Supplementary materials for manuscript :
Metabolomic analysis reveals distinct profiles in the plasma and
urine of rats fed a high protein diet
Chunlong Mu1, Yuxiang Yang1, Zhen Luo, Weiyun Zhu*
Laboratory of Gastrointestinal Microbiology, College of Animal Science and
Technology, Nanjing Agricultural University, Nanjing 210095, China
Corresponding Author
* Corresponding Author:
Weiyun Zhu, Phone: +86 25 84395523. Fax: +86 25
84395314. E-mail: zhuweiyun@njau.edu.cn
Table 1. Composition and nutrient concentrations of the experimental diets
Items
Ingredients (%, as-fed basis)
Casein
Sucrose
wheat starch
Amylodextrin
Cellulose
Colza oil
DL-Methionine
Salt
Calcium hydrophosphate
Calcium phosphate
choline chloride
Vitamin-mineral mix*
Total
Chemical composition (g/100g DM)
Protein
Available carbohydrates
Acid hydrolyzed fat
Total digestible fiber
Ash
Protein/ CHO
Energy (kcal/g)
ME (kcal/100 g DM)
Macronutrients on energy basis (% of ME)
Protein
NFE (carbohydrate)
Acid hydrolyzed fat
* AIN-93G mineral and vitamin mixes.
MPMC diet
HPLC diet
20
10
40.4
13.2
5
7
0.3
0.2
1
1.3
0.1
1.5
100
54
10
6.6
13.2
5
7
0.1
0.2
1
1.3
0.1
1.5
100
20.96
55.54
7.18
4.7
2.53
0.38
3.54
328.8
45.04
29.92
7.7
4.7
3.54
1.5
3.35
327.8
22
59
19
48
32
20
Table 2. Body weight, food intake and plasma biochemical parameters in MPMC and HPLC rats
MPMC
HPLC
P value
Body weight
365.11±11.07
264.97±8.92
<0.001
Body weight gain (g)
136.3 ± 9.07
42.42 ± 6.72
<0.001
Liver weight (g)
15.63 ± 0.7
13.41 ± 0.63
0.014
4.25±0.13
5.03±0.12
<0.001
0.62 ± 0.03
0.6 ± 0.03
0.659
0.17±0.01
0.23±0.01
<0.001
4.737 ± 0.25
2.54 ± 0.2
<0.001
1.29±0.07
0.95±0.08
<0.001
5.21 ± 0.29
2.41 ± 0.14
<0.001
0.72±0.05
0.45±0.04
<0.001
Food intake (g/d)
17.45 ± 0.47
13.87 ± 0.38
<0.001
Energy intake (kcal/d)
61.76 ± 1.66
46.48 ± 1.29
<0.001
Total protein (g/L)
44.27 ± 2.81
45.54 ± 1.41
0.631
Albumin (g/L)
21.84 ± 1.37
23.42 ± 0.66
0.579
Globulin (g/L)
22.93 ± 1.54
22.12 ± 0.9
0.315
Glucose (mmol/L)
14.62 ± 1.33
10.48 ± 0.71
0.023
Triglyceride (mmol/L)
2.31 ± 0.26
0.73 ± 0.07
<0.001
Total cholesterol (mmol/L)
1.17 ± 0.1
1.3 ± 0.06
0.247
HDLC (mmol/L)
0.76 ± 0.05
0.95 ± 0.03
0.003
LDLC (mmol/L)
0.15 ± 0.02
0.16 ± 0.01
0.436
Urea (mmol/L)
5.43 ± 0.39
11.1 ± 1.05
<0.001
Uric acid (μmol/L)
207.7 ± 17.53
173.4 ± 16.92
0.247
Creatinine (μmol/L)
23.5 ± 1.35
25.4 ± 2.08
0.631
Glutamic-pyruvic transaminase (U/L)
22.55 ± 2.76
32.23 ± 7.25
0.684
Glutamic-oxalacetic transaminase (U/L)
20.55 ± 1.72
25.35 ± 2.69
0.280
Insulin (μU/mL)
53.74 ± 8.99
19.98 ± 2.26
0.008
Glucagon (pg/mL)
65.03 ± 8.53
74.91 ± 9.28
0.4
Insulin/Glugacon
0.85 ± 0.13
0.3 ± 0.04
<0.001
thyroxine (ng/mL)
19.36 ± 3.28
22.24 ± 4.06
0.73
triiodothyronine (ng/mL)
0.32 ± 0.03
0.28 ± 0.02
0.34
Growth hormone (ng/mL)
0.91 ± 0.09
1.02 ± 0.08
0.315
Gastrin (pg/mL)
20.31 ± 3.41
24.53 ± 2.78
0.356
HOMA-IR
37.007±7.27
9.514±1.615
<0.001
IAI
-6.47±0.27
-5.24±0.187
<0.001
Expressed as %body weight
Spleen weight (g)
Expressed as %body weight
Mesenteric adipose (g)
Expressed as %body weight
Epididymal adipose (g)
Expressed as %body weight
Values are expressed as mean ± SEM, n = 10 for each group.
Table 3. Quantitative concentrations of selected urinary metabolites in MPMC and HPLC groups
MPMC
Glucose (mmol/L)
Uric acid (mg/L)
Creatinine (mmol/L)
0.64±0.064
295.93±33.79
5.32±0.516
HPLC
0.276±0.055
138.03±17.1
1.8±0.285
Data were expressed as mean ± SEM, n = 10 each group.
P-value
<0.001
<0.001
<0.001
Table 4. Parameter description of plasma metabolite pair-wise correlation network in MPMC (M)
and HPLC (H) group.
Degree 1
Alanine
β-Amino Isobutyrate
Glutamine
Glycine
Isoleucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
Galactose
Lyxose
Mannonate-γ-Lactone
Threonic Acid
2-Ketoglutarate
Citrate
Fumarate
Lactate
Malate
Oxalate
Pyruvate
11-Cis-Octadecenoate
β-Hydroxybutyrate
Cis-4,7,10,13,16,19-Docosahexaenoate
Glycerol
Linoleate
Palmitate
Uric Acid
1
M
3
2
5
1
1
0
3
3
2
5
2
3
4
0
3
1
3
2
0
4
2
5
2
5
1
5
2
5
4
4
4
6
H
13
5
1
9
5
7
8
10
7
11
9
1
3
8
6
5
6
3
7
5
10
8
5
1
1
5
2
1
2
4
5
4
Betweenness
Centrality 2
M
H
8.00
162.59
15.00
0.00
6.03
0.00
0.00
44.18
0.00
37.00
0.00
1.24
18.00
25.34
28.00
176.40
8.00
0.83
20.00
60.73
14.00
5.36
8.33
0.00
42.08
37.00
0.00
3.63
0.00
86.80
0.00
40.84
0.00
86.80
0.00
75.47
0.00
15.21
0.67
64.53
0.00
176.40
12.07
369.47
0.00
37.00
39.35
0.00
0.00
0.00
14.40
9.12
0.00
6.53
25.98
0.00
2.63
0.00
1.80
9.00
1.80
3.24
14.60
37.00
Eigenvector
Centrality 3
M
H
0.12
1.00
0.06
0.46
0.81
0.05
0.18
0.58
0.02
0.39
0.00
0.65
0.13
0.75
0.15
0.74
0.03
0.64
0.17
0.97
0.06
0.76
0.17
0.03
0.28
0.19
0.00
0.68
0.41
0.13
0.01
0.33
0.41
0.13
0.09
0.05
0.00
0.55
0.75
0.07
0.04
0.74
0.80
0.31
0.04
0.34
0.54
0.00
0.13
0.00
0.67
0.30
0.04
0.02
0.67
0.05
0.66
0.01
0.57
0.16
0.57
0.25
1.00
0.07
The degree of a vertex of a graph is the number of edges incident to the vertex
Betweenness centrality quantifies the number of times a node acts as a bridge along the shortest path
between two other nodes
3
Eigenvector Centrality assigns relative scores to all nodes in the network based on the concept that
connections to high-scoring nodes contribute more to the score of the node in question than equal
connections to low-scoring nodes.
2
Table 5. Parameter description of plasma-urine metabolite pair-wise correlation network in
MPMC or HPLCgroup.
Betweenness Eigenvector
Label
Degree1
Centrality2
Centrality3
MPMC group
Plasma metabolite
Valine
Pyruvate
Alpha-Tocopherol
Adipate
Cis-4,7,10,13,16,19-Docosahexaenoate
Urine metabolite
Serine
D-Fructose
D-Turanose
Raffinose
Ribitol
9
6
5
4
3
52.50
27.00
22.50
6.00
9.00
1.00
0.38
0.55
0.11
0.21
2
2
3
2
2
7.00
7.00
13.00
7.00
5.00
0.48
0.48
0.26
0.48
0.22
HPLC group
Plasma metabolite
2-oxoisocaproate
4
9.00
β-Hydroxybutyrate
4
12.50
Citrate
3
5.50
Tyrosine
3
3.00
Palmitate
2
6.00
Stearate
2
4.00
Urine metabolite
L-Norleucine
4
13.67
Erythrose
2
4.00
Pentitol
2
6.00
Raffinose
2
4.00
3,4-Dihydroxybutanoate
3
7.67
1
The degree of a vertex of a graph is the number of edges incident to the vertex
2
Betweenness centrality quantifies the number of times a node acts as a bridge along the shortest path
between two other nodes
3
Eigenvector Centrality assigns relative scores to all nodes in the network based on the concept that
connections to high-scoring nodes contribute more to the score of the node in question than equal
connections to low-scoring nodes.
0.32
1.00
0.87
0.47
0.14
0.12
0.93
0.12
0.14
0.20
0.78
(x100,000,000)
a
1.1
MPMC
11-cis-octadecenoic acid
1.0
Citric acid
Urea
Lactic acid
Threonine
Ornithine
0.9
β-hydroxybutyric
0.8
acid
Pyruvic acid
Malic acid
HPLC
Glucose
Glutamate
Cholesterol
Mannose
Adipic acid
Oxalic acid
Palmitic acid
0.6
0.5
Stearic acid
Palmitoleic acid
Glutamine
Proline
Serine
0.7
Frucose
Arachidonic acid
Pyroglutamic acid
0.4
Valine
0.3
2-ketoglutaric acid
0.2
0.1
0.0
-0.1
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
(x100,000,000)
b
3.25
MPMC
2.75
HPLC
Maltose
3.00
Lactate
D-glucose
Succinic acid
Pseudo uridine
Aconitic acid
2.50
2.25
Sorbitol
Glycolic acid
Raffinose
Citric acid
Creatinine
2.00
Hippuric acid
Pyruvic acid
1.75
Glucoronic acid
Urea
1.50
Stearic acid
1.25
2-ketoglutaric acid
1.00
Erythrose
Palmitic acid
Taurine
0.75
0.50
0.25
0.00
-0.25
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
Retention Time (min)
Supplementary Fig 1. GC/MS chromatogram overlay of MPMC and HPLC group rats depicting the plasma (a) and urinary (b) metabolite peaks.
a
b
Fig.2 (a) The positive correlation between energy-metabolism relatives and some amino acids in plasma of HPLC rats. (b)
Positive correlation between plasma amino acids. The black line indicated the unique pair-wise correlation in MPMC rats,
blue line indicated the shared correlation both in MPMC and HPLC rats, and red line indicated the unque pair-wise
correlation in HPLC rats.
Fig.3 Plasma-urinary metabolite pair-wise correlation network using data from MPMC and HPLC group. Correlations with a p
value less than 0.01 and coefficient more than 0.8 were used to construct the network. Edge color in red indicated positive
correlation and blue indicated negative correlation. The node color of certain metabolite was the same as its source metabolite
property. Red circle shape indicated plasma metabolites while black circle indicated urinary metabolites.