Targeted Metabolomics Analysis of Serum Amino Acids in T2DM Patients

Figures & data

Table 1 Baseline Characteristics of Healthy Controls and T2DM Patients

	Health Group (n=30)	T2DM Group (n=30)
Age (years)	48.53±12.29	54.37±13.04
Sex (%Male)	53.00% (16/30)	60.00% (18/30)
Course of disease (year)	--	10.6±7.54
BMI (Kg/m^2)	21.76±1.14	25.42±4.81***
Height (cm)	166.8±8.86	167.57±10.81
Weight (kg)	60.78±7.73	71.63±19.41*
Fasting plasma glucose (mmol/L)	5.29±0.35	8.07±3.32***
2h-post load glucose (mmol/L)	5.44±0.29	16.67±4.85***
HbA1c	--	9.21±2.27
Total cholesterol (mmol/L)	3.79±0.54	4.89±1.24***
HDL-cholesterol (mmol/L)	1.71±0.28	1.05±0.22***
LDL-cholesterol (mmol/L)	2.24±0.68	2.81±0.76**
Triglyceride (mmol/L)	1.01±0.25	2.04±1.16***

Notes: All data were presented with Means±SEM, *P<0.05, **P<0.01, ***P<0.001 compared with Health group.

Figure 1 Representative MRM chromatography from amino acid quantification of Healthy group (A) and T2DM group (B).

Figure 2 (A) PCA score plots (n = 30) of the healthy group and T2DM group. (B) Heat maps of normalized amino acids concentrations in serum sample. Columns represent the samples (Healthy and T2DM groups), and rows represent amino acids.

Table 2 Standard Curve Linearity Data Obtained for the Analyses

Amino Acid	Abbreviation	Linear Equation	r^2	Linearity Range (μmol/L)
Threonine	THR	Y=9189.81+9722.12	0.996720	1–400
Leucine	LEU	Y=0.0271421X+0.0136813	0.996680	1–100
Isoleucine	ILE	Y=25807.5931x+2085.8635	0.996254	1–100
Valine	VAL	Y=0.0152271X+0.00292939	0.997734	1–200
Phenylalanine	PHE	Y=0.0388753X+0.00787353	0.990535	1–100
Methionine	MET	Y=0.130294X+0.0220339	0.991864	1–200
Tryptophan	TRP	Y=0.132323X+0.0249838	0.992632	1–100
Lysine	LYS	Y=0.0881003X+0.0275966	0.994684	1–200
δ-Hydroxylysine	OH-LYS	Y=5395.27X+794.596	0.996051	1–100
Histidine	HIS	Y=471.025X-27.9178	0.987669	1–400
1-Methylhistidine	1-MEHIS	Y=298.254X-52.7013	0.985188	1–400
3-Methylhistidine	3-MEHIS	Y=218.769X-35.5509	0.981883	1–400
Glycine	GLY	Y=0.00250766X+0.144617	0.991037	2–200
Sarcosine	SAR	Y=17037.8X+10485	0.994777	1–100
Tyrosine	TYR	Y=0.100472X+0.0230359	0.995043	1–100
β-Alanine	ALA_B	Y=2700.95X+7954.14	0.989546	1–100
α-Alanine	ALA	Y=0.00126224X+0.000715378	0.994383	1–200
Cysteine	CYS	Y=3843.73X+1982.53	0.990827	1–400
Aspartic acid	ASP	Y=0.0654146X+0.0304666	0.990312	1–200
Proline	PRO	Y=0.00709628X+0.00134925	0.998382	1–200
Serine	SER	Y=0.0309731X+0.0540789	0.984065	1–200
Glutamic acid	GLU	Y=0.0162808X+0.00381015	0.991416	1–200
Arginine	ARG	Y=224.395X-14.4734	0.984827	1–400
Citrulline	CIT	Y=3457.03X+2110.79	0.980604	1–200
Ornithine	ORN	Y=2182.65+430.424	0.994599	1–100
Asparagine	ASN	Y=0.0216939X+0.0201025	0.993572	1–200
Glutamine	GLN	Y=5534.14X-2200.6258	0.996794	1–400
α-Aminobutyric acid	α-GABA	Y=24399.5X+4298.92	0.992687	1–100
β-Aminobutyric acid	β-GABA	Y=27933.8X+2842.25	0.989107	1–100
γ-Aminobutyric acid	γ-GABA	Y=23135.9X+3004.45	0.992405	1–100
Anserine	ANS	Y=460.044X-12.3337	0.980254	1–200
Taurine	TAU	Y=48.1016X-14.2997	0.996904	1–400
Ethanolamine	ETH	Y=8821.76X+6399.15	0.983612	1–200
α-Aminoadipic acid	AMADP	Y=16775.6X+3103.56	0.986693	1–200

Table 3 Amino Acid Concentration in Serum of Healthy Volunteers and T2DM Patients

	Amino Acid	Healthy	T2DM
1	THR	369.59±16.81	328.47± 14.60
2	LEU	50.59±2.59	63.86±1.82***
3	ILE	104.36±5.78	128.00±4.90**
4	VAL	198.85±4.32	228.98±6.68***
5	PHE	69.64±1.73	66.49±1.51
6	MET	24.61±0.95	26.72±1.16
7	TRP	53.83±2.53	50.52±3.57
8	LYS	198.80±11.04	217.76±10.27
9	OH-LYS	7.72±0.69	6.65±0.51
10	GLY	425.86±48.08	262.97±33.98**
11	TYR	66.16±2.11	62.91±3.79
12	HIS	96.62±14.29	102.75±4.17
13	1-MEHIS	3.82±0.24	4.94±0.33**
14	3-MEHIS	4.21±0.37	5.64±0.54*
15	ALA_B	21.41±0.75*	14.00±0.50***
16	ALA	668.50±28.21	537.93±26.95**
17	CYS	52.85±3.59	60.57±4.98
18	ASP	9.57±0.85	3.99±0.23***
19	PRO	206.09±10.69	257.78±19.73*
20	SER	163.42±5.33	153.32±5.02
21	GLU	90.98±2.84	37.25±2.11***
22	ARG	16.52±2.08	47.50±5.06***
23	ASN	63.03±2.42	68.81±2.47
24	GLN	837.11±21.89	537.57±28.35***
25	α-GABA	0.81±0.06	0.16±0.03***
26	β-GABA	1.74±0.22	1.55±0.17
27	γ-GABA	27.21±1.70	39.77±2.69***
28	ANS	0.096±0.02	0.15±0.03
29	TAU	1105.616±63.42	188.80±16.46***
30	ETH	38.20±1.78	17.50±0.89***
31	CIT	38.13±2.40	36.14±2.79
32	SAR	489.45±8.74	454.24±7.55**
33	AMADP	1.04±0.12	1.31±0.13
34	ORN	1190.06±59.90	769.36±63.53***

Notes: All data were presented with Means±SEM, *P<0.05, **P<0.01, ***P<0.001 compared with Health group.

Figure 3 Quantitative analysis of 8 significant changed amino acids. A two-tailed Mann–Whitney U-test was applied to test for statistical significance; The asterisk denotes a significance, ****p < 0.0001. The content of ARG (A), LEU (B), TAU (C), ETH (D), GLU (E), GLN (F), beta-ALA (G) AND alfa-GABA (H) in the serum of healthy volunteers and T2DM patients.

Figure 4 The heatmap of association between physiological parameters and 14 significantly changed amino acids. Red indicates positive correlations, and blue indicates negative correlations. The asterisk denotes a significance, *p < 0.05, **p < 0.01.