (CHEBI:16530) C5H8O3 [1.09 .. 1.13] d 1.11 C1CC(=O)OC1CO InChI=1S/C5H8O3/c6-3-4-1-2-5(7)8-4/h4,6H,1-3H2/t4-/m0/s1 0.3841 0.44603 0.45971 0.40812 0.43029 0.44967 0.42433 0.42904 0.34552 0.37077 0.46425 0.46797 0.6004 0.41551 0.40045 0.90097 0.63821 0.55322 0.37936 0.37669 0.36033 0.43332 0.44989 0.35089 0.47622 0.44308 0.4517 0.4518 0.50616 0.5386 0.46486 0.73078 0.6052 0.54294 0.31732 0.3232 0.36521 0.45255 0.44277 0.48473 0.42378 0.45155 0.54116 0.52507 0.52542 0.41999 0.43199 0.43958 0.38041 0.38005 0.36326 0.34043 0.34167 0.337 0.36546 0.34857 0.32372 0.37879 0.3048 0.3441 0.36223 0.33856 0.30621 0.32417 0.30555 0.27728 0.31908 0.30371 0.29977 0.28778 0.38179 0.3811 0.31582 0.35126 0.38876 0.34888 0.3342 0.35098 0.39406 0.31888 0.33946 0.33403 0.33484 0.49369 0.48864 0.50144 0.39364 0.38305 0.41657 0.42897 0.35129 0.3627 0.3794 0.34347 0.37401 0.38879 0.42101 0.37497 0.43388 0.42048 0.32086 0.38445 0.43871 0.40794 0.32791 0.36121 0.43292 0.32946 0.32251 0.39796 0.50321 0.36466 0.4007 0.44061 0.42875 0.41932 0.45087 0.45885 0.39083 0.41719 0.37581 0.39023 0.39345 0.4188 0.42829 0.35279 0.37736 0.32044 0.27824 0.30233 0.3478 0.34799
ethanol (CHEBI:16236) C2H6O [1.09 .. 1.13] t 1.11 CCO InChI=1S/C2H6O/c1-2-3/h3H,2H2,1H3 0.3841 0.44603 0.45971 0.40812 0.43029 0.44967 0.42433 0.42904 0.34552 0.37077 0.46425 0.46797 0.6004 0.41551 0.40045 0.90097 0.63821 0.55322 0.37936 0.37669 0.36033 0.43332 0.44989 0.35089 0.47622 0.44308 0.4517 0.4518 0.50616 0.5386 0.46486 0.73078 0.6052 0.54294 0.31732 0.3232 0.36521 0.45255 0.44277 0.48473 0.42378 0.45155 0.54116 0.52507 0.52542 0.41999 0.43199 0.43958 0.38041 0.38005 0.36326 0.34043 0.34167 0.337 0.36546 0.34857 0.32372 0.37879 0.3048 0.3441 0.36223 0.33856 0.30621 0.32417 0.30555 0.27728 0.31908 0.30371 0.29977 0.28778 0.38179 0.3811 0.31582 0.35126 0.38876 0.34888 0.3342 0.35098 0.39406 0.31888 0.33946 0.33403 0.33484 0.49369 0.48864 0.50144 0.39364 0.38305 0.41657 0.42897 0.35129 0.3627 0.3794 0.34347 0.37401 0.38879 0.42101 0.37497 0.43388 0.42048 0.32086 0.38445 0.43871 0.40794 0.32791 0.36121 0.43292 0.32946 0.32251 0.39796 0.50321 0.36466 0.4007 0.44061 0.42875 0.41932 0.45087 0.45885 0.39083 0.41719 0.37581 0.39023 0.39345 0.4188 0.42829 0.35279 0.37736 0.32044 0.27824 0.30233 0.3478 0.34799