Metabolomics studies identify novel diagnostic and prognostic indicators in patients with alcoholic hepatitis

Table 1 Characteristic precursor-product transitions

	Name	Precursor	Product
Analytes	Trimethylamine N-oxide	76	58
	Choline	104	60
	Betaine	118	59
	Valine	118	72
	Leucine	86	43
	Isoleucine	86	56
	Ornithine	133	70
	Crotonobetaine	144	59
	Butyrobetaine	146	60
	Lysine	147	84
	Methyl-lysine	161	84
	Carnitine	162	60
	Phenylalanine	166	120
	Arginine	175	70
	Citrulline	176	70
	Tyrosine	182	136
	Methyl-arginine	189	70
	Symmetric dimethyl-arginine	203	70
	Asymmetric dimethylarginine	203	70
	Acetyl-carnitine	204	85
	Propionyl-carnitine	218	85
	Butyryl-carnitine	232	85
	Pentanoyl-carntine	246	85
	Hexanoyl-carnitine	260	85
	Octenoyl-carnitine	286	85
Internal standard	Trimethylamine N-oxide-d9	85	66
	Choline-trimethyl-d9	113	69
	Betaine-trimethyl-d9	127	68
	Valine-13C5, 15N1	124	77
	Leucine-13C6, 15N1	139	92
	Ornithine 3, 3, 4, 4, 5, 5-d6	139	76
	Crotonobetaine-trimethyl-d9	153	68
	Butyrobetaine-trimethyl-d9	155	69
	Lysine-u-13C6, 15N2	155	90
	Phenylalanine-13C6	172	126
	Citrulline 2, 3, 4, 5-d4	180	74
	Arginine-13C6	181	74
	Tyrosine-u-13C9, 15N1	192	145
	Asymmetric dimethylarginine 2, 3, 3, 4, 4, 5, 5-d7	210	77
	Acetyl-carnitine-d3	207	85
	Propionyl-carnitine-d3	221	85
	butyryll-carnitine-d3	235	85
	Pentanoyl-carnitine-d9	246	85
	Hexanoyl-carnitine-d3	263	85

Table 2 Patient characteristics

Factor	Cirrhosis with acute decompensation from etiologies other than alcohol (n = 23)		Alcoholic hepatitis (n = 25)		P-value
	n	Summary	n	Summary
Age (yr)	14	53.8 ± 9.8	20	51.5 ± 10.4	0.511
Male	21	13 (61.9)	23	10 (43.5)	0.223
Caucasian	10	10 (100.0)	19	16 (84.2)	0.534
AST	23	40.0 (33.0, 75.0)	25	138.0 (88.0, 161.0)	< 0.0012b
ALT	23	21.0 (15.0, 30.0)	25	51.0 (42.0, 71.0)	< 0.0012b
Bilirubin	23	3.8 (1.4, 6.0)	25	9.4 (6.8, 21.7)	0.0052b
Albumin	23	2.8 ± 0.66	25	2.8 ± 0.68	0.861
INR	23	1.5 ± 0.41	25	1.7 ± 0.59	0.161
PT	23	16.4 ± 4.5	25	19.1 ± 6.1	0.0951
Creatinine	23	0.94 (0.74, 1.5)	25	0.72 (0.57, 1.04)	0.0622
MELD score	23	16.4 ± 8.8	25	20.5 ± 10.0	0.141
Maddrey's score	16	22.2 (16.0, 37.0)	17	43.5 (34.0, 60.6)	0.0282a
Ascites	23		25		0.142
None		9 (39.1)		14 (56.0)
Small		4 (17.4)		6 (24.0)
Large		9 (39.1)		4 (16.0)
Severe		1 (4.3)		1 (4.0)
HE	23		25		0.942
None		2 (8.7)		6 (24.0)
Mild		12 (52.2)		7 (28.0)
Severe		9 (39.1)		12 (48.0)
Steroids	23	2 (8.7)	25	13 (52.0)	0.0013b
Trental	23	3 (13.0)	25	9 (36.0)	0.0673
OLT	23	2 (8.7)	25	1 (4.0)	0.604
Deceased	23	6 (26.1)	25	8 (32.0)	0.653

P-values were calculated using the test corresponding to superscript characters:

¹ANOVA;

²Kruskal-Wallis test;

³Pearson’s χ² test;

⁴Fisher’s exact test.

^aP < 0.05 and

^bP < 0.01. AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; MELD: Model for end-stage liver disease; OLT: Orthotopic liver transplantation; PT: Prothrombin time; INR: International normalized ratio; HE: Hepatic encephalopathy.

Table 3 Model for end-stage liver disease-adjusted average biomarker levels

Biomarker (μmol/L)	Cirrhosis with acute decompensation from etiologies other than alcohol (n = 23)	Alcoholic hepatitis (n = 25)	P-value
Choline	5.8 (4.7, 7.2)	7.0 (5.7, 8.6)	0.22
TMAO	0.74 (0.34, 1.6)	0.87 (0.42, 1.8)	0.76
Carnitine	37.2 (32.1, 43.1)	32.6 (28.3, 37.5)	0.2
Betaine	83.6 (64.7, 108.2)	134.0 (104.7, 171.5)	0.012a
Butyrobetaine	1.6 (1.3, 1.8)	1.7 (1.4, 2.0)	0.46
Crotonobetaine	0.12 (0.10, 0.15)	0.14 (0.12, 0.18)	0.35
Creatinine	92.0 (75.0, 112.9)	59.3 (48.8, 72.1)	0.003b
Ornithine	71.6 (59.1, 86.9)	62.0 (51.5, 74.6)	0.29
Lysine	131.3 (112.7, 153.1)	134.8 (116.4, 156.2)	0.81
Methyl-lysine	4.0 (2.9, 5.6)	3.4 (2.5, 4.7)	0.46
Argine	61.7 (52.0, 73.2)	61.8 (52.4, 72.8)	0.99
Citrulline	40.2 (33.2, 48.6)	23.7 (19.7, 28.5)	< 0.001b
MMA	0.24 (0.20, 0.27)	0.21 (0.19, 0.24)	0.32
Homocitrulline	0.73 (0.55, 0.97)	0.37 (0.28, 0.48)	0.001b
Leucine	52.8 (44.0, 63.4)	48.8 (40.9, 58.1)	0.54
Iso-leucine	27.1 (21.7, 33.8)	28.0 (22.6, 34.6)	0.83
Valine	115.4 (100.4, 132.7)	101.8 (89.1, 116.4)	0.2
Phenylalanine	90.4 (78.0, 104.7)	60.2 (52.3, 69.3)	< 0.001b
Tyrosine	166.0 (126.8, 217.3)	107.4 (83.0, 139.1)	0.025a
Acetyl-carnitine	17.8 (14.9, 21.1)	16.9 (14.3, 20.0)	0.69
Propionyl-carnitine	1.02 (0.76, 1.4)	1.2 (0.88, 1.5)	0.5
Butyryl-carnitine	2.0 (1.6, 2.5)	2.2 (1.8, 2.7)	0.58
Trimethyl-Lysine	1.00 (0.81, 1.2)	1.1 (0.92, 1.4)	0.42
SDMA	0.81 (0.65, 1.02)	0.58 (0.47, 0.72)	0.042a
Dimethyl-Lysine	0.92 (0.72, 1.2)	0.74 (0.59, 0.94)	0.22
ADMA	0.90 (0.78, 1.03)	0.82 (0.72, 0.93)	0.32
Pentanoyl-carnitine	0.25 (0.20, 0.31)	0.27 (0.22, 0.34)	0.48
Hexanoyl-carnitine	0.69 (0.56, 0.85)	0.61 (0.50, 0.74)	0.38
Octenoyl-carnitine	0.05 (0.02, 0.12)	0.01 (0.00, 0.02)	0.009b

Values presented as mean (95%CI) and P-values obtained from analysis of covariance. The natural logarithm of each biomarker was modeled as the outcome variable with disease group and MELD as the independent variables.

^aP < 0.05,

^bP < 0.01. ADMA: Asymmetric dimethylarginine; SDMA: Symmetric dimethylarginine; MMA: Monomethylarginine; TMAO: Trimethylamine N-oxide.

Table 4 Utility of biomarkers in differentiating cirrhosis with alcoholic hepatitis from cirrhosis with acute decompensation from etiologies other than alcohol: Receiver operating characteristics analysis

Biomarker	AUC (95%CI)
Citrulline and betaine	0.835 (0.747, 0.978)
Betaine and phenylalanine	0.810 (0.684, 0.937)
Citrulline and phenylalanine	0.758 (0.609, 0.907)
Citrulline	0.758 (0.610, 0.907)
Betaine	0.732 (0.588, 0.877)
Phenylalanine	0.715 (0.567, 0.863)
Crotonobetaine	0.663 (0.498, 0.827)
Tyrosine	0.650 (0.484, 0.817)
Butyrobetaine	0.649 (0.489, 0.808)
Creatinine	0.645 (0.486, 0.805)
Octenoyl-carnitine	0.631 (0.488, 0.774)
Propionyl-carnitine	0.620 (0.453, 0.787)
Homocitrulline	0.618 (0.457, 0.779)
Trimethyl-lysine	0.616 (0.447, 0.784)
Butyryl-carnitine	0.615 (0.451, 0.778)
Pentanoyl-carnitine	0.613 (0.455, 0.771)
Choline	0.597 (0.432, 0.762)
Valine	0.588 (0.424, 0.752)
Lysine	0.559 (0.393, 0.725)
Methyl-lysine	0.552 (0.383, 0.721)
Iso-leucine	0.548 (0.379, 0.716)
Acetyl-carnitine	0.543 (0.375, 0.710)
Hexanoyl-carnitine	0.529 (0.362, 0.696)
TMAO	0.521 (0.347, 0.695)
Argine	0.507 (0.339, 0.675)
ADMA	0.481 (0.314, 0.647)
Leucine	0.477 (0.309, 0.644)
Carnitine	0.470 (0.301, 0.638)
MMA	0.453 (0.286, 0.620)
SDMA	0.447 (0.277, 0.617)
Dimethyl-lysine	0.417 (0.243, 0.592)
Ornithine	0.351 (0.186, 0.517)

AUC: Area under receiver operating characteristics curve; ADMA: Asymmetric dimethylarginine; SDMA: Symmetric dimethylarginine; MMA: Monomethylarginine; TMAO: Trimethylamine N-oxide.

Table 5 Correlations between biomarkers and model for end-stage liver disease and Maddrey’s score in patients with alcoholic hepatitis

Biomarker	Alcoholic hepatitis
	MELD		Maddrey’s score
	rho (95%CI)	P-value	rho (95%CI)	P-value
Choline	0.28 (-0.13, 0.69)	0.18	0.02 (-0.53, 0.57)	0.95
TMAO	-0.25 (-0.67, 0.17)	0.23	0.29 (-0.24, 0.82)	0.26
Carnitine	0.54 (0.18, 0.91)	0.005b	0.48 (-0.01, 0.96)	0.054
Betaine	0.24 (-0.18, 0.65)	0.26	-0.00 (-0.55, 0.55)	0.99
Butyrobetaine	0.30 (-0.11, 0.71)	0.15	0.29 (-0.24, 0.81)	0.26
Crotonobetaine	0.07 (-0.36, 0.50)	0.74	-0.03 (-0.58, 0.52)	0.91
Creatinine	0.44 (0.05, 0.83)	0.027a	0.48 (-0.00, 0.96)	0.052
Ornithine	0.28 (-0.13, 0.70)	0.17	0.26 (-0.27, 0.79)	0.32
Lysine	0.37 (-0.03, 0.77)	0.068	0.27 (-0.25, 0.80)	0.29
Methyl-lysine	0.09 (-0.34, 0.52)	0.67	-0.01 (-0.56, 0.54)	0.96
Argine	0.00 (-0.43, 0.44)	0.98	0.42 (-0.08, 0.92)	0.095
Citrulline	0.09 (-0.34, 0.52)	0.66	0.05 (-0.50, 0.60)	0.86
MMA	0.34 (-0.07, 0.74)	0.098	0.39 (-0.11, 0.90)	0.12
Homocitrulline	0.66 (0.34, 0.99)	< 0.001b	0.59 (0.14, 1.00)	0.014a
Leucine	0.03 (-0.40, 0.46)	0.89	0.50 (0.02, 0.97)	0.043
Iso-leucine	0.11 (-0.32, 0.54)	0.59	0.29 (-0.24, 0.82)	0.26
Valine	0.20 (-0.22, 0.62)	0.34	0.54 (0.08, 1.00)	0.025a
Phenylalanine	0.34 (-0.06, 0.75)	0.092	0.56 (0.11, 1.00)	0.018a
Tyrosine	0.30 (-0.11, 0.71)	0.14	0.44 (-0.05, 0.94)	0.074
Acetyl-carnitine	0.49 (0.11, 0.86)	0.014a	0.50 (0.03, 0.98)	0.04a
Propionyl-carnitine	0.40 (0.01, 0.80)	0.046a	0.28 (-0.25, 0.81)	0.28
Butyryl-carnitine	0.48 (0.10, 0.86)	0.016a	0.25 (-0.29, 0.78)	0.34
Trimethyl-lysine	0.48 (0.11, 0.86)	0.014a	0.38 (-0.13, 0.89)	0.14
SDMA	0.38 (-0.02, 0.78)	0.064	0.55 (0.09, 1.00)	0.023a
Dimethyl-lysine	0.31 (-0.10, 0.72)	0.13	0.18 (-0.37, 0.72)	0.5
ADMA	0.42 (0.03, 0.81)	0.037a	0.60 (0.16, 1.00)	0.011a
Pentanoyl-carnitine	0.53 (0.16, 0.90)	0.007b	0.31 (-0.22, 0.83)	0.23
Hexanoyl-carnitine	0.49 (0.12, 0.87)	0.013a	0.40 (-0.11, 0.90)	0.11
Octenoyl-carnitine	0.45 (0.07, 0.84)	0.024a	0.23 (-0.30, 0.77)	0.37

Values presented as mean (95%CI) and P-values obtained from analysis of covariance. The natural logarithm of each biomarker was modeled as the outcome variable with disease group and MELD as the independent variables. A superscript of a indicates

^aP < 0.05,

^bP < 0.01. rho: Spearman’s correlation; MELD: Model for end-stage liver disease; ADMA: Asymmetric dimethylarginine; SDMA: Symmetric dimethylarginine; MMA: Monomethylarginine; TMAO: Trimethylamine N-oxide.

Table 6 Model for end-stage liver disease-adjusted average biomarker levels

Biomarker (μmol/L)	Alcoholic cirrhosis (n = 23)	Alcoholic hepatitis (n = 25)	Holm-bonferroni corrected P-value
Citrulline	40.2 (33.2, 48.6)	23.7 (19.7, 28.5)	0.009b
Phenylalanine	90.4 (78.0, 104.7)	60.2 (52.3, 69.3)	0.009b
Homocitrulline	0.73 (0.55, 0.97)	0.37 (0.28, 0.48)	0.029a
Creatinine	92.0 (75.0, 112.9)	59.3 (48.8, 72.1)	0.087
Octenoyl-carnitine	0.05 (0.02, 0.12)	0.01 (0.00, 0.02)	0.26
Betaine	83.6 (64.7, 108.2)	134.0 (104.7, 171.5)	0.35
Tyrosine	166.0 (126.8, 217.3)	107.4 (83.0, 139.1)	0.73
SDMA	0.81 (0.65, 1.02)	0.58 (0.47, 0.72)	0.99
Carnitine	37.2 (32.1, 43.1)	32.6 (28.3, 37.5)	0.99
Valine	115.4 (100.4, 132.7)	101.8 (89.1, 116.4)	0.99
Choline	5.8 (4.7, 7.2)	7.0 (5.7, 8.6)	0.99
Dimethyl-lysine	0.92 (0.72, 1.2)	0.74 (0.59, 0.94)	0.99
Ornithine	71.6 (59.1, 86.9)	62.0 (51.5, 74.6)	0.99
MMA	0.24 (0.20, 0.27)	0.21 (0.19, 0.24)	0.99
ADMA	0.90 (0.78, 1.03)	0.82 (0.72, 0.93)	0.99
Crotonobetaine	0.12 (0.10, 0.15)	0.14 (0.12, 0.18)	0.99
Hexanoyl-carnitine	0.69 (0.56, 0.85)	0.61 (0.50, 0.74)	0.99
Trimethyl-lysine	1.00 (0.81, 1.2)	1.1 (0.92, 1.4)	0.99
Butyrobetaine	1.6 (1.3, 1.8)	1.7 (1.4, 2.0)	0.99
Methyl-lysine	4.0 (2.9, 5.6)	3.4 (2.5, 4.7)	0.99
Pentanoyl-carnitine	0.25 (0.20, 0.31)	0.27 (0.22, 0.34)	0.99
Propionyl-carnitine	1.02 (0.76, 1.4)	1.2 (0.88, 1.5)	0.99
Leucine	52.8 (44.0, 63.4)	48.8 (40.9, 58.1)	0.99
Butyryl-carnitine	2.0 (1.6, 2.5)	2.2 (1.8, 2.7)	0.99
Acetyl-carnitine	17.8 (14.9, 21.1)	16.9 (14.3, 20.0)	0.99
TMAO	0.74 (0.34, 1.6)	0.87 (0.42, 1.8)	0.99
Lysine	131.3 (112.7, 153.1)	134.8 (116.4, 156.2)	0.99
Iso-leucine	27.1 (21.7, 33.8)	28.0 (22.6, 34.6)	0.99
Argine	61.7 (52.0, 73.2)	61.8 (52.4, 72.8)	0.99

Values presented as mean (95%CI) and P-values obtained from analysis of covariance. The natural logarithm of each biomarker was modeled as the outcome variable with disease group and MELD as the independent variables. A superscript of a indicates

^aP < 0.05,

^bP < 0.01. ADMA: Asymmetric dimethylarginine; SDMA: Symmetric dimethylarginine; MMA: Monomethylarginine; TMAO: Trimethylamine N-oxide.

Table 7 Utility of biomarkers in predicting transplant-free survival

Biomarker	3-mo survival	6-mo survival
(μmol/L)	AUC (95%CI)	AUC (95%CI)
MELD	0.82 (0.69, 0.95)	0.77 (0.62, 0.92)
Tyrosine	0.91 (0.74, 1.0)	0.89 (0.74, 1.0)
Phenylalanine	0.77 (0.56, 0.97)	0.79 (0.61, 0.98)
Carnitine	0.73 (0.53, 0.93)	0.74 (0.56, 0.93)
ADMA	0.72 (0.49, 0.96)	0.70 (0.48, 0.92)
MMA	0.71 (0.47, 0.94)	0.70 (0.49, 0.91)

AUC: Area under receiver operating characteristics curve; MELD: Model for end-stage liver disease; ADMA: Asymmetric dimethylarginine; MMA: Monomethylarginine.