Differentiation of minimal and grade 1 hepatic encephalopathy using neurophysiological, neuropsychological, magnetic resonance spectroscopic, and systemic inflammatory parameters

Table 1 Baseline characteristics, n (%)/mean ± SD

Characteristic	No HE (n = 50)	Minimal HE (n = 50)	Grade 1 HE (n = 50)	P value
Age, years	44.28 ± 9.15	46.7 ± 11.02	44.18 ± 11.29	0.401
Gender, male	41 (82)	42 (84)	45 (90)
Etiology
Alcohol	18 (36)	17 (34)	26 (52)	0.264
MASLD	9 (18)	10 (20)	5 (10)
HBV	3 (6)	10 (20)	4 (8)
Idiopathic	5 (10)	3 (6)	4 (8)
HCV	4 (8)	2 (4)	4 (8)
Others	11 (22)	8 (16)	7 (14)
Baseline investigations
Hemoglobin (g/dL)	9.32 ± 2.17	8.81 ± 1.62	8.91 ± 1.86	0.363
TLC (cells/μL)	5612.6 ± 2970	6606 ± 4144	7804 ± 6057	0.582
Platelet count (cells/μL)	97180 ± 67191	100240 ± 47215	100200 ± 53333	0.549
Blood urea (mg/dL)	31.58 ± 11.15	37.24 ± 12.95	38.26 ± 12.63	0.015
Serum creatinine (mg/dL)	0.89 ± 0.23	1.02 ± 0.25	1.03 ± 0.3	0.013
Total protein (g/dL)	7.4 ± 0.66	6.82 ± 0.67	6.65 ± 0.91	< 0.001
Serum albumin (g/dL)	3.3 ± 0.57	2.86 ± 0.49	2.79 ± 0.51	< 0.001
Total bilirubin (mg/dL)	2.14 ± 2.26	4.1 ± 3.74	4.42 ± 3.89	< 0.001
AST (U/L)	86.14 ± 82.93	89.64 ± 40.5	110.8 ± 76.02	0.005
ALT (U/L)	46.3 ± 54.15	43.54 ± 18.73	49.84 ± 31.1	0.188
ALP (U/L)	157.96 ± 56.93	161.82 ± 86.38	160.52 ± 62.11	0.943
Sodium (mEq/L)	134.66 ± 5.02	133.54 ± 5.09	131.9 ± 4.59	0.02
Potassium (mEq/L)	4.15 ± 0.42	4.16 ± 0.62	4.15 ± 0.62	0.997
INR	1.23 ± 0.21	1.48 ± 0.37	1.65 ± 0.55	< 0.001
Serum ammonia (μg/dL)	87.18 ± 19.12	123.42 ± 19.97	164.08 ± 30.12	< 0.001
Baseline prognostic scores, PHES and CFF
MELD	11.28 ± 3.17	15.8 ± 4.36	17.58 ± 5.8	0.001
CTP score	7.9 ± 1.09	10.3 ± 1.56	11.16 ± 1.5	0.001
CTP grade (A/B/C), n	1/44/5	0/12/38	0/7/43	0.001
PHES, median (interquartile range)	-1 (-2 to 1)	-5 (-6 to -5)	-8 (-9 to -7)	< 0.001
CFF (Hz)	42.82 ± 2.99	37.52 ± 2.07	34 ± 3.55	< 0.001
Inflammatory markers
TNF-α (pg/mL)	25.33 ± 10.81	47.08 ± 13.99	64.31 ± 28,39	< 0.001
IL-6 (pg/mL)	22.68 ± 16.79	69.92 ± 42.23	113.92 ± 46.13	< 0.001
IL-18 (pg/mL)	171.53 ± 61.58	348.01 ± 86.26	519.41 ± 178.71	< 0.001
Endotoxin (EU/mL)	0.37 ± 0.05	0.47 ± 0.04	0.6 ± 0.05	< 0.001
IL-10 (pg/mL)	8.19 ± 2.19	13.72 ±2	16.43 ± 1.86	< 0.001
IL-12 (pg/mL)	6.9 ± 2.23	12.97 ± 2.11	17.77 ± 2.74	< 0.001
IL-1β (pg/mL)	5.1 ± 15.6	10.62 ± 1.81	14.95 ± 2.27	< 0.001
MRS peaks
Myoinositol (mmol/L), median (interquartile range)	0.1 (0.02-0.2)	-0.11 (-0.2 to -0.08)	-0.2 (-0.3 to -0.16)	< 0.001
Choline (mmol/L)	0.71 ± 0.18	0.42 ± 0.18	0.38 ± 0.33	< 0.001
Glutamine (mmol/L)	0.41 ± 0.15	0.73 ± 0.23	1.01 ± 0.38	< 0.001
Bispectral index
Bispectral index value	91.18 ± 3.43	81.48 ± 3.9	72.9 ± 5.49	< 0.001
Spectral EEG analysis
MDF (Hz)	12.9 ± 4.66	7.89 ±1.19	5.13 ± 1.57	< 0.001
Relative power of frequency bands
Beta (%)	24.54 ± 8.73	12.76 ± 5.97	10.3 ± 5.91	< 0.001
Alpha (%)	48.5 ± 13.16	24 ± 10.6	16 ± 8.15	< 0.001
Theta (%)	11.6 ± 4.34	30.08 ± 12.98	24.7 ±12.9	< 0.001
Delta (%)	13.74 ± 6.73	31.42 ± 14.23	46.96 ± 17.56	< 0.001
Grade of EEG alteration
Grade 0	50 (100)	23 (46)	3 (6)	< 0.001
Grade 1	0 (0)	11 (22)	0 (0)
Grade 2	0 (0)	12 (24)	17 (34)
Grade 3	0 (0)	4 (8)	30 (60)

HE: Hepatic encephalopathy; MASLD: Metabolic dysfunction-associated steatotic liver disease; HBV: Hepatitis B virus; HCV: Hepatitis C virus; TLC: Total leukocyte count; ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; INR: International normalized ratio; MELD: Model for end-stage liver disease; CTP: Child Turcotte Pugh; PHES: Psychometric hepatic encephalopathy score; CFF: Critical flicker frequency; TNF: Tumor necrosis factor; IL: Interleukin; MRS: Magnetic resonance spectroscopy; EEG: Electroencephalogram; MDF: Mean dominant frequency.

Table 2 Complications and mortality over 1 year, n (%)

	No HE (n = 50)	Minimal HE (n = 50)	Grade 1 HE (n = 50)	P value
GI bleed	10 (20)	11 (22)	15 (30)	< 0.001
Infections	4 (8)	7 (14)	13 (26)	< 0.001
Progression of HE (to grade ≥ 2)	10 (20)	24 (48)	31 (62)	< 0.001
More than one complication	8 (16)	17 (34)	30 (60)	< 0.001
Hospitalisation	14 (28)	22 (44)	34 (68)	< 0.001
Death	5 (10)	14 (28)	21 (42)	< 0.001

GI: Gastrointestinal; HE: Hepatic encephalopathy.

Table 3 Univariate and multivariate regression analysis to identify significant risk factors of complications

Parameters	Univariate hazard ratio by the Cox regression model		Multivariate hazard ratio
	HR (95%CI)	P value	HR (95%CI)	P value
Age	0.989 (0.968-1.011)	0.328
Hemoglobin	0.966 (0.865-1.080)	0.545
TLC	1.000 (1.000-1.000)	0.026	1.000 (1.000-1.000)	0.597
Platelets	1.000 (1.000-1.000)	0.223
Blood urea	1.017 (1.000-1.035)	0.051
Creatinine	4.429 (2.159-9.086)	< 0.0001	1.434 (0.546-3.765)	0.464
Total protein	0.631 (0.486-0.818)	0.001	0.920 (0.660-1.283)	0.623
Serum albumin	0.489 (0.327-0.732)	0.001	0.950 (0.539-1.676)	0.860
Total bilirubin	1.085 (1.041-1.131)	0.0001	0.954 (0.866-1.052)	0.345
AST	1.006 (1.003-1.008)	< 0.0001	1.004 (0.998-1.010)	0.166
ALT	1.008 (1.002-1.014)	0.007	0.998 (0.986-1.010)	0.998
ALP	1.001 (0.999-1.004)	0.279
Na	0.949 (0.908-0.991)	0.018	0.971 (0.920-1.025)	0.283
K	1.014 (0.678-1.515)	0.947
INR	1.716 (1.253-2.350)	0.001	0.349 (0.159-0.766)	0.009
Serum ammonia	1.008 (1.003-1.014)	0.002	0.993 (0.983-1.004)	0.233
CFF	0.911 (0.868-0.975)	0.0002	0.933 (0.855-1.019)	0.122
MELD	1.110 (1.076-1.145)	< 0.0001	1.209 (1.104-1.322)	< 0.0001
Myoinositol peak	0.154 (0.045-0.525)	0.003	2.330 (0.212-25.657)	0.490
Choline peak	0.334 (0.126-0.887)	0.028	0.994 (0.291-3.396)	0.992
Glutamine peak	2.004 (1.170-3.433)	0.011	0.949 (0.392-2.300)	0.908
TNF-α	1.013 (1.006-1.021)	0.0003	0.995 (0.981-1.010)	0.517
IL-6	1.005 (1.001-1.09)	0.012	0.998 (0.992-1.004)	0.513
IL-18	1.001 (1.000-1.002)	0.008	1.000 (0.997-1.003)	0.939
Endotoxin	43.315 (6.576-285.318)	0.0001	3.181 (0.002-4085.938)	0.751
IL-10	1.136 (1.071-1.204)	< 0.0001	1.145 (0.950-1.381)	0.156
IL-12	1.083 (1.038-1.130)	0.0002	0.823 (0.676-1.002)	0.053
IL-1β	1.101 (1.049-1.156)	0.0001	1.171 (0.967-1.418)	0.107
Gender
Female	1
Male	1.598 (0.799-3.196)	0.185
CTP grade
A	1
B	1654.812 (0.000-4.83E+60)	0.913
C	5289.119 (0.000-1.54E+61)	0.899
Grade of HE
No HE	1
Minimal HE	1.756 (0.957-3.223)	0.069
Grade 1 HE	3.018 (1.696-5.373)	0.0002	3 (1.25-7.20)	0.014

HR: Hazard ratio; CI: Confidence interval; TLC: Total leukocyte count; ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; INR: International normalized ratio; CFF: Critical flicker frequency; MELD: Model for end-stage liver disease; TNF: Tumor necrosis factor; IL: Interleukin; CTP: Child Turcotte Pugh; HE: Hepatic encephalopathy.

Table 4 Univariate and multivariate regression analysis to identify significant risk factors of progression of hepatic encephalopathy (hepatic encephalopathy ≥ grade 2)

Parameters	Univariate hazard ratio by the Cox regression model		Multivariate hazard ratio
	HR (95%CI)	P value	HR (95%CI)	P value
Age	1.009 (0.986-1.032)	0.441
Hemoglobin	1.005 (0.888-1.138)	0.937
TLC	1.000 (1.000-1.000)	0.055
Platelets	1.000 (1.000-1.000)	0.163
Blood urea	1.019 (1.000-1.038)	0.056
Creatinine	3.800 (1.760-8.208)	0.0007	1.293 (0.431-3.880)	0.646
Total protein	0.634 (0.475-0.845)	0.002	0.951 (0.647-1.399)	0.800
Serum albumin	0.497 (0.315-0.784)	0.003	1.123 (0.590-2.139)	0.724
Total bilirubin	1.092 (1.043-1.144)	0.0002	0.924 (0.825-1.034)	0.169
AST	1.006 (1.003-1.008)	0.0002	1.004 (0.999-1.008)	0.523
ALT	1.003 (0.998-1.009)	0.221
ALP	1.002 (0.99-1.005)	0.279
Na	0.945 (0.901-0.992)	0.022	0.969 (0.914-1.028)	0.294
K	0.981 (0.625-1.540)	0.934
INR	1.925 (1.390-2.664)	0.0001	0.443 (0.202-0.969)	0.041
Serum ammonia	1.010 (1.004-1.017)	0.001	0.996 (0.985-1.008)	0.523
CFF	0.909 (0.862-0.959)	0.0004	0.942 (0.855-1.039)	0.232
MELD	1.122 (1.083-1.161)	< 0.0001	1.211	< 0.0001
Myoinositol peak	0.059 (0.015-0.234)	0.0001	0.284 (0.023-3.549)	0.329
Choline peak	0.211 (0.069-0.645)	0.006	0.687 (0.165-2.858)	0.606
Glutamine peak	2.227 (1.244-3.986)	0.007	0.740 (0.287-1.904)	0.532
TNF-α	1.012 (1.005-1.020)	0.002	0.997 (0.981-1.012)	0.668
IL-6	1.005 (1.001-1.010)	0.012	0.998 (0.991-1.004)	0.521
IL-18	1.001 (1.000-1.003)	0.014	1.000 (0.997-1.003)	0.972
Endotoxin	58.094 (6.844-493.151)	0.0002	0.731 (0.000-1717.320)	0.937
IL-10	1.149 (1.075-1.229)	< 0.0001	1.122 (0.906-1.388)	0.291
IL-12	1.089 (1.038-1.142)	0.0004	0.786 (0.623-0.990)	0.041
IL-1β	1.116 (1.057-1.179)	0.0001	1.142 (0.926-1.410)	0.215
Gender
Female	1
Male	1.357 (0.647-2.844)	00.419
CTP grade
A	1
B	1.46E+03 (0.000-1.20E+71)	0.927
C	5.57E+03 (0.000-4.57E+71)	0.914
Grade of HE
No HE	1
Minimal HE	2.783 (1.330-5.822)	0.007	1.499 (0.327-6.878)	0.603
Grade 1 HE	4.200 (2.056-8.580)	0.0001	2.23	00.02

HR: Hazard ratio; CI: Confidence interval; TLC: Total leukocyte count; ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; INR: International normalized ratio; CFF: Critical flicker frequency; MELD: Model for end-stage liver disease; TNF: Tumor necrosis factor; IL: Interleukin; CTP: Child Turcotte Pugh; HE: Hepatic encephalopathy.

Table 5 Univariate and multivariate regression analysis to identify significant risk factors of mortality

Parameters	Univariate hazard ratio by the Cox regression model		Multivariate hazard ratio
	HR (95%CI)	P value	HR (95%CI)	P value
Age	1.025 (0.997-1.054)	0.081
Hemoglobin	0.917 (0.782-1.076)	0.289
TLC	1.000 (1.000-1.000)	0.473
Platelets	1.000 (1.000-1.000)	0.158
Blood urea	1.049 (1.025-1.074)	0.0001	1.008 (0.963-1.056)	0.726
Creatinine	7.596 (3.170-18.205)	< 0.0001	0.842 (0.133-5.346)	0.856
Total Protein	0.367 (0.251-0.537)	< 0.0001	0.495 (0.279-0.881)	0.017
Serum albumin	0.359 (0.197-0.655)	0.001	1.243 (0.502-3.082)	0.638
Total bilirubin	1.073 (1.008-1.143)	0.028	0.865 (0.725-1.031)	0.106
AST	1.003 (1.000-1.007)	0.039	1.001 (0.995-1.07)	0.740
ALT	1.002 (0.996-1.009)	0.517
ALP	0.998 (0.993-1.03)	0.544
Na	0.906 (0.852-0.963)	0.002	0.920 (0.852-0.994)	0.035
K	1.678 (0.994-2.834)	0.053
INR	1.948 (1.281-2.963)	0.002	0.338 (0.121-0.942)	0.038
Serum ammonia	1.012 (1.005-1.020)	0.001	0.994 (0.977-1.010)	0.449
CFF	0.895 (0.836-0.959)	0.002	0.895 (0.773-1.038)	0.142
MELD	1.132 (1.084-1.183)	< 0.0001	1.263	0.000
Myoinositol peak	0.080 (0.014-1.183)	0.005	0.759 (0.015-39.025)	0.891
Choline peak	0.245 (0.059-1.017)	0.053
Glutamine peak	2.521 (1.201-5.295)	0.015	0.692 (0.180-2.662)	0.593
TNF-α	1.015 (1.005-1.024)	0.003	1.003 (0.984-1.022)	0.777
IL-6	1.006 (1.000-1.011)	0.036	0.999 (0.990-1.007)	0.776
IL-18	1.001 (1.00-1.003)	0.137
Endotoxin	123.151 (7.777-1.95E+03)	0.0006	9445.964 (0.220-3.97E+08)	0.093
IL-10	1.149 (1.053-1.255)	0.002	1.022 (0.757-1.381)	0.886
IL-12	1.085 (1.021-1.154)	0.009	0.713 (0.532-0.956)	0.024
IL-1β	1.115 (1.040-1.195)	0.002	1.206 (0.901-1.614)	0.208
Gender
Female	1
Male	1.044 (0.438-2.486)	0.923
CTP grade
A	1
B	1180.167 (0.000-6.66E+95)	0.948
C	5819.475 (3.28E+96)	0.937
Grade of HE
No HE	1
Minimal HE	3.069 (1.105-8.524)	0.031	1.333 (0.165-10.745)	0.787
Grade 1 HE	5.155 (1.942-13.683)	0.001	2.34	0.008

HR: Hazard ratio; CI: Confidence interval; TLC: Total leukocyte count; ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; INR: International normalized ratio; CFF: Critical flicker frequency; MELD: Model for end-stage liver disease; TNF: Tumor necrosis factor; IL: Interleukin; CTP: Child Turcotte Pugh; HE: Hepatic encephalopathy.

Table 6 Univariate and multivariate regression analysis to identify significant risk factors of number of hospitalisations

Parameters	Univariate incidence rate ratio by the Poisson regression model		Multivariate incidence rate ratio
	Incidence rate ratio (95%CI)	P value	Incidence rate ratio (95%CI)	P value
Age	0.994 (0.973-1.015)	0.574
Hemoglobin	0.949 (0.846-1.063)	0.365
TLC	1.000 (1.000-1.000)	0.115
Platelet count	1.000 (1.000-1.000)	0.323
Blood urea	1.021 (1.004-1.039)	0.017	1.003 (0.979-1.028)	0.817
Creatinine	3.012 (1.502-6.039)	0.002	1.267 (0.433-3.709)	0.666
Total Protein	0.650 (0.501-0.843)	0.001	0.934 (0.667-1.309)	0.692
Serum albumin	0.548 (0.364-0.826)	0.004	0.825 (0.487-1.395)	0.472
Total bilirubin	1.056 (1.008-1.107)	0.023	0.989 (0.892-1.097)	0.836
AST	1.002 (1.000-1.005)	0.097
ALT	1.002 (0.997-1.007)	0.356
ALP	1.001 (0.998-1.004)	0.418
Na	0.961 (0.922-1.001)	0.056
K	0.923 (0.622-1.368)	0.689
INR	1.573 (1.094-2.262)	0.014
Serum ammonia	1.008 (1.003-1.013)	0.003	0.999 (0.990-1.009)	0.917
CFF	0.919 (0.876-0.965)	0.001	0.848 (0.875-1.026)	0.186
MELD	1.069 (1.035-1.104)	0.0001	1.068 (0.975-1.171)	0.157
Myoinositol peak	0.206 (0.059-0.721)	0.013	4.348 (0.411-46.003)	0.222
Choline peak	0.513 (0.206-1.280)	0.152
Glutamine peak	2.053 (1.190-3.542)	0.010	1.081 (0.496-2.357)	0.844
TNF-α	1.011 (1.005-1.017)	0.0004	1.005 (0.995-1.014)	0.345
IL-6	1.006 (1.002-1.009)	0.003	1.002 (0.997-1.07)	0.378
IL-18	1.001 (1.000-1.002)	0.007	1.000 (0.997-1.002)	0.817
Endotoxin	31.751 (4.610-218.657)	0.0004	0.356 (0.001-245.301)	0.757
IL-10	1.123 (1.056-1.194)	0.0002	1.035 (0.872-1.228)	0.698
IL-12	1.085 (1.038-1.134)	0.0003	0.713 (0.754-1.072)	0.024
IL-1β	1.103 (1.050-1.159)	0.0001	1.170 (0.971-1.411)	0.100
Gender
Female
Male	0.981 (0.514-1.873)	0.954
CTP grade
A
B	1.72E+08 (0.000-7.12E+88)	0.841
C	3.44E+08 (0.000-1.71E+101)	0.857
Grade of HE
No HE
Minimal HE	1.056 (0.467-2.386)	0.896
Grade 1 HE	2.45 (1.22-4.90)	0.012	2.1 (1.24-3.56)	0.006

HR: Hazard ratio; CI: Confidence interval; TLC: Total leukocyte count; ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; INR: International normalized ratio; CFF: Critical flicker frequency; MELD: Model for end-stage liver disease; TNF: Tumor necrosis factor; IL: Interleukin; CTP: Child Turcotte Pugh; HE: Hepatic encephalopathy.