Ijms 24 03563
Ijms 24 03563
Ijms 24 03563
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
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).
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.
Table 2. Differences in metabolic profile between fast progressors and nonfast progressors.
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.
Table 4. Differences in metabolic profile between fast progressors and nonfast progressors in patients
who had a liver biopsy.
Table 4. Cont.
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.
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.
Using binary logistic regression, a formula was generated to predict the presence of
cirrhosis, combining the metabolites that were significant on multivariate analysis:
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
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].
4.4. 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]
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