Ijms 24 03563

Download as pdf or txt
Download as pdf or txt
You are on page 1of 12

International Journal of

Molecular Sciences

Article
Metabolic Profile Reflects Stages of Fibrosis in Patients with
Non-Alcoholic Fatty Liver Disease
Nila Jambulingam 1,† , Roberta Forlano 1,† , Benjamin Preston 1 , Benjamin H. Mullish 1 , Greta Portone 1 ,
Yama Baheer 1 , Michael Yee 2 , Robert D. Goldin 3 , Mark R. Thursz 1 and Pinelopi Manousou 1, *

1 Liver Unit, Division of Digestive Diseases, Department of Metabolism, Digestion and Reproduction,
Faculty of Medicine, Imperial College London, London W2 1NY, UK
2 Section of Endocrinology and Metabolic Medicine, St Mary’s Hospital, Imperial College NHS Trust,
London W2 1NY, UK
3 Department of Cellular Pathology, Faculty of Medicine, Imperial College London, London W2 1NY, UK
* Correspondence: p.manousou@imperial.ac.uk
† These authors contributed equally to this work.

Abstract: Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease
worldwide, with fibrosis stage being the main predictor for clinical outcomes. Here, we present
the metabolic profile of NAFLD patients with regards to fibrosis progression. We included all
consecutive new referrals for NAFLD services between 2011 and 2019. Demographic, anthropometric
and clinical features and noninvasive markers of fibrosis were recorded at baseline and at follow-
up. Significant and advanced fibrosis were defined using liver stiffness measurement (LSM) as
LSM ≥ 8.1 kPa and LSM ≥ 12.1 kPa, respectively. Cirrhosis was diagnosed either histologically or
clinically. Fast progressors of fibrosis were defined as those with delta stiffness ≥ 1.03 kPa/year
(25% upper quartile of delta stiffness distribution). Targeted and untargeted metabolic profiles were
analysed on fasting serum samples using Proton nuclear magnetic resonance (1 H NMR). A total of
189 patients were included in the study; 111 (58.7%) underwent liver biopsy. Overall, 11.1% patients
Citation: Jambulingam, N.; Forlano, R.;
were diagnosed with cirrhosis, while 23.8% were classified as fast progressors. A combination of
Preston, B.; Mullish, B.H.; Portone, G.;
metabolites and lipoproteins could identify the fast fibrosis progressors (AUROC 0.788, 95% CI:
Baheer, Y.; Yee, M.; Goldin, R.D.;
0.703–0.874, p < 0.001) and performed better than noninvasive markers. Specific metabolic profiles
Thursz, M.R.; Manousou, P. Metabolic
Profile Reflects Stages of Fibrosis in
predict fibrosis progression in patients with nonalcoholic fatty liver disease. Algorithms combining
Patients with Non-Alcoholic Fatty metabolites and lipids could be integrated in the risk-stratification of these patients.
Liver Disease. Int. J. Mol. Sci. 2023,
24, 3563. https://doi.org/10.3390/ Keywords: NAFLD; fibrosis; metabolic profile; fast fibrosers
ijms24043563

Academic Editors: Mariapia Vairetti,


Giuseppe Colucci and Andrea
1. Introduction
Ferrigno
Nonalcoholic Fatty Liver Disease (NAFLD) affects a third of the general population in
Received: 11 January 2023 Western countries [1] and represents the most common cause of abnormal liver function
Revised: 7 February 2023
tests. NAFLD includes a spectrum of pathological disorders, from simple steatosis to
Accepted: 8 February 2023
nonalcoholic steato-hepatitis (NASH) with inflammation and ballooning, and a certain
Published: 10 February 2023
degree of fibrosis up to cirrhosis [2]. The growing prevalence of NAFLD mirrors the
epidemic of metabolic syndrome, mainly type-2 diabetes and obesity, to which it is closely
associated [1]. Fibrosis stage represents the main predictor of clinical outcomes—liver- and
Copyright: © 2023 by the authors.
nonliver-related—in this population [3]. As such, developing significant and advanced
Licensee MDPI, Basel, Switzerland. fibrosis marks a crucial point in the pathogenesis of the disease.
This article is an open access article Currently, liver histology represents the gold standard for staging fibrosis in this
distributed under the terms and population [4,5]. However, due to well-identified limitations of liver biopsy, such as the
conditions of the Creative Commons bleeding risk and the cost, it is unfeasible for all the patients to undergo such investigation.
Attribution (CC BY) license (https:// A plethora of noninvasive markers, such as ELF score, FIB-4, NAFLD fibrosis score and
creativecommons.org/licenses/by/ transient elastography, has therefore been developed over the last few years in an attempt
4.0/). to predict fibrosis stage [6], fast fibrosers and clinical events [7–9].

Int. J. Mol. Sci. 2023, 24, 3563. https://doi.org/10.3390/ijms24043563 https://www.mdpi.com/journal/ijms


Int. J. Mol. Sci. 2023, 24, 3563 2 of 12

Over the last decade, metabolomic profiling has gained much popularity in the field
of translational hepatology. There has been an increasing body of evidence hinting at
a possible role of circulating aromatic amino acids as noninvasive markers for NAFLD
severity. In a recent study, hepatocellular ballooning and inflammation, assessed by the
NASH CRN scoring system, were associated with increased branched chain amino acids
and aromatic amino acids, while fibrosis stages could be predicted by a combination
of glutamate, serine and glycine [10]. Moreover, plasma branched chain amino acids
correlated with NAFLD severity, more pronouncedly in women compared to men [11].
In another study, a combination of glycocholic acid, taurocholic acid, phenylalanine and
branched chain amino acids could predict the presence of NASH accurately [12].
In this study, we analysed the metabolic profile of a well-phenotyped cohort of NAFLD
patients and investigated its association with fast progressors of fibrosis.

2. Results
2.1. Study Population
A total of 189 patients were included in the study, with a median follow-up of
72 months (54–106) between their first clinic date to the end of the study and a median time
between fibroscan of 15 months (11–22). Median age was 52 (41–60) years. Median BMI
was 30.25 (27.7–34.35) kg/m2 . Non-Hispanic white patients comprised 40.7% (77/189),
South Asians 18% (34/189), African/Afro-Caribbeans 6.3% (12/189), White Hispanics 5.8%
(11/189), Arabs 5.8% (11/189) and East Asians 4.8% (9/189). In terms of comorbidities,
49.7% (94/189) of the patients had T2DM, 42.9% (81/189) hypertension, 42.9% (81/189)
dyslipidaemia and 8.5% (16/189) hypothyroidism (Table 1).

Table 1. Clinical and demographic features of the study population.

Study Population (n = 189)


Demographics
Male Sex, N (%) 122 (64.6)
Ethnicities, N (%) White Non-Hispanic 77 (40.7)
White Hispanic 11 (5.8)
Arab 11 (5.8)
South Asian 34 (18)
East Asia 9 (4.8)
African/Afro-Caribbean 12 (6.3)
Other 35 (18.5)
Age, median (IQR) 52 (41–60)
Comorbidities
T2DM, N (%) 94 (49.7)
Dyslipidaemia, N (%) 81 (42.9)
Hypertension, N (%) 81 (42.9)
Hypothyroidism, N (%) 16 (8.5)
Biochemistry
AST (IU/L), median (IQR) 40 (31–54.5)
ALP (IU/L), median (IQR) 81 (65–104)
GGT (IU/L), median (IQR) 56 (35–105)
PLT (×109 /L), median (IQR) 225 (187–271)
HbA1c (mmol/L), median (IQR) 44 (37.25–54)
Abbreviations: T2DM: type-2 diabetes mellitus, AST: Aspartate transaminase, ALP: Alkaline Phosphatase, GGT:
Gamma Glutamyl transferase, U/L: units per Litre, L: litre, mmol/L: millimoles per litre.

In total, 111 (58.7%) patients underwent a liver biopsy. Overall, 26.7% (50/189) of
patients had LSM between 8.1 and 12 kPa, and 18.7% (35/189) of patients had a LSM of
more than 12 kPa. A total of 11.1% (21/189) patients were diagnosed with cirrhosis. Based
Int. J. Mol. Sci. 2023, 24, 3563 3 of 12

on delta stiffness, 38 (23.8%) patients were classified as fast progressors and 112 (76%)
nonfast progressors.

2.2. Metabolic Profile in Fast Progressors vs. Nonfast Progressors


When comparing metabolites between fast progressors and nonfast progressors,
38 metabolites were significantly different (Table 2).

Table 2. Differences in metabolic profile between fast progressors and nonfast progressors.

Fast Progressors Nonfast Progressors


Metabolite p Value
(n = 38) (n = 112)
Glutamic Acid
0.2 0.15 0.045
(mmol/L)
Proline (mmol/L) 0.24 0.19 0.035
Valine (mmol/L) 0.31 0.28 0.024
H2A2 (mg/dL) 3.6 3.09 0.042
H3TG (mg/dL) 2.51 2.14 p < 0.0001
IDAB (mg/dL) 6.01 5.34 0.039
IDPN (nmol/L) 109.17 97.09 0.039
IDTG (mg/dL) 18.38 13.59 0.024
L3FC (mg/dL) 2.72 3.84 0.034
L4FC (mg/dL) 3.45 3.97 0.042
TPTG (mg/dL) 163.30 140.48 0.022
V1CH (mg/dL) 9.42 7.06 0.013
V1FC (mg/dL) 4.32 3.22 0.014
V1PL (mg/dL) 9.96 7.89 0.04
V1TG (mg/dL) 57.02 45.59 0.044
V2CH (mg/dL) 3.76 2.93 0.018
V2FC (mg/dL) 1.77 1.43 0.014
V2PL (mg/dL) 4.93 3.95 0.03
V2TG (mg/dL) 19.43 16.08 0.031
V3CH (mg/dL) 4.42 3.84 0.034
V3FC (mg/dL) 2.35 1.86 0.033
V3PL (mg/dL) 5.58 4.69 0.04
V4FC (mg/dL) 2.85 2.22 0.019
VLAB (mg/dL) 11.60 9.69 0.04
VLCH (mg/dL) 28.86 22.58 0.016
VLFC (mg/dL) 12.96 10.49 0.033
VLPL (mg/dL) 28.76 24.96 0.042
VLPN (nmol/L) 210.90 176.11 0.04
VLTG (mg/dL) 115.59 92.33 0.036
H1TG (mg/dL) 4.31 3.02 0.002
H2TG (mg/dL) 2.23 1.82 p < 0.0001
HDTG (mg/dL) 12.34 10.45 0.001
L5FC (mg/dL) 3.49 4.04 0.021
H4FC (mg/dL) 2.68 3.47 0.006
H4CH (mg/dL) 15.92 18.75 0.002
H4A1 (mg/dL) 64.81 71.96 0.005
H4A2 (mg/dL) 16.46 18.58 0.012
H4PL (mg/dL) 23.28 26.42 0.011
Abbreviations: H2A2, HDL-2 Apolipoprotein A2; H3TG, HDL-3 triglyceride; IDAB, IDL apolipoprotein B100;
IDPN, IDL particle number; IDTG, IDL triglyceride; L3FC, LDL-3 free cholesterol; L4FC, LDL-4 free cholesterol;
TPTG, total plasma triglycerides; V1CH, VLDL-1 cholesterol; V1FC, VLDL-1 free cholesterol; V1PL, VLDL-1
phospholipid; V1TG, VLDL-1 triglycerides; V2CH, VLDL-2 cholesterol; V2FC, VLDL-2 free cholesterol; V2PL,
VLDL-2 phospholipid; V2TG, VLDL-2 triglyceride; V3CH, VLDL-3 cholesterol; V3FC, VLDL-3 free cholesterol;
V3PL, VLDL-3 phospholipid; V4FC, VLDL-4 free cholesterol; VLAB, VLDL class apolipoprotein B100; VLCH,
VLDL class cholesterol; VLFC, VLDL class free cholesterol; VLPL, VLDL class phospholipid; VLPN, VLDL class
particle number; VLTG, VLDL class triglycerides; H1TG, HDL-1 triglyceride; H2TG, HDL-2 Triglyceride; HDTG,
HDL triglyceride; L5FC, LDL-5 free cholesterol; H4FC, HDL-4 free cholesterol; H4CH, HDL-4 cholesterol; H4A1,
HDL-4 apolipoprotein A1; H4A2, HDL-4 apolipoprotein A2; H4PL, HDL-4 phospholipid.
Int. J. Mol. Sci. 2023, 24, 3563 4 of 12

On multivariate analysis, only 14 metabolites were significantly associated with fi-


brosis progression (Table 3). Using binary logistic regression, a formula was generated to
predict the progression of fibrosis, based on these metabolites:

30.485 × H2TG (mg⁄dL) + 0.706 × H4A1 (mg⁄dL) + 1.586 × H4PL (mg⁄dL) +


1.989 × H4A2 (mg/dL) + 0.063 × V4FC (mg/dL) + 0.719 × IDTG (mg/dL) +
12.94 × VLFC (mg/dL) + 0.103 × V3CH (mg/dL) + 42.376 × Proline (mmol/L) + (1)
3.18 × IDAB (mg/dL) + 0.284 × VLAB (mg/dL) + 44.243 × V3PL (mg/dL) +
0.532 × VLPL (mg/dL) + 0.319 × H2A2 (mg/dL) + 0.339

The metabolic profile generated from the above formula showed the ability to predict
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW
fibrosis progression in this population, with an AUROC of 0.788 (95% CI: 0.703–0.874, 5 of 13
p < 0.001). A cut-off of 0.274 gave a sensitivity of 68% and specificity of 76% (Youden’s
J Statistic 0.443). Metabolic profile performed better than other markers for predicting
fibrosisALT
fibrosis progression: progression:
(AUROCALT (AUROC
0.59, 95% CI:0.59, 95% CI:p0.48–0.71,
0.48–0.71, p = 0.08),
= 0.08), AST AST (AUROC
(AUROC 0.65, 0.65,
95% CI: 0.52–0.76, p = 0.006), FIB-4 (AUROC 0.5, 95% CI: 0.39–0.61, p = 0.9), NAFLD fibrosis fibrosis
95% CI: 0.52–0.76, p = 0.006), FIB-4 (AUROC 0.5, 95% CI: 0.39–0.61, p = 0.9), NAFLD
score 95%
score (AUROC 0.56, (AUROC 0.56, 95%pCI:
CI: 0.44–0.67, 0.44–0.67,
= 0.26) p = 0.26)
and LSM and LSM(AUROC
at baseline at baseline (AUROC
0.43, 0.43, 95%
95% CI:
CI: 0.31–0.55, p = 0.2) (Figure 1).
0.31–0.55, p = 0.2) (Figure 1).

Figure
Figure 1. Diagnostic 1. Diagnostic
performance ofperformance of metabolic
metabolic profile profile forfibrosis
for predicting predicting fibrosis progression
progression comparedcompared
to ALT, AST, NAFLD fibrosis score, FIB-4 and LSM. Abbreviations: ALT, alanine aminotransferase;
to ALT, AST, NAFLD fibrosis score, FIB-4 and LSM. Abbreviations: ALT, alanine aminotransferase;
AST, aspartate aminotransferase; LSM: liver stiffness measurement.
AST, aspartate aminotransferase; LSM: liver stiffness measurement.
2.3. Subgroup with Liver Histology
Table 3. Multivariate analysis of metabolites
Among those independently
who underwent biopsy,associated
15 (14.6%)with
hadfibrosis
stage 0,progression.
22 (21.4%) had stage 1,
19 (18.4%) had stage 2, 35 (34%) had stage 3 and 12 (11.7%) had stage 4 fibrosis. In this
Metabolite OR (95% CI) Significance
cohort, 33 (31.7%) had NASH.
H2TG (mg/dL) When compared to30.48those(4.37–212.57) <0.001
without NASH, only phenylalanine was significantly lower
H4A1 (mg/dL)
in those with NASH (0.07 mmol/L (0.06–0.08) vs. 0.08 mmol/L0.001
0.706 (0.57–0.88) (0.06–0.09); p = 0.048). The
H4PL (mg/dL)
AUROC of phenylalanine 1.586
for (1.09–2.32)
predicting the presence of NASH 0.017
was 0.381 (95% CI 0.269–
H4A2 (mg/dL)
0.493, p = 0.051), which is1.989 (1.99–1.18)
in keeping 0.009
with poor diagnostic performance.
V4FC (mg/dL) 0.063 (0.06–0.007)
When comparing metabolites 0.014nonfast progressors in
between fast progressors and
IDTG (mg/dL) 0.719 (0.72–0.57) 0.004
those who had a biopsy, 37 metabolites were significantly different (Table 4).
VLFC (mg/dL) 12.94 (1.92–87.15) 0.009
V3CH (mg/dL) 0.103 (0.01–1.02) 0.052
Table 4. Differences in metabolic profile between fast progressors and nonfast progressors in
Proline (mmol/L) 42.376 (2.34–767.97)
patients who had a liver biopsy.
0.011

Nonfast Progressors (n =
Metabolite Fast Progressors (n = 22) p Value
63)
Two Oxoglutaric Acid (mmol/L) 0 0 <0.001
H4CH (mg/dL) 15.545 18.99 0.002
H4PL (mg/dL) 22.28 26.14 0.004
Int. J. Mol. Sci. 2023, 24, 3563 5 of 12

Table 3. Cont.

Metabolite OR (95% CI) Significance


IDAB (mg/dL) 3.18 (1.38–7.32) 0.006
VLAB (mg/dL) 0.284 (0.09–0.92) 0.036
V3PL (mg/dL) 44.243 (1.66–1179.2) 0.024
VLPL (mg/dL) 0.532 (0.30–0.95) 0.032
H2A2 (mg/dL) 0.319 (0.12–0.83) 0.019
Abbreviations: H2TG, HDL 2 Triglyceride; H4A1, HDL 4 Apolipoprotein 1; H4PL, HDL 4 Phospholipid; H4A2,
HDL 4 Apolipoprotein A2; V4FC, VLDL 4 free cholesterol; IDTG, IDL class triglyceride; VLFC, VLDL free
cholesterol; V3CH, VLDL 3 cholesterol; IDAB, IDL class Apolipoprotein B100; VLAB, VLDL class Apolipoprotein
B100; V3PL, VLDL 3 Phopholipid; VLPL, VLDL Phospholipid; H2A2, HDL 2 Apolipoprotein A2.

2.3. Subgroup with Liver Histology


Among those who underwent biopsy, 15 (14.6%) had stage 0, 22 (21.4%) had stage 1,
19 (18.4%) had stage 2, 35 (34%) had stage 3 and 12 (11.7%) had stage 4 fibrosis. In this
cohort, 33 (31.7%) had NASH.
When compared to those without NASH, only phenylalanine was significantly lower
in those with NASH (0.07 mmol/L (0.06–0.08) vs. 0.08 mmol/L (0.06–0.09); p = 0.048).
The AUROC of phenylalanine for predicting the presence of NASH was 0.381 (95% CI
0.269–0.493, p = 0.051), which is in keeping with poor diagnostic performance.
When comparing metabolites between fast progressors and nonfast progressors in
those who had a biopsy, 37 metabolites were significantly different (Table 4).

Table 4. Differences in metabolic profile between fast progressors and nonfast progressors in patients
who had a liver biopsy.

Fast Progressors Nonfast Progressors


Metabolite (n = 22) p Value
(n = 63)
Two Oxoglutaric Acid
0 0 <0.001
(mmol/L)
H4CH (mg/dL) 15.545 18.99 0.002
H4PL (mg/dL) 22.28 26.14 0.004
H4FC (mg/dL) 2.52 3.31 0.006
H4A1 (mg/dL) 62.62 71.85 0.009
V1CH (mg/dL) 9.42 6 0.01
H2TG (mg/dL) 2.225 1.82 0.01
V2FC (mg/dL) 1.765 1.21 0.012
V2CH (mg/dL) 3.67 2.81 0.012
VLCH (mg/dL) 28.25 21.46 0.012
V1FC (mg/dL) 4.32 2.69 0.013
H4A2 (mg/dL) 15.9 18.46 0.014
VLFC (mg/dL) 12.9 9.72 0.017
H3TG (mg/dL) 2.53 2.22 0.017
HDTG (mg/dL) 12.52 10.46 0.02
HDFC (mg/dL) 9.28 11.02 0.02
V4FC (mg/dL) 3.135 2.13 0.021
VLPL (mg/dL) 28.34 23.94 0.021
VLPN (mg/dL) 206.75 161.42 0.023
VLAB (mg/dL) 11.37 8.88 0.024
TPA2 (mg/dL) 28.405 30.78 0.025
V3FC (mg/dL) 2.335 1.63 0.027
V2PL (mg/dL) 4.765 3.65 0.028
TPTG (mg/dL) 163.59 127.42 0.028
V2TG (mg/dL) 19 14.99 0.029
V3CH (mg/dL) 4.415 3.54 0.029
H3CH (mg/dL) 8.465 9.21 0.034
VLTG (mg/dL) 112.85 87.7 0.034
HDA1 (mg/dL) 123.85 133.81 0.037
V1PL (mg/dL) 9.275 7.02 0.038
L3FC (mg/dL) 2.69 3.63 0.041
V4CH (mg/dL) 6.18 4.77 0.041
IDTG (mg/dL) 18.005 11.73 0.042
HDA2 (mg/dL) 29.005 31.37 0.044
Int. J. Mol. Sci. 2023, 24, 3563 6 of 12

Table 4. Cont.

Fast Progressors Nonfast Progressors


Metabolite p Value
(n = 22) (n = 63)
IDCH (mg/dL) 14.98 11.66 0.045
V3PL (mg/dL) 5.58 4.13 0.049
IDFC (mg/dL) 4.265 3.58 0.049
Abbreviations: H4CH, HDL-4 cholesterol; H4PL, HDL-4 phospholipid; H4FC, HDL-4 free cholesterol; H4A1, HDL-
4 apolipoprotein A1; V1CH, VLDL-1 cholesterol; H2TG, HDL-2 triglyceride; V2FC, VLDL-2 free cholesterol; V2CH,
VLDL-2 cholesterol; VLCH, VLDL class cholesterol; V1FC, VLDL-1 free cholesterol; H4A2, HDL-4 apolipoprotein
A2; VLFC, VLDL class free cholesterol; H3TG, HDL-3 triglyceride; HDTG, HDL class triglyceride; HDFC, HDL
class free cholesterol; V4FC, VLDL-4 free cholesterol; VLPL, VLDL class phospholipid; VLPN, VLDL class particle
number; VLAB, VLDL class apolipoprotein B100; TPA2, Total Plasma apolipoprotein A2; V3FC, VLDL-3 free
cholesterol; V2PL, VLDL-2 phospholipid; TPTG, Total Plasma trigylceride; V2TG, VLDL-2 triglyceride; V3CH,
VLDL-3 cholesterol; H3CH, HDL-3 cholesterol; VLTG, VLDL class triglyceride; HDA1, HDL class apolipoprotein
A1; V1PL, VLDL-1 phospholipid; L3FC, LDL-3 free cholesterol; V4CH, VLDL-4 cholesterol; IDTG, IDL class
triglyceride; HDA2, HDL class apolipoprotein A2; IDCH, IDL class cholesterol; V3PL, VLDL-3 phospholipid;
IDFC, IDL class free cholesterol.

On multivariate analysis, only one of these metabolites was significantly associated


with fibrosis progression: H4CH, with an OR of 0.818 (95% CI: 0.722–0.927, p = 0.002). Using
binary logistic regression and including baseline fibrosis stage, a formula was generated to
predict the progression of fibrosis:

0.818 × H4CH (mg/dL) + 0.793 × Baseline Fibrosis Stage + 13.437 (2)

The metabolic profile generated from the above formula showed the ability of predic-
tion of fibrosis progression in this population, with an AUROC of 0.744 (95% CI: 0.618–0.870,
p = 0.002). A cut-off of 0.203 gave a sensitivity of 85% and specificity of 58% (Youden’s
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 of 13
J
Statistic 0.433). Metabolic profile performed similar or better than other markers for pre-
dicting fibrosis progression: ALT (AUROC 0.69, 95% CI: 0.55–0.84, p = 0.016), AST (AUROC
0.75, 95% CI: 0.62–0.89,
AST (AUROC p = 0.75,
0.001),
95%FIB-4 (AUROC
CI: 0.62–0.89, 0.55, 95%
p = 0.001), FIB-4CI: 0.40–0.71,
(AUROC p = CI:
0.55, 95% 0.5), NAFLD
0.40–0.71, p
fibrosis score (AUROC 0.42,fibrosis
= 0.5), NAFLD 95% CI: 0.26–0.58,
score p = 0.32)
(AUROC 0.42, 95% CI:and LSM at
0.26–0.58, p =baseline
0.32) and (AUROC 0.41,
LSM at baseline
95% CI: 0.25–0.59, (AUROC 0.41,(Figure
p = 0.3) 95% CI: 0.25–0.59,
2). p = 0.3) (Figure 2).

Figure
Figure 2. Diagnostic 2. Diagnostic performance
performance of metabolicofprofile
metabolic
forprofile for predicting
predicting fibrosisfibrosis progression
progression compared
compared to
to ALT, AST, NAFLD fibrosis score, FIB-4 and LSM in patient who had had a liver biopsy. Abbre-
ALT, AST, NAFLD fibrosis score, FIB-4 and LSM in patient who had had a liver biopsy. Abbreviations:
viations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; LSM: liver stiffness
ALT, alanine aminotransferase;
measurement. AST, aspartate aminotransferase; LSM: liver stiffness measurement.

2.4. Metabolic Profile in Cirrhotic vs. Noncirrhotic Patients


In this cohort, 21 (11.1%) patients had cirrhosis. When comparing to the noncirrhot-
ics, 10 metabolites were significantly higher in the cirrhotic group (Table 5). Conversely,
10 different metabolites were significantly lower in the cirrhotic group (Table 5).
Int. J. Mol. Sci. 2023, 24, 3563 7 of 12

2.4. Metabolic Profile in Cirrhotic vs. Noncirrhotic Patients


In this cohort, 21 (11.1%) patients had cirrhosis. When comparing to the noncirrhotics,
10 metabolites were significantly higher in the cirrhotic group (Table 5). Conversely,
10 different metabolites were significantly lower in the cirrhotic group (Table 5).

Table 5. Differences in metabolic profile between cirrhotic and noncirrhotic patients.

Noncirrhotics
Metabolite Cirrhotics (n = 21) p Value
(n = 162)
H1A1 (mg/dL) 33.48 16.95 0.007
H1A2 (mg/dL) 3.49 1.83 0.016
H1CH (mg/dL) 20.43 13.64 0.014
H1PL (mg/dL) 28.42 15.86 0.017
H1FC (mg/dL) 4.29 3.15 0.039
L2TG (mg/dL) 2.73 2.00 0.015
3OHB (mg/dL) 0.09 0.06 0.012
H1TG (mg/dL) 5.3 3.25 0.001
H2TG (mg/dL) 2.48 1.96 0.021
HDTG (mg/dL) 14.57 11.15 0.02
L5FC (mg/dL) 3.35 3.91 0.027
H4FC (mg/dL) 2.4 3.32 0.014
H4CH (mg/dL) 13.35 18.03 0.006
H4A1 (mg/dL) 59.23 70.09 0.014
H4A2 (mg/dL) 15.8 18.46 0.001
H4PL (mg/dL) 19.67 26.09 0.004
HDA2 (mg/dL) 28.09 30.61 0.01
TPA2 (mg/dL) 27.62 30.09 0.012
Creatine (mmol/L) 0.01 0.02 0.036
Lysine (mmol/L) 0.18 0.23 0.001
Abbreviations: H1A1, HDL 1 Apolipoprotein A1; H1A2, HDL 1 Apolipoprotein A2; H1CH, HDL 1 cholesterol;
H1PL, HDL 1 Phospholipid; H1FC, HDL 1 free cholesterol; L2TG, LDL 2 triglyceride; 3OHB, 3 hydroxybutyric
acid; H1TG, HDL 1 triglyceride; H2TG, HDL 2 triglyceride; HDTG HDL class triglyceride; L5FC, LDL 5 free
cholesterol; H4FC, HDL 4 free cholesterol; H4CH, HDL 4 cholesterol; H4A1, HDL 4 Apolipoprotein A1; H4A2,
HDL 4 Apolipoprotein A2; H4PL, HDL 4 Phospholipid; HDA2, HDL class Apolipoprotein A2; TPA2, total plasma
Apolipoprotein A2.

On multivariate analysis, only lysine (OR 0.001, 95% CI 0.000002, 0.13, p = 0.0008),
HDA2 (OR 0.8, 95% CI 0.77, 0.94, p = 0.002), H1A2 (OR 1.6, 95% CI 1.2, 2.27, p = 0.002)
and creatine (OR 6.8 × 10−15 , 95% CI 8.0 × 10−29 , 0.58, p = 0.046) remained significantly
associated with the presence of cirrhosis (Table 6).

Table 6. Multivariate analysis of the metabolites independently associated with the presence of cirrhosis.

Metabolite OR (95% CI) p-Value


Lysine (mmol/L) 0.001 (0.000002–0.137) 0.008
HDA2 (mg/dL) 0.856 (0.77–0.94) 0.002
H1A2 (mg/dL) 1.654 (1.2–2.27) 0.002
Creatine (mmol/L) 6.8 (0.008–0.58) 0.046
Abbreviations: OR: odds ratio, 95% CI: 95% confidence interval, HDA2: HDL class Apolipoprotein A2, H1A2:
HDL-1 Apolipoprotein A2.

Using binary logistic regression, a formula was generated to predict the presence of
cirrhosis, combining the metabolites that were significant on multivariate analysis:

0.001 × Lysine (mmol/L) + 0.8 × HDA2 (mg/dL) + 1.6 × H1A2 (mg/dL)


(3)
+ 6.827 × 10−15 × Creatine (mmol/L) + 27.18

The metabolic profile generated from the above formula showed an excellent pre-
diction of the presence of cirrhosis in this population, with an AUROC of 0.84 (95% CI:
Int. J. Mol. Sci. 2023, 24, 3563 8 of 12

0.752, 0.934, p < 0.001). A cut-off of 0.127 gave a sensitivity of 76% and a specificity of
82% (Youden’s J statistic 0.583). Metabolic profile performed similar to the NAFLD fibrosis
score and FIB4 but better than other markers for predicting cirrhosis in our cohort: ALT
(AUROC 0.33, 95% CI: 0.2–0.46, p = 0.02), AST (AUROC 0.44, 95% CI: 0.3–0.59, p = 0.48),
Int. J. Mol. Sci. 2023, 24, x FOR PEERFIB-4 (AUROC 0.78, 95% CI: 0.66–0.9, p < 0.001), NAFLD fibrosis score (AUROC 0.84,9 95%
REVIEW of 13
CI: 0.74–0.91, p < 0.001) and LSM at baseline (AUROC 0.76, 95% CI: 0.62–0.9, p < 0.001)
(Figure 3).

Figure3.
Figure 3. Diagnostic
Diagnostic performance
performance of
of aa combination
combination of
of metabolites
metabolites to
to predict
predict the
the presence
presence of
of cirrhosis.
cirrhosis.
Abbreviations:ALT,
Abbreviations: ALT,alanine
alanineaminotransferase;
aminotransferase;AST,
AST,aspartate
aspartateaminotransferase.
aminotransferase.

3.
3. Discussion
Discussion
NAFLD
NAFLD has has become
become thethe most
most common
common cause
cause ofof liver
liver disease
disease in in the
the Western
Westernworld
world
and
and the fastest growing cause for liver transplantation [13]. Despite the bigbig
the fastest growing cause for liver transplantation [13]. Despite the burden
burden of
of the
the disease, identifying patients at risk of progression remains a challenge
disease, identifying patients at risk of progression remains a challenge [14]. Of note, the [14]. Of note,
the recent
recent advances
advances in in metabolomics
metabolomics andlipidomics
and lipidomicsmay mayprovide
provide useful
useful insights
insights into
into the
the
pathogenesis of the condition as well as new predictive tools for clinical
pathogenesis of the condition as well as new predictive tools for clinical outcomes in this outcomes in this
population
population [15].
[15]. In
In this
this study,
study, wewe analysed
analysed thethe metabolic
metabolic profile
profile of
of aawell-phenotyped
well-phenotyped
group
group of NAFLD patients from a tertiary care centre. We then evaluatedtheir
of NAFLD patients from a tertiary care centre. We then evaluated theirmetabolic
metabolic
profile against fibrosis progression and severity.
profile against fibrosis progression and severity.
Fibrosis stage represents the main predictor of clinical outcomes in patients with
Fibrosis stage represents the main predictor of clinical outcomes in patients with
NAFLD [3]. Moreover, a faster progression of the liver disease translates into an earlier
NAFLD [3]. Moreover, a faster progression of the liver disease translates into an earlier
development of both hepatic and nonhepatic clinical outcomes [16]. As such, identifying
development of both hepatic and nonhepatic clinical outcomes [16]. As such, identifying
those who are at higher risk for fibrosis progression may be clinically important in NAFLD
those who are at higher risk for fibrosis progression may be clinically important in
patients. In this cohort, faster progressors presented a peculiar lipid profile, characterised by
NAFLD patients. In this cohort, faster progressors presented a peculiar lipid profile, char-
higher levels of very-low density lipoproteins (VLDL) (VLFC and V3PL) and triglycerides
acterised by higher levels of very-low density lipoproteins (VLDL) (VLFC and V3PL) and
(H2TG) and low HDL (H4CH) (Table 3), which were not observed in cirrhotics (Table 6).
triglycerides (H2TG) and low HDL (H4CH) (Table 3), which were not observed in cirrhot-
Overall, worsening insulin resistance is known to be characterised by elevated VLDL and
ics (Table 6). Overall, worsening insulin resistance is known to be characterised by ele-
triglycerides secondary to an impaired hepatic and systemic lipid metabolism [17]. On an
vated VLDL and triglycerides secondary to an impaired hepatic and systemic lipid me-
opposite trend, sera from patients with cirrhosis were particularly enriched in low-density
tabolism [17]. On an opposite trend, sera from patients with cirrhosis were particularly
HDL1 lipoproteins (H1PL, H1CH) and apolipoprotein A2 (HDA2 and TPA2). Specifically,
enriched
lower in low-density
levels of VLDL and HDL1 lipoproteins
triglycerides may (H1PL,
reflectH1CH)
reduced andhepatic
apolipoprotein
syntheticA2 (HDA2
function,
and TPA2). Specifically, lower levels of VLDL and triglycerides may reflect reduced he-
patic synthetic function, greater porto-systemic shunting and relative malnutrition [18].
Moreover, an impaired cholesterol efflux capacity [19], as well as lower lipoprotein scav-
enger activity [20], may be responsible for elevated HDL and apolipoproteins in those
with cirrhosis. There was no difference in terms of statin treatment, suggesting that
Int. J. Mol. Sci. 2023, 24, 3563 9 of 12

greater porto-systemic shunting and relative malnutrition [18]. Moreover, an impaired


cholesterol efflux capacity [19], as well as lower lipoprotein scavenger activity [20], may be
responsible for elevated HDL and apolipoproteins in those with cirrhosis. There was no
difference in terms of statin treatment, suggesting that changes in metabolic profile were
due to primary disturbances. Hence, lipid profile may reflect, to some extent, the course of
the progression of the liver disease, moving from a phase of florid metabolic dysfunction
with fibrosis progression to a less atherogenic profile with established cirrhosis.
Among the metabolites, proline was independently associated with fibrosis progres-
sion in this population (Table 3). Proline and its derivate hydroxyproline represent a major
player in the collagen synthesis of 30% of the body proteins [21]. Proline also acts as a
stabilizer for the helical structure of collagen fibres in the liver [22]. Moreover, previous
studies demonstrated that proline uptake increases in early stages of acute steatohepatitis
and is proportional to collagenogenesis in animal models [23]. On a similar note, lysine, an
essential amino acid mainly catabolised by the liver, was independently associated with
the presence of cirrhosis. Previous studies have associated lower lysine levels with collagen
disturbances as a result of overexpression of the enzyme lysil oxidases [24]. Under physio-
logical conditions, lysil oxidases deaminate lysine residues for maintaining the structural
integrity of the extra-cellular matrix. In pathological conditions such as fibrogenesis, such
an enzyme is overexpressed and promotes collagen cross-linking and stabilisation against
proteolytic degradation, maintaining hepatic stellate cells in an activated state [25]. More-
over, higher levels of pipecolic acid, one of lysine’s catabolites, were previously described in
patients with chronic liver disease and cirrhosis [26]. Taken together, these results suggest
a potential role in measuring serum proline and lysine as a biomarker of hepatic collagen
turnover in patients with NAFLD. Further prospective studies are required to explore their
role as predictors of liver-related events in this population.
Finally, a combination of lipoproteins and metabolites gave an excellent prediction of
fibrosis progression (Figure 2) and presence of cirrhosis (Figure 3). With regards to fibrosis
progression, metabolic profile performed equal or better than FIB-4, NAFLD fibrosis score
and liver functions tests, which are currently used to stratify patients at risk for more severe
liver disease in clinical practice [4]. Moreover, while previous studies demonstrated that
specific metabolic profiles may distinguish those with simple steatosis from those with
NASH [27], this is the first study exploring the association of metabolomics with fibrosis
progression and severity. Unfortunately, the small number of events in this population
did not allow for an internal validation, while an external cohort was not available to test
these findings at the time of the work. Future work should focus validating these results in
longitudinal studies as well as external cohorts.
Further studies are required to validate these results in longitudinal studies as well as
external cohorts.
In this study, we demonstrated that specific metabolic profile could predict fast fibrosis
progression in a cohort of patients with nonalcoholic fatty liver disease. Metabolic profile
performed better than traditional noninvasive markers of fibrosis. In an era of precision
medicine, algorithms combining metabolites and lipids may provide comprehensive tools
to stratify patients with NAFLD. Integrating clinical features and multiomics results may
lead to a better understanding of the phenotypes of the patients and may allow for the
capture of the complexity of the disease.

4. Materials and Methods


4.1. Study Population
This study included all consecutive new referrals to the specialist NAFLD clinic at
St Mary’s Hospital (Imperial College Healthcare NHS Trust, London, UK) between 2011
and 2019. Exclusion criteria were the use of steatogenic drugs, excess alcohol consumption
(defined as alcohol consumption greater than 14 UI per week) as well as other concomitant
liver diseases.
Int. J. Mol. Sci. 2023, 24, 3563 10 of 12

Demographic, anthropometric and biochemical data were collected at the time of the
baseline fibroscan or at the time of the liver biopsy. Ethnicities were clustered into 6 groups
(Table 1). If ethnicity was not specified by the patient, it was classified as Other. All comor-
bidities, such as hypertension, type-2 diabetes mellitus (T2DM) and hypercholesterolaemia,
were recorded. Transient elastography (TE) was performed by an experienced physician
after 4 h fasting and allowed for the assessment of liver stiffness measurement (LSM) and
controlled attenuation parameter (CAP). A cut-off of LSM ≥ 8.1 kPa was considered to
be significant fibrosis, while LSM ≥ 12.1 kPa was considered to be advanced fibrosis [4].
All patients were monitored every 6 months for more than one year, with clinical data
documented at subsequent consultation. Liver biopsies were performed when clinically
indicated. Liver biopsy specimens were formalin-fixed, paraffin-embedded, stained and
scored by an expert liver pathologist as per the NASH CRN scoring system [2]. NASH was
defined based on NAS score ≥ 5.
Cirrhosis was diagnosed either histologically or clinically, as a combination of biochem-
ical, imaging and elastographic features. Delta stiffness was calculated as the difference in
LSM over a set time period divided by number of months. Patients were divided into fast
progressors and nonfast progressors when delta stiffness was more than 1.03 kPa/year or
less than 1.03 kPa/year, respectively. This cut off was identified using the top 25% of our
cohort, which is in line with previous literature [28].

4.2. Metabolic Profile


Fasting serum samples were collected for all patients included in the study. They were
then centrifuged and stored at −80 ◦ C in the Imperial Hepatology and Gastroenterology
Biobank (Imperial College London, London, UK). Targeted and untargeted metabolomic
profiles were carried out using proton nuclear magnetic resonance (1 H NMR). Overall,
values for 27 small metabolites and 112 lipoproteins were obtained for each serum sample,
as per published protocol [29].

4.3. Statistical Analysis


Distribution of the variables was identified using the Shapiro–Wilk normality test,
which suggested a nonparametric distribution of the data, and therefore, nonparametric
analyses were applied. Descriptive statistics were presented by the median and interquartile
range for continuous variables or number and percentage for categorical variables. Differ-
ence between groups was measured using the Mann–Whitney U test and Kruskal–Wallis
for continuous variables, while Pearson’s chi-squared was used for categorical variables. A
Bonferroni-corrected Dunn’s test was used for pairwise analysis of variables within which
there were multiple groups. Significant variables were carried forward to multivariate
analysis to identify odds ratio (OR) of the variables associated with clinical outcomes.
Binary logistic regression was then used to generate a formula collating the variables which
were significantly associated with the clinical outcomes on multivariate analysis. ROC
(receiver operating characteristic) curves were used to assess the diagnostic performance
of the combination of variables identified with the analysis. Sensitivity, specificity and
Youden indexes were estimated for a given cut-off. All tests were two-sided, and a p-value
0.05 was considered significant.
Statistical analysis was performed using SPSS (version 28.0; SPSS Inc. Chicago, IBM,
Chicago, IL, USA).

4.4. Ethics
This study was retrospective and included only fully anonymised data from inves-
tigations and assessments performed as per standard of care. As such, ethical approval
was not required, as stated by the UK policy framework for health and social care. Liver
tissue and plasma were stored at Imperial Hepatology Gastroenterology Biobank, which
was fully REC approved by Oxford C Research and Ethics Committee under REC reference
16/SC/0021.
Int. J. Mol. Sci. 2023, 24, 3563 11 of 12

Author Contributions: N.J.: data curation, formal analysis, software and writing—original draft. R.F.:
data curation, formal analysis, investigation, methodology, project administration, visualisation and
writing—original draft. B.P.: data curation and writing—review and editing. B.H.M.: writing—review
and editing. G.P.: data curation and writing—review and editing. Y.B.: data curation and writing—
review and editing. M.Y.: writing—review and editing. R.D.G.: writing—review and editing. M.R.T.:
writing—review and editing. P.M.: conceptualisation, funding acquisition, supervision and writing—
review and editing. All the authors have reviewed and approved the final draft. All authors have
read and agreed to the published version of the manuscript.
Funding: The Division of Digestive Diseases receives financial support from the National Institute of
Health Research (NIHR) Imperial Biomedical Research Centre (BRC).
Data Availability Statement: Data are contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Riazi, K.; Azhari, H.; Charette, J.H.; Underwood, F.E.; King, J.A.; Afshar, E.E.; Swain, M.G.; Congly, S.E.; Kaplan, G.G.; Shaheen,
A.-A. The prevalence and incidence of NAFLD worldwide: A systematic review and meta-analysis. Lancet Gastroenterol. Hepatol.
2022, 7, 851–861. [CrossRef] [PubMed]
2. Kleiner, D.E.; Brunt, E.M.; Van Natta, M.; Behling, C.; Contos, M.J.; Cummings, O.W.; Ferrell, L.D.; Liu, Y.-C.; Torbenson, M.S.;
Unalp-Arida, A.; et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005,
41, 1313–1321. [CrossRef] [PubMed]
3. Ekstedt, M.; Hagstrom, H.; Nasr, P.; Fredrikson, M.; Stal, P.; Kechagias, S.; Hultcrantz, R. Fibrosis Stage Is the Strongest Predictor
for Dis-ease-Specific Mortality in NAFLD After Up to 33Years of Follow-Up. Hepatology 2015, 61, 1547–1554. [CrossRef]
4. Berzigotti, A.; Tsochatzis, E.; Boursier, J.; Castera, L.; Cazzagon, N.; Friedrich-Rust, M.; Petta, S.; Thiele, M. EASL Clinical Practice
Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis—2021 update. J. Hepatol. 2021, 75, 659–689.
[CrossRef] [PubMed]
5. National Institute for Health and Care Excellence. Non-Alcoholic Fatty Liver Disease (NAFLD): Assessment and Management
NG49. 2016. Available online: https://www.nice.org.uk/guidance/ng49 (accessed on 10 January 2023).
6. Zhang, X.; Wong, G.L.-H.; Wong, V.W.-S. Application of transient elastography in nonalcoholic fatty liver disease. Clin. Mol.
Hepatol. 2020, 26, 128–141. [CrossRef]
7. Lurie, Y.; Webb, M.; Cytter-Kuint, R.; Shteingart, S.; Lederkremer, G.Z. Non-invasive diagnosis of liver fibrosis and cirrhosis.
World J. Gastroenterol. 2015, 21, 11567–11583. [CrossRef]
8. Younes, R.; Caviglia, G.P.; Govaere, O.; Rosso, C.; Armandi, A.; Sanavia, T.; Pennisi, G.; Liguori, A.; Francione, P.; Gallego-Durán,
R.; et al. Long-term outcomes and predictive ability of non-invasive scoring systems in patients with non-alcoholic fatty liver
disease. J. Hepatol. 2021, 75, 786–794. [CrossRef]
9. Loosen, S.H.; Kostev, K.; Keitel, V.; Tacke, F.; Roderburg, C.; Luedde, T. An elevated FIB-4 score predicts liver cancer development:
A longitudinal analysis from 29,999 patients with NAFLD. J. Hepatol. 2021, 76, 247–248. [CrossRef]
10. Gaggini, M.; Carli, F.; Bugianesi, E.; Gastaldelli, A.; Rosso, C.; Buzzigoli, E.; Marietti, M.; Della Latta, V.; Ciociaro, D.; Abate,
M.L.; et al. Altered amino acid concentrations in NAFLD: Impact of obesity and insulin resistance. Hepatology 2018, 67, 145–158.
[CrossRef]
11. Grzych, G.; Vonghia, L.; Bout, M.-A.; Weyler, J.; Verrijken, A.; Dirinck, E.; Curt, M.J.C.; Van Gaal, L.; Paumelle, R.; Francque,
S.; et al. Plasma BCAA Changes in Patients with NAFLD Are Sex Dependent. J. Clin. Endocrinol. Metab. 2020, 105, 2311–2321.
[CrossRef]
12. Masarone, M.; Troisi, J.; Aglitti, A.; Torre, P.; Colucci, A.; Dallio, M.; Federico, A.; Balsano, C.; Persico, M. Untargeted metabolomics
as a diagnostic tool in NAFLD: Discrimination of steatosis, steatohepatitis and cirrhosis. Metabolomics 2021, 17, 12. [CrossRef]
[PubMed]
13. Younossi, Z.; Stepanova, M.; Ong, J.P.; Jacobson, I.M.; Bugianesi, E.; Duseja, A.; Eguchi, Y.; Wong, V.W.; Negro, F.; Yilmaz, Y.; et al.
Nonalcoholic Steatohepatitis Is the Fastest Growing Cause of Hepatocellular Carcinoma in Liver Transplant Candidates. Clin.
Gastroenterol. Hepatol. 2018, 17, 748–755.e3. [CrossRef]
14. Kaswala, D.H.; Lai, M.; Afdhal, N.H. Fibrosis Assessment in Nonalcoholic Fatty Liver Disease (NAFLD) in 2016. Dig. Dis. Sci.
2016, 61, 1356–1364. [CrossRef] [PubMed]
15. Masoodi, M.; Gastaldelli, A.; Hyötyläinen, T.; Arretxe, E.; Alonso, C.; Gaggini, M.; Brosnan, J.; Anstee, Q.M.; Millet, O.; Ortiz, P.;
et al. Metabolomics and lipidomics in NAFLD: Biomarkers and non-invasive diagnostic tests. Nat. Rev. Gastroenterol. Hepatol.
2021, 18, 835–856. [CrossRef] [PubMed]
16. Singh, S.; Allen, A.M.; Wang, Z.; Prokop, L.J.; Murad, M.H.; Loomba, R. Fibrosis Progression in Nonalcoholic Fatty Liver vs
Nonalcoholic Steatohepatitis: A Systematic Review and Meta-analysis of Paired-Biopsy Studies. Clin. Gastroenterol. Hepatol. 2014,
13, 643–654.e9. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 3563 12 of 12

17. Sparks, J.D.; Sparks, C.E.; Adeli, K. Selective Hepatic Insulin Resistance, VLDL Overproduction, and Hypertriglyceridemia. Arter.
Thromb. Vasc. Biol. 2012, 32, 2104–2112. [CrossRef]
18. Patel, S.; Siddiqui, M.B.; Chandrakumaran, A.; Rodriguez, V.A.; Faridnia, M.; Roman, J.H.; Zhang, E.; Patrone, M.V.; Kakiyama,
G.; Walker, C.; et al. Progression to Cirrhosis Leads to Improvement in Atherogenic Milieu. Dig. Dis. Sci. 2020, 66, 263–272.
[CrossRef]
19. Buzzetti, E.; Pinzani, M.; Tsochatzis, E.A. The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metabolism
2016, 65, 1038–1048. [CrossRef]
20. McCullough, A.; Previs, S.F.; Dasarathy, J.; Lee, K.; Osme, A.; Kim, C.; Ilchenko, S.; Lorkowski, S.W.; Smith, J.D.; Dasarathy, S.;
et al. HDL flux is higher in patients with nonalcoholic fatty liver disease. Am. J. Physiol. Metab. 2019, 317, E852–E862. [CrossRef]
21. Hu, C.-A.A.; Khalil, S.; Zhaorigetu, S.; Liu, Z.; Tyler, M.; Wan, G.; Valle, D. Human ∆1-pyrroline-5-carboxylate synthase: Function
and regulation. Amino Acids 2008, 35, 665–672. [CrossRef]
22. Gordon, M.K.; Hahn, R.A. Collagens. Cell Tissue Res. 2010, 339, 247–257. [CrossRef] [PubMed]
23. Cao, Q.; Lu, X.; Azad, B.B.; Pomper, M.; Smith, M.; He, J.; Pi, L.; Ren, B.; Ying, Z.; Sichani, B.S.; et al. cis-4-[18F]fluoro-L-proline
Molecular Imaging Experimental Liver Fibrosis. Front. Mol. Biosci. 2020, 7, 90. [CrossRef] [PubMed]
24. Ortiz, C.; Schierwagen, R.; Schaefer, L.; Klein, S.; Trepat, X.; Trebicka, J. Extracellular Matrix Remodeling in Chronic Liver Disease.
Curr. Tissue Microenviron. Rep. 2021, 2, 41–52. [CrossRef] [PubMed]
25. Kagan, H.M.; Li, W. Lysyl oxidase: Properties, specificity, and biological roles inside and outside of the cell. J. Cell. Biochem. 2003,
88, 660–672. [CrossRef]
26. Kawasaki, H.; Hori, T.; Nakajima, M.; Takeshita, K. Plasma levels of pipecolic acid in patients with chronic liver disease. Hepatology
1988, 8, 286–289. [CrossRef]
27. Mayo, R.; Crespo, J.; Martínez-Arranz, I.; Banales, J.M.; Arias, M.; Mincholé, I.; de la Fuente, R.A.; Jimenez-Agüero, R.; Alonso, C.;
de Luis, D.A.; et al. Metabolomic-based noninvasive serum test to diagnose nonalcoholic steatohepatitis: Results from discovery
and validation cohorts. Hepatol. Commun. 2018, 2, 807–820. [CrossRef]
28. Polyzos, S.A.; Perakakis, N.; Boutari, C.; Kountouras, J.; Ghaly, W.; Anastasilakis, A.D.; Karagiannis, A.; Mantzoros, C.S. Targeted
Analysis of Three Hormonal Systems Identifies Molecules Associated with the Presence and Severity of NAFLD. J. Clin. Endocrinol.
Metab. 2019, 105, e390–e400. [CrossRef]
29. Beckonert, O.; Keun, H.C.; Ebbels, T.M.D.; Bundy, J.; Holmes, E.; Lindon, J.C.; Nicholson, J.K. Metabolic profiling, metabolomic
and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. Nat. Protoc. 2007, 2, 2692–2703.
[CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.

You might also like