Transcript Uric acid

Urine extract
N-acetyl-D-phenylalanine
TOF MSMS 206.08ES-
TOF MSMS 206.08ES-
Supplemenatry Figure S1-A. Determination of the chemical structure of up-regulated metabolites in urine
sample from diabetic subjects by tandem mass spectrometry. Top panel shows the MS/MS fragmentation of the
ion with m/z 206.0804 from urine samples in the negative electrospray ion mode. The bottom panel shows the
fragmentation for standard N-acetyl-D-phenylalanine under the same conditions.
Urine extract
2-Ketobutyric acid
TOF MSMS 101.03ES-
TOF MSMS 101.03ES-
Supplementary Figure S1-B. Determination of the chemical structure of down regulated metabolite in urine samples of diabetic
subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 101.03 from urine extract in
the negative ion mode. The bottom panel shows the fragmentation for standard 2-Ketobutyric acid under the same conditions.
Urine extract
2-Ketoglutaric acid
TOF MSMS 145.02ES-
TOF MSMS 145.02ES-
Supplementary Figure S1-C. Determination of the chemical structure of down regulated metabolite in urine samples of Diabetic
subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 145.02 from urine extract in
the negative ion mode. The bottom panel shows the fragmentation for standard 2-Ketoglutaric acid under the same conditions.
Urine extract
1-Methylhistidine
TOF MSMS 168.08 ES-
TOF MSMS 168.08 ES-
Supplementary Figure S1-D. Determination of the chemical structure of down regulated metabolite in urine samples of Diabetic
subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 168.08 from urine extract in
the negative ion mode. The bottom panel shows the fragmentation for standard 1-Methylhistidine under the same conditions.
Urine extract
TOF MSMS 188.04 ES-
Kynurenic acid
TOF MSMS 188.04 ES-
Supplementary Figure S1-E. Determination of the chemical structure of down regulated metabolite in plasma samples of
Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 188.04 from urine
extract in the negative ion mode. The bottom panel shows the fragmentation for standard Kynurenic acid under the same conditions.
Urine extract
Xanthurenic acid
TOF MSMS 206.08ES-
TOF MSMS 206.08ES-
Supplementary Figure S1-F. Determination of the chemical structure of down regulated metabolite in plasma samples of
Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 206.08 from urine
extract in the positive ion mode. The bottom panel shows the fragmentation for standard Xanthurenic acid under the same conditions.
Plasma extract
Taurine
TOF MSMS 124.02ES-
TOF MSMS 124.02ES-
Supplementary Figure S1-G : Determination of the chemical structure of down regulated metabolite in plasma samples of
Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 124.02 from plasma
extract in the negative ion mode. The bottom panel shows the fragmentation for standard Taurine under the same conditions.
Plasma extract
Uric acid
TOF MSMS 167.03ES-
TOF MSMS 167.03ES-
Supplementary Figure S1-H. Determination of the chemical structure of down regulated metabolite in plasma samples of
Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 167.03 from urine
extract in the negative ion mode. The bottom panel shows the fragmentation for standard Uric acid under the same conditions.
Plasma extract
Inosine
TOF MSMS 267.07ES-
TOF MSMS 267.07ES-
Supplementary Figure S1-I. Determination of the chemical structure of downregulated metabolite in plasma samples of Diabetic
subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 267.07 from plasma extract in the
negative ion mode. The bottom panel shows the fragmentation for standard Inosine under the same conditions.
Urine extract
Serotonin
TOF MSMS 177.10 ES+
TOF MSMS 177.10 ES+
Supplementary Figure S1-J. Determination of the chemical structure of upegulated metabolite in urine samples of Diabetic
subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 177.10 from plasma extract
in the positive ion mode. The bottom panel shows the fragmentation for standard Serotonin under the same conditions.
Urine extract
Pyruvic acid
TOF MSMS 87.00 ES-
TOF MSMS 87.00 ES-
Supplementary Figure S1-K. Determination of the chemical structure of upregulated metabolite in plasma samples of
Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 87.00 from urine
extract in the negative ion mode. The bottom panel shows the fragmentation for standard Pyruvic acid under the same conditions.
Urine extract
Leucine
TOF MSMS 130.08 ES-
TOF MSMS 130.08 ES-
Supplementary Figure S1-K. Determination of the chemical structure of upregulated metabolite in plasma samples of
Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 130.08 from
urine extract in the negative ion mode. The bottom panel shows the fragmentation for standard Leucine under the same conditions.
Plasma extract
Sphingosine-1-Phosphate
TOF MSMS 380.2552 ES+
TOF MSMS 380.2552 ES+
Supplementary Figure S1-L. Determination of the chemical structure of up-regulated metabolite in plasma samples
of by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 380.2552 from plasma
extract in the positive ion mode. The bottom panel shows the fragmentation for standard Sphingosine-1-phosphate under the
same conditions.
Plasma extract
TOF MSMS 117.0189 ES-
Succinate
TOF MSMS 117.0189 ES-
Supplementary Figure S1-M. Determination of the chemical structure of downregulated metabolite in plasma samples by
tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 117.0189 from plasma extract in the
positive ion mode. The bottom panel shows the fragmentation for standard Succinate under the same conditions.
MS/MS spectra of PC(18:0/0:0)
Plasma Extract
Lipid maps m/z 524.37 ES+
TOF MSMS 524.37 ES+
H
18:0 LPC + H
18:0 LPC –H2 O + H
H
Supplementary Figure S1-N. Determination of the chemical structure of upegulated metabolite in plasma samples of Diabetic
subjects by tandem mass spectrometry and matching the fragments with standard fragmentation patern . The top panel shows the
fragmentation for standard PC(18:0/0:0) taken from Lipid maps. The bottom panel shows the MS/MS fragmentation of the ion with m/z
524.37 from plasma extract in the positive ion mode.
Supplementary Figure S1-O. Determination of the chemical structure of down regulated metabolite in plasma samples via
tandem mass spectrometry. The top panel shows the fragmentation for standard PE(P-16:0/22:6). The bottom panel shows the
MS/MS fragmentation of the ion with m/z 746.51 from plasma extract in the negative ion mode.
MS/MS spectra of PG(18:0/18:1)
36:1GPGro -H
TOF MSMS 775.54 ES-
(281) 18:1 FA-H
(283) 18:0 FA-H
Plasma Extract
Lipid maps m/z 775.54 ES-
Supplementary Figure S1-P. Determination of the chemical structure of up-regulated metabolite in plasma samples by tandem mass
spectrometry. The top panel shows the fragmentation for standard PG(18:0/18:1) taken from Lipid maps. The bottom panel shows the
MS/MS fragmentation of the ion with m/z 775.54 from plasma extract in the negative ion mode.
Urine
Metabolites
Ionization Mode Compound Name
ES+
Xanthurenic Acid
Xanthurenic Acid
ESKyuneric acid
Kyuneric acid
ESCi2- Ketoglutaric Acid
ES+
Serotonin
Serotonin
ESSuccinate
Succinate
ESPyruvic Acid
Parent (m/z)
206.0694
206.0694
188.0745
188.0745
145.0534
177.1025
177.1025
117.0189
117.0189
87.0071
Transition (m/z)Dwell (s)
131.9828
0.043
159.9844
0.043
116.0088
0.043
144.0125
0.043
100.9649
0.217
132.1017
0.025
115.7523
0.025
99.922
0.217
73.0332
0.217
43.001
0.217
Cone (V)
36
36
18
18
16
40
40
16
16
16
Collision (V)
Molecular Formula
28
C10H7NO4
18
C10H7NO4
26
C10H7NO3
16
C10H7NO3
8
C5H6O5
16
C10H12N20
20
C10H12N20
8
C4H6O4
8
C4H6O4
8
C3H4O3
Plasma
Metabolites
Ionization Mode Compound Name
ES+
Sphingosine- 1- phosphate
ES+
PC(18:0/0:0)
PC(18:0/0:0)
ESPG(18:0/18:1)
PG(18:0/18:1)
Parent (m/z)
380.1125
524.3717
524.3717
775.5504
775.5504
Transition (m/z)Dwell (s)
264.199
0.03
506.3611
0.037
184.0739
0.037
419.6802
0.031
281.2859
0.031
Cone (V)
60
20
20
25
25
Collision (V)
Molecular Formula
23
C18H38NO5P
38
C26H55NO7P
22
C26H55NO7P
24
C42H81O10P
24
C42H81O10P
Supplementary Table ST1. UPLC-MRM-MS Parameters used for quantitative measurement of metabolites in plasma and
urine samples. Metabolites were quantified using Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer
(Xevo TQ, Waters). For each metabolite, a MRM method was optimized using the “IntelliStart” function on the Xevo TQ for
determining the optimal cone voltages, collision energy and dwell time for maximal sensitivity and ion transmission for the selection
of Q1/Q3 transitions.
Ionization Compond Name
Mode
Parent (m/z)
Transition (m/z) Dwell (s)
Cone (V) Collision (V)
ES-
D6 - Succinate
121.0319
121.0319
76.9714
101.9437
0.011
0.011
22
22
10
10
ES-
D5- Kynurenic Acid
193.0957
120.997
0.011
18
26
193.0957
149.0005
0.011
18
16
ES-
13C3 - Pyruvic Acid
89.9442
45.0122
0.011
22
8
ESCi-
D6 - α-Ketoglutaric Acid
149.0681
104.969
0.011
16
8
ES+
D7-Sphingosine - 1- Phosphate
387.34
387.34
81.95
271.31
0.05
0.05
18
18
30
18
Supplementary Table ST2. UPLC-MRM-MS Parameters of stable-isotope-labeled standards used for SID-MRMMS in plasma and urine samples. For each metabolite, a MRM method was optimized using the “IntelliStart” function
on the Xevo TQ for determining the optimal cone voltages, collision energy and dwell time for maximal sensitivity and
ion transmission.
Pyruvic acid
m/z 87.00
43.00
13 C
3 -Pyruvic acid
m/z 89.94
45.01
Kynurenic acid
m/z 188.07
144.01
D5-Kynurenic acid
m/z 193.09
149.00
Succinate
m/z 117.01
73.03
D6-Succinate
m/z 121.03
76.97
2-Ketoglutaric acid
m/z 145.05
100.96
D6-Ketoglutaric acid
m/z 149.06
104.96
Spingosine-1-phosphate
m/z 380.11
264.19
D7-Spingosine-1-phosphate
m/z 387.34
271.31
Supplementary Figure S2. Multiple
reaction monitoring yields ion traces
of stable isotope labeled and
unlabeled metabolites that show
retention time reproducibility. The
urine and plasma samples were
processed by spiking a known
concentration of the stable isotope
labeled metabolites and injected for
UPLC-SID-MRM-MS analysis as
described in the Methods section.
MRM runs were performed by
monitoring Q1/Q3 transitions. The
peak area of the endogenous
metabolite was normalized to that of
the respective isotope labeled standard
and the relative ratios in T2DM and
controls were calculated.
B
A
T2DM
Control
T2DM
Control
Supplementary figure S3. Random forests analysis of T2DM and Control subjects. Panel A: Heat map visualization of
the top 50 features rankings comparing relative levels in control and T2DM in plasma samples. Panel B. Heatmap of the
top 50 features in urine samples. Each row represents a unique feature with a characteristic mass to charge and retention
time (minutes).
S.No.
m/z (mode)
RT(min)
Relative Fold Change
(T2DM/Control)
P-value
1
399.1639(-)
3.4176
52.2
0.002
2
429.0847(-)
3.0324
27.7
0.01
3
404.0421(-)
2.8961
14.5
0.01
4
455.0450(-)
2.9946
9.3
0.02
5
391.0627(-)
3.5508
6.6
0.03
6
273.0720(-)
0.4020
6.04
0.01
7
557.0609(-)
3.9133
5.6
0.006
8
171.0646(-)
2.541
0.4
0.02
9
10
1.6.0286
176.934(-)
2.2618
0.3436
0.4
0.3
0.01
0.02
11
236.0644(-)
0.2644
0.1
0.01
12
429.0820(-)
3.5082
0.04
0.03
13
470.2229(+)
3.0051
44.7
0.005
14
308.0307(+)
3.2817
22.6
0.04
15
121.0290(+)
1.6804
21.9
0.02
16
113.0819(+)
0.3239
17.4
0.001
17
148.041(+)
3.1763
0.4
0.002
18
144.0438(+)
1.8838
0.2
0.02
19
399.0439(+)
431.0959(+)
2.831
3.4517
0.2
0.04
0.01
0.02
20
Supplementary Table ST3: Unidentified putative markers of diabetes
Supplementary Figure S4. KEGG pathway for tryptophan metabolism. Functional pathway analysis for changes in
metabolite levels in diabetes revealed tryptophan metabolism to be one of the major pathways involved with a significant
representation of metabolites participating in this pathway. (Green: down-regulated in diabetes; Red: up-regulated in
diabetes)
(T2DM)
(Control)
Supplementary Figure S5: OPLSDA plot generated for the nine target metabolites.