Profiling analysis of amino acids from hyperlipidaemic rats treated with Gynostemma pentaphyllum and atorvastatin

Figures & data

Figure 1. Representative UPLC/DAD chromatograms. (A) Blank solvent, (B) 12 amino acid derivatives of standard solution, (C) amino acid derivatives in a representative plasma sample, (D) amino acid derivatives in a representative liver homogenate sample. (1) Glutamic acid; (2) serine; (3) glycine; (4) arginine; (5) is; (6) tyrosine; (7) valine; (8) methionine; (9) isoleucine; (10) leucine; (11) phenylalanine; (12) tryptophan; (13) lysine.

Table 1. Calibration curves of 12 amino acids derivatives for plasma.

Amino acids	Linear equation	r	Linear range, μg/mL	LOQ, μg/mL
Glutamic acid	y = 4.54x + 0.411	0.995	0.47–30	0.47
Serine	y = 5.80x − 0.258	0.997	4.30–275	4.30
Glycine	y = 6.74x + 0.822	0.992	3.91–250	3.91
Arginine	y = 7.97x − 0.801	0.996	3.52–225	3.52
Tyrosine	y = 15.1x + 3.49	0.999	11.52–738	11.52
Valine	y = 7.15x − 0.644	0.992	5.85–375	5.85
Methionine	y = 6.42x + 0.571	0.991	2.54–162	2.54
Isoleucine	y = 5.74x + 0.642	0.992	3.9–250	3.9
Leucine	y = 5.21x − 0.248	0.993	5.86–374	5.86
Phenylalanine	y = 7.64x + 0.843	0.998	3.9–250	3.9
Tryptophan	y = 4.45x − 0.254	0.990	0.97–62.4	0.97
Lysine	y = 13.2x + 1.11	0.992	0.78–50	0.78

Table 2. Calibration curves of 12 amino acids derivatives for liver.

Amino acids	Linear equation	r	Linear range, μg/mL	LOQ, μg/mL
Glutamic acid	y = 4.64x + 0.221	0.992	0.47–30	0.47
Serine	y = 5.45x − 0.323	0.993	4.30–275	4.30
Glycine	y = 6.44x + 0.121	0.994	3.91–250	3.91
Arginine	y = 7.42x − 0.081	0.990	3.52–225	3.52
Tyrosine	y = 14.8x + 2.04	0.993	11.52–738	11.52
Valine	y = 7.24x − 0.721	0.991	5.85–375	5.85
Methionine	y = 6.53x + 0.621	0.992	2.54–162	2.54
Isoleucine	y = 5.34x + 0.387	0.998	3.9–250	3.9
Leucine	y = 5.30x − 0.568	0.997	5.86–374	5.86
Phenylalanine	y = 7.04x + 0.541	0.992	3.9–250	3.9
Tryptophan	y = 4.95x − 0.742	0.993	0.97–62.4	0.97
Lysine	y = 13.6x + 1.41	0.998	0.78–50	0.78

Figure 2. PR analysis of the UPLC/DAD spectra of rat plasma. (A) PCA analysis of the spectra of plasma from normal and model rats (R²X = 0.752, Q²=0.574). (B) Scores plot of the PLS-DA analysis of the spectra from the plasma of normal and hyperlipidaemic rats (R²X = 0.749, R²Y = 0.939, Q²=0.92). (C) Scores plot of the PLS-DA analysis of the spectra from the plasma of normal, hyperlipidaemic and GP-treated rats (R²X = 0.776, R²Y = 0.822, Q²=0.738). (D) Scores plot of the PLS-DA analysis of the spectra from the plasma of normal, hyperlipidaemic and atorvastatin-treated rats (R²X = 0.777, R²Y = 0.758, Q²=0.668). (E) Loading plot of the PLS-DA analysis of the spectra from the plasma of normal and hyperlipidaemic rats (box: control group; dot: model group; diamond: GP-treated group; star: atorvastatin-treated group).

Figure 3. PR analysis of the UPLC/DAD spectra of rat liver. (A) PCA analysis of the spectra of liver from normal and model rats (R²X = 0.907, Q²=0.555). (B) Scores plot of the PLS-DA analysis of the spectra from the liver of normal and hyperlipidaemic rats (R²X = 0.777, R²Y = 0.934, Q²=0.902). (C) Scores plot of the PLS-DA analysis of the spectra from the liver of normal, hyperlipidaemic and GP-treated rats (R²X = 0.828, R²Y = 0.811, Q²=0.581). (D) Scores plot of the PLS-DA analysis of the spectra from the liver of normal, hyperlipidaemic and atorvastatin-treated rats (R²X = 0.755, R²Y = 0.773, Q²=0.569). (E) Loading plot of the PLS-DA analysis of the spectra from the liver of normal and hyperlipidaemic rats. (box: control group; dot: model group; diamond: GP-treated group; star: atorvastatin-treated group).

Table 3. Quantitative analysis of amino acids of plasma in four groups of rats (x  ± s, μg/mL).

					Hyperlipidemia versus control		GP versus hyperlipidaemia		Atorvastatin versus hyperlipidaemia
Amino acids	Control	Hyperlipidaemia	GP	Atorvastatin	Change	p value	Change	p value	Change	p value
Glu	3.24 ± 0.5	3.55 ± 0.7	3.17 ± 0.6	3.46 ± 0.7	–	0.168	–	0.258	–	0.321
Ser	30.8 ± 4.8	31.9 ± 5.0	32.5 ± 5.5	32.1 ± 4.7	–	0.285	–	0.542	–	0.551
Gly	27.5 ± 4.0	19.5 ± 3.6	22.8 ± 4.1	21.4 ± 4.4	↓	<0.001	↑	0.036	↑	0.022
Arg	25.5 ± 3.8	26.9 ± 4.2	27.1 ± 3.4	2.63 ± 4.3	–	0.452	–	0.345		0.655
Tyr	89.5 ± 11	134 ± 12.1	98.6 ± 9.5	122 ± 19.2	↑	<0.001	↓	0.039	↓	0.045
Val	43.4 ± 6.8	38.6 ± 4.8	35.8 ± 5.8	42.1 ± 6.2	↓	0.028	–	0.064	↑	0.042
Met	14.8 ± 2.1	15.1 ± 3.2	14.6 ± 3.5	15.9 ± 2.8	–	0.098	–	0.341	–	0.574
Ile	23.4 ± 4.0	20.6 ± 4.8	22.8 ± 5.4	21.0 ± 5.1	↓	0.031	–	0.539	–	0.416
Leu	40.8 ± 7.9	45.8 ± 8.2	43.2 ± 9.5	41.4 ± 7.1	↓	0.041	–	0.485	–	0.320
Phe	26.7 ± 3.5	28.4 ± 4.7	29.6 ± 3.6	27.5 ± 4.3	–	0.095	–	0.157	–	0.183
Try	6.51 ± 1.0	6.08 ± 1.1	6.40 ± 1.2	5.88 ± 1.5	–	0.145	–	0.275	–	0.315
Lys	4.81 ± 1.1	3.85 ± 1.1	4.03 ± 0.9	3.95 ± 1.0	↓	0.031	–	0.896	↑	0.026

“↑” and “↓” represented the Amino acid is up- or down-regulated, “–” represent the compound has no significant change.

P-values determined using paired-sample t-test, p-values less than 0.05 were considered significant.

Table 4. Quantitative analysis of amino acids of liver in four groups of rats (x  ± s, μg/mL).

							GP versus hyperlipidaemia		Atorvastatin versus hyperlipidaemia
Amino acids	Control	Hyperlipidaemia	GP	Atorvastatin	Change	p value	Change	p value	Change	p value
Glu	18.1 ± 3.5	15.8 ± 2.8	14.8 ± 3.6	12.9 ± 3.4	↓	0.041	–	0.819	–	0.140
Ser	153 ± 34.2	159 ± 30.2	165 ± 38.9	161 ± 35.3	–	0.072	–	0.124	–	0.251
Gly	138 ± 25.4	130 ± 22.9	131 ± 24.0	135 ± 21.8	–	0.285	–	0.584	–	0.621
Arg	104 ± 20.4	88.9 ± 16.9	92.8 ± 18.2	98 ± 16.1	↓	0.032	–	0.904	↑	0.032
Tyr	443 ± 78.8	521 ± 89.1	511 ± 95.8	514 ± 101	↑	0.003	–	0.379	–	0.818
Val	217 ± 39.0	152 ± 29.5	162 ± 33.4	173 ± 41.4	↓	0.006	–	0.150	↑	0.029
Met	71.2 ± 15.1	75.4 ± 14.5	80.1 ± 22.1	72.5 ± 21.8	–	0.364	–	0.712	–	0.854
Iso	140 ± 25.6	136 ± 30.4	133 ± 28.0	131 ± 29.4	–	0.188	–	0.615	–	0.369
Leu	193 ± 40.6	186 ± 42.8	181 ± 35.6	183 ± 38.5	–	0.280	–	0.654	–	0.341
Phe	134 ± 24.1	86.8 ± 14.5	91.3 ± 19.8	124 ± 20.1	↓	0.002	–	0.090	↑	0.032
Try	32.3 ± 6.4	36.4 ± 8.2	38.2 ± 7.9	39.1 ± 7.5	–	0.351	–	0.360	–	0.695
Lys	21.6 ± 6.4	18.1 ± 4.1	18.6 ± 5.0	19.2 ± 5.4	↓	0.021	–	0.082	–	0.632

“↑” and “↓” represented the Amino acid is up- or down-regulated; “–” represent the compound has no significant change. p values determined using paired-sample t-test, p values less than 0.05 were considered significant.

Figure 4. A summary of the related metabolic pathways for amino acids that changed significantly in hyperlipidaemic group. “↑” and “↓” represent the compound is up- and down-regulated compared with the control group.