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\begin{document}

\title{The gut-liver axis in nonalcoholic fatty liver disease: association of
intestinal permeability with disease severity and treatment outcomes}



\author[1]{Yu-Pei Zhuang}%
\author[1]{Yi-Ting Zhang}%
\author[1]{Hao-Jie Zhong}%
\author[1]{Xing-Xiang He}%
\affil[1]{The First Affiliated Hospital of Guangdong Pharmaceutical University}%


\vspace{-1em}



  
  \date{June 16, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Objective To investigate the association between intestinal permeability
and severity of nonalcoholic fatty liver disease (NAFLD), and the value
of intestinal permeability in predicting the efficacy of metabolic
therapy for NAFLD. Methods Disease severity was compared between
patients with normal and elevated intestinal permeability; correlations
between D-lactate and different NAFLD parameters were analyzed; and the
effects of metabolic therapy on NAFLD patients with normal and elevated
intestinal permeability were evaluated. Results A total of 190 patients
with NAFLD were enrolled. NAFLD patients with elevated intestinal
permeability had significantly higher levels of liver test parameters,
liver ultrasonographic fat attenuation parameter, triglyceride,
homeostasis model assessment of insulin resistance value and diamine
oxidase (all P<\selectlanguage{english}0.05) than NAFLD patients with normal intestinal
permeability. Further, serum D-lactate levels were positively correlated
with alanine transaminase, aspartate transaminase, gamma-glutamyl
transpeptidase, total bilirubin, indirect bilirubin, fat attenuation
parameter, triglyceride, and diamine oxidase (all P<\selectlanguage{english}0.05). Moreover,
NAFLD patients with elevated intestinal permeability showed less
improvement in TG levels (P=0.014) after metabolic therapy. Conclusion
Intestinal permeability correlates with the disease severity in patients
with NAFLD. Moreover, intestinal permeability may have value for
predicting the efficacy of metabolic therapy for NAFLD patients.%
\end{abstract}%



\sloppy


Article type: Original Paper

The gut-liver axis in nonalcoholic fatty liver disease: association of
intestinal permeability with disease severity and treatment outcomes

Running Title: Nonalcoholic fatty liver disease and intestinal
permeability

\textbf{Abstract}

\textbf{Objective}To investigate the association between intestinal
permeability and severity of nonalcoholic fatty liver disease (NAFLD),
and the value of intestinal permeability in predicting the efficacy of
metabolic therapy for NAFLD.

\textbf{Methods} Disease severity was compared between patients with
normal and elevated intestinal permeability; correlations between
D-lactate and different NAFLD parameters were analyzed; and the effects
of metabolic therapy on NAFLD patients with normal and elevated
intestinal permeability were evaluated.

\textbf{Results} A total of 190 patients with NAFLD were enrolled. NAFLD
patients with elevated intestinal permeability had significantly higher
levels of liver test parameters, liver ultrasonographic fat attenuation
parameter, triglyceride, homeostasis model assessment of insulin
resistance value and diamine oxidase (all P<\selectlanguage{english}0.05) than NAFLD patients
with normal intestinal permeability. Further, serum D-lactate levels
were positively correlated with alanine transaminase, aspartate
transaminase, gamma-glutamyl transpeptidase, total bilirubin, indirect
bilirubin, fat attenuation parameter, triglyceride, and diamine oxidase
(all P<\selectlanguage{english}0.05). Moreover, NAFLD patients with elevated intestinal
permeability showed less improvement in TG levels (P=0.014) after
metabolic therapy.

\textbf{Conclusion} Intestinal permeability correlates with the disease
severity in patients with NAFLD. Moreover, intestinal permeability may
have value for predicting the efficacy of metabolic therapy for NAFLD
patients.

\textbf{Keywords:} nonalcoholic fatty liver disease; intestinal
permeability; disease severity; patients; treatment effectiveness

\textbf{WHAT'S KNOWN}

\begin{itemize}
\tightlist
\item
  Nonalcoholic fatty liver disease (NAFLD) has emerged as the major
  cause of chronic liver disease.
\item
  Many intestinal factors (such as intestinal permeability) have been
  found to be associated with the pathogenesis, progression, and
  severity of liver diseases.
\item
  The association between intestinal permeability and disease severity
  remains controversial.
\end{itemize}

\textbf{WHAT'S NEW}

Intestinal permeability correlates with the disease severity in patients
with NAFLD.

Intestinal permeability may have value for predicting the efficacy of
metabolic therapy for NAFLD patients.



\textbf{Introduction}

Nonalcoholic fatty liver disease (NAFLD) has emerged as the major cause
of chronic liver disease worldwide, and its global prevalence is
estimated to be 25\% {[}1, 2{]}. NAFLD encompasses a spectrum of
pathological changes, ranging from steatosis and nonalcoholic
steatohepatitis (NASH), to liver cirrhosis and even hepatocellular
carcinoma {[}3{]}. Considerable progress has been made toward
understanding the pathogenesis of NAFLD, including the contributions of
insulin resistance, inflammation, and oxidative stress {[}4-6{]};
however, the factors contributing to disease severity and progression
have not been completely clarified.

There is evidence for a close interaction between the gut and the liver,
known as the ``gut-liver axis'' {[}7, 8{]}, and gut microbiota,
microbial metabolites, and immune responses are associated with NAFLD
pathogenesis {[}8-10{]}. Further, recent studies have suggested a role
for intestinal barrier dysfunction in the progression of NAFLD
{[}11-13{]}. In animal studies, increased intestinal permeability can be
detected in mice with NAFLD induced by high-fat or choline-deficient
diets {[}13, 14{]}. In addition, clinical studies have demonstrated
increased intestinal permeability in patients with NAFLD relative to
healthy controls {[}12, 13{]}. Moreover, a previous study reported a
correlation between the lactulose/mannitol ratio and pathologic severity
of NAFLD, indicating that intestinal permeability might correlate with
the severity of NAFLD {[}12{]}. However, another investigation detected
no significant differences in liver transaminases or triglycerides
between NAFLD patients with normal and increased intestinal permeability
{[}15{]}. Hence, further investigation is needed to fully understand
whether intestinal permeability is associated with disease severity in
NAFLD patients, especially with respect to liver test parameters and
blood lipid levels. Further, recent studies have shown that some
therapies can ameliorate NAFLD by improving the gut barrier
permeability, indicating a potential role for addressing intestinal
permeability in NAFLD treatment approaches {[}16, 17{]}. Nonetheless, it
is still not known whether the treatment effect of NAFLD is affected by
intestinal permeability.

Polyethylene glycol, \textsuperscript{51}Cr-labelled ethylene diamine
tetraacetate acid (\textsuperscript{51}Cr-EDTA), and a number of
non-invasive tests (i.e., urinary recovery of orally administered
sugars) are widely used to measure intestinal permeability in humans
{[}18{]}. More recently, D-lactate, which is only produced by intestinal
bacteria, has been also introduced as a convenient and well-accepted
biomarker for intestinal permeability {[}19, 20{]}. Given that this
compound is found in small concentrations in human blood, elevated level
of serum D-lactate indicates increased intestinal permeability.

This study was designed to investigate the association between
intestinal permeability and severity of NAFLD and the value of
intestinal permeability for predicting the efficacy of metabolic therapy
for NAFLD with the use of serum D-lactate.

\textbf{Methods}

\emph{Participants}

This retrospective study was performed in accordance with the ethical
guidelines of the Declaration of Helsinki and approved by the ethics
committee of The First Affiliated Hospital of Guangdong Pharmaceutical
University (No. 20176601). Informed consent was obtained from all
patients. We followed the STROBE (Strengthening the Reporting of
Observational Studies in Epidemiology) statement in reporting this
study. The sample size was estimated using an online software (Power and
Sample Size Calculation; HyLown Consulting LLC, Atlanta, GA, USA).

Patients with NAFLD who were hospitalized at The First Affiliated
Hospital of Guangdong Pharmaceutical University for metabolic therapy
between October 2017 and October 2019 and tested for D-lactate were
eligible for inclusion. Patients with carcinoma; severe heart, brain, or
kidney disease; other chronic liver diseases such as viral, alcoholic,
autoimmune, and Wilson's disease; and those with missing important
medical data were excluded.

Data of all enrolled patients were used to analyze the association
between intestinal permeability and severity of NAFLD. Data of those
patients who completed a one-month follow-up after metabolic therapy
were used to analyze the value of intestinal permeability for predicting
the efficacy of metabolic therapy for NAFLD (\textbf{Figure 1} ).

\emph{Data collection}

Medical records were reviewed and the following information extracted:
demographic characteristics; smoking status; comorbidity; liver
parameters (alanine transaminase {[}ALT{]}, aspartate transaminase
{[}AST{]}, gamma-glutamyl transpeptidase {[}GGT{]}, total bilirubin
{[}TBIL{]}, direct bilirubin {[}DBIL{]}, and indirect bilirubin
{[}IBIL{]}); metabolic parameters (triglyceride {[}TG{]}, total
cholesterol {[}TC{]}, high-density lipoprotein {[}HDL{]}, low-density
lipoprotein {[}LDL{]}, and homeostasis model assessment of insulin
resistance {[}HOMA-IR{]} value); ultrasonographic parameters (liver
stiffness measurement {[}LSM{]} and fat attenuation parameter
{[}FAP{]}); and intestinal parameters (serum D-lactate, diamine oxidase
{[}DAO{]} and lipopolysaccharide {[}LPS{]}).

\emph{Definitions}

NAFLD was defined as hepatic steatosis proven by imaging or histology,
lack of secondary causes of hepatic fat accumulation, and no significant
alcohol consumption (weekly alcohol intake [?]210 g in men and [?]140 g in
women) {[}21{]}. Hypertension was defined by blood pressure [?]140/90
mmHg, use of anti-hypertensive medication, or a self-reported history of
hypertension. Diabetes was defined by fasting glucose [?]7.0 mmol/L, 2-h
glucose [?]11.1 mmol/L during an oral glucose tolerance test, use of
anti-diabetic medication, or a self-reported history of diabetes.
Elevated intestinal permeability was defined by elevated serum D-lactate
([?]15 U/L). HOMA-IR value was calculated as previously described
{[}22{]}. Metabolic therapy included lipid-lowering medicines (such as
simvastatin and metformin) and lifestyle modifications (healthy eating
and regular exercise).

\emph{Intestinal mucosal barrier function measurement}

Blood samples were collected after patients had fasted for 8 h. The
parameters of intestinal mucosal barrier function including D-lactate,
DAO, and LPS were analyzed using the Intestinal Mucosal Barrier
Biochemical Index Analysis System (JY-DLT, Beijing Zhongsheng Jinyu
Diagnostic Technology Co., Ltd., China) according to the manufacturer's
protocols {[}23, 24{]}.

\emph{Statistical analysis}

All statistical analyses were conducted using SPSS 22.0 (IBM Corp.,
Armonk, NY, USA). Continuous variables are presented as mean\selectlanguage{ngerman}±standard
deviation for those with normal distributions and as median and
interquartile range for those with skewed distributions. Categorical
variables are presented as numbers with percentages. Continuous
variables were compared using the Student's\emph{t} -test or
Mann--Whitney U test, and categorical variables were compared using the
chi-squared or Fisher's exact tests, as applicable. Correlations between
D-lactate and clinical parameters in patients with NAFLD were determined
using Pearson's or Spearman's correlation, as applicable. P\selectlanguage{english}<0.05
(two-sided) was considered to indicate statistical significance.

\textbf{Results}

\emph{Characteristics of participants}

We identified 190 eligible patients with NAFLD based on the inclusion
and exclusion criteria. Of these patients, 79 (41.58\%) had elevated
intestinal permeability (D-lactate, [?]15 U/L) and 111 (58.42\%) had
normal intestinal permeability (D-lactate, \textless{}15 U/L). First, we
compared the characteristics between these two groups of patients. There
were no significant intergroup differences in demographic
characteristics, smoking status, or comorbidities (\textbf{Table 1} ).

\emph{Disease severity in NAFLD patients with normal or elevated
intestinal permeability}

Next, we analyzed disease severity in NAFLD patients with normal or
elevated intestinal permeability. NAFLD patients with elevated
intestinal permeability (D-lactate: 19.00 {[}16.48--23.23{]} U/L) had
significantly higher levels of liver test parameters ALT, AST, GGT,
TBIL, and IBIL (all P<\selectlanguage{english}0.05); higher levels of the liver ultrasonographic
parameter, FAP (P=0.001); higher levels of TG (P=0.023) and an elevated
HOMA-IR value (P=0.020) among metabolic parameters; and a higher level
of the intestinal parameter, DAO (P=0.025), than those with normal
intestinal permeability (D-lactate, 9.35 {[}6.47--12.40{]} U/L)
(\textbf{Table 2} ).

\emph{Correlation between D-lactate and disease severity in patients
with NAFLD}

Next, we assessed the correlations between D-lactate level and the
clinical parameters identified as differing in NAFLD patients with and
without elevated intestinal permeability. As shown in Table 3, D-lactate
was positively correlated with ALT (r=0.312, P<\selectlanguage{english}0.001); AST (r=0.303,
P<\selectlanguage{english}0.001); GGT (r=0.190, P=0.017); TBIL (r=0.214, P=0.004); IBIL
(r=0.247, P=0.001); FAP (r=0.252, P=0.001); TG (r=0.173, P=0.021); and
DAO (r=0.218, P=0.002). A positive correlation was also detected between
D-lactate and LPS, although it was not statistically significant
(r=0.132, P=0.069). However, D-lactate did not correlate with DBIL, LSM,
TC, HDL, LDL, and HOMA-IR values (\textbf{Table 3} ).

\emph{Efficacy of metabolic therapy in NAFLD patients with normal or
elevated intestinal permeability}

Finally, we explored whether intestinal permeability can predict the
efficacy of metabolic therapy in patients with NAFLD. Thirty patients
with NAFLD completed a one-month follow-up after metabolic therapy. The
effects of metabolic therapy were assessed by determining the
improvements in blood lipids as follows: \selectlanguage{greek}Δ\selectlanguage{english}lipid=baseline levels--levels
one month after treatment.

NAFLD patients with elevated intestinal permeability had a lower \selectlanguage{greek}Δ\selectlanguage{english}TG
value one month after metabolic therapy, at -0.10 (-0.39--0.39) vs. 1.00
(0.90--1.30) (P=0.014) than those with normal intestinal permeability.
Besides, patients with normal intestinal permeability seemed to have a
better improvement of TC, HDL, and LDL after one month of metabolic
therapy, although the differences were not statistically significant
(\textbf{Table 4} ). However, the changes in clinical characteristics
(BMI); liver test parameters (ALT, AST, GGT, TBIL, DBIL, and IBIL); and
liver ultrasonographic parameters (LSM and FAP) showed no significant
differences between the two groups after one month of metabolic therapy.

\textbf{Discussion}

Our study showed that NAFLD patients with elevated intestinal
permeability have more severe disease status, manifested as more serious
liver dysfunction, hyperlipidemia, liver fat deposition, insulin
resistance, and intestinal barrier damage. Our data also showed that
serum D-lactate is positively correlated with parameters indicative of
disease severity, including ALT, AST, GGT, TBIL, IBIL, FAP, and TG.
These findings reveal a clear association between intestinal
permeability and disease severity in patients with NAFLD, which add to
accumulating evidence supporting the ``gut-liver axis.'' Hence,
intestinal manifestations warrant increased attention in patients with
severe NAFLD such as NASH and NAFLD-associated cirrhosis.

However, a previous study with a small sample size (35 NAFLD patients
and 24 controls) reported no significant differences in liver
transaminases and TG between NAFLD patients with normal and increased
intestinal permeability, measured by \textsuperscript{51}Cr-EDTA
excretion testing {[}15{]}; while another study reported a correlation
between intestinal permeability and pathologic severity (portal
inflammation, fibrosis, and ballooning of hepatocytes) using the
lactulose-mannitol bowel permeability test, in line with our findings
{[}12{]}. Further, zonulin, a moderator of intestinal permeability, was
found to be positively correlated with some parameters of disease
severity, such as ALT, TG, HOMA-IR, and liver histopathology in patients
with NAFLD, hence indicating a correlation between intestinal
permeability and NAFLD severity {[}25{]}. In addition to the small
sample size of the previous study being likely inadequate to show the
relationship between intestinal permeability and disease severity in
patients with NAFLD, using different methods to detect intestinal
permeability may also lead to inconsistent results. In addition
to\textsuperscript{51}Cr-EDTA excretion testing and lactulose-mannitol
bowel permeability test, testing for D-lactate levels has become a
widely used method for intestinal permeability detection in recent years
{[}23, 24{]}. Owing to the simplicity of this method, the bias caused by
multiple operations can be reduced. However, given that the criteria for
defining intestinal permeability have not reached certain consensus,
other tests to evaluate intestinal permeability should be used to
confirm these findings by further studies.

There are two plausible reasons for the observed association between
intestinal permeability and severity of NAFLD. First, elevated
intestinal permeability can cause pathogenesis and progression of NAFLD.
Animal research has shown that increased intestinal permeability induced
by dextran sulfate sodium (DSS) enhances high-fat diet-induced hepatic
inflammation and steatosis in mice {[}26{]}. Subsequent to the increase
of intestinal permeability, bacterial components, particularly LPS, can
translocate into the portal vein and thus the liver, resulting in liver
inflammation and injury {[}27, 28{]}. In support of this hypothesis, a
clinical study showed that plasma antibodies against LPS were increased
in patients with NASH compared with healthy controls, and increased with
aggravated inflammation in NASH, indicating an association between LPS
exposure and the severity of NASH in humans {[}29{]}. Consistent with
this finding, our data showed a positive correlation between D-lactate
and LPS, although the difference did not reach the threshold for
statistical significance (P=0.069), suggesting that increased intestinal
permeability may lead to LPS translocation, and consequently enhanced
liver injury. The alternative explanation is that NAFLD may contribute
to the increase in intestinal permeability, as disruption of the
intestinal epithelial barrier and gut vascular barrier can be detected
in NAFLD mice induced by high-fat diet {[}14{]}.

Recent studies have found that improvement of intestinal permeability by
fecal microbiota transplantation or probiotics had a therapeutic effect
on NAFLD {[}16, 17{]}. These results suggest that improvement of
intestinal permeability may promote the lipid-lowering effect of
metabolic therapy in patients with NAFLD. Accordingly, our data revealed
that NAFLD patients with elevated intestinal permeability present with
less substantial improvement in TG levels after metabolic therapy. This
study has provided evidence for the role of elevated intestinal
permeability in NAFLD progression and suggested that a combination of
treatment to improve intestinal barrier and metabolic therapy may have
better therapeutic effects on NAFLD patients, especially those with
elevated intestinal permeability.

However, we found that the improvement of clinical characteristics,
liver test parameters, and liver ultrasonographic parameters were not
significantly different between NAFLD patients with elevated and normal
intestinal permeability after one month of metabolic treatment. As these
patients received metabolic therapy, the improvement of blood lipids was
more obvious, while other parameters of disease severity did not show
significant improvement. In addition, we only analyzed the results of
the one-month-long metabolic therapy, thus the unexceptional improvement
in other parameters could likely be attributed to the short treatment
time. Therefore, a prospective and long-term follow-up study is needed
to assess whether intestinal permeability affects the improvement of
other parameters in patients with NAFLD after treatment.

The present study has some limitations. First, intestinal biopsies were
not performed as standard in our investigation, as most enrolled
patients had no gastrointestinal symptoms. Second, because this is a
retrospective study, some potential confounders (e.g., use of medication
with liver toxicity) that may lead to liver injury and steatosis were
not recorded. Third, although our sample size was larger than those of
previous studies investigating the association between intestinal
permeability and NAFLD, this was a single-center study with a limited
sample size. Only 30 patients' clinical data were available for
analyzing the efficacy of metabolic therapy in NAFLD patients with
normal or elevated intestinal permeability; moreover, we could not
analyze whether intestinal permeability affects the improvement of other
parameters in patients with NAFLD after treatment. Therefore, our
results should be interpreted with caution, and future studies are
needed to confirm these findings.

In summary, intestinal permeability correlates with the severity of
liver dysfunction, hyperlipidemia, liver fat deposition, insulin
resistance, and intestinal barrier damage in patients with NAFLD.
Moreover, intestinal permeability may be valuable for predicting the
efficacy of metabolic therapy in patients with NAFLD.

~\textbf{Acknowledgement}

The manuscript has been presented as a preprint.



\textbf{References}

{[}1{]} Lazarus JV, Ekstedt M, Marchesini G, et al. A cross-sectional
study of the public health response to non-alcoholic fatty liver disease
in Europe. J Hepatol 2020, 72(1):14-24.

{[}2{]} Younossi ZM, Koenig AB, Abdelatif D, et al. Global epidemiology
of nonalcoholic fatty liver disease-Meta-analytic assessment of
prevalence, incidence, and outcomes. Hepatology 2016, 64(1):73-84.

{[}3{]} Sanyal AJ. Past, present and future perspectives in nonalcoholic
fatty liver disease. Nature reviews Gastroenterology \& hepatology 2019,
16(6):377-386.

{[}4{]} Brunt EM, Wong VW, Nobili V, et al. Nonalcoholic fatty liver
disease. Nat Rev Dis Primers 2015, 1:15080.

{[}5{]} Tilg H, Adolph TE, Moschen AR. Multiple parallel hits hypothesis
in NAFLD - revisited after a decade. Hepatology 2020.

{[}6{]} Cai J, Zhang XJ, Li H. Role of innate immune signaling in
non-alcoholic fatty liver disease. Trends Endocrinol Metab 2018,
29(10):712-722.

{[}7{]} Tilg H, Burcelin R, Tremaroli V. Liver tissue microbiome in
NAFLD: next step in understanding the gut-liver axis? Gut 2020,
69(8):1373-1374.

{[}8{]} Albillos A, de Gottardi A, Rescigno M. The gut-liver axis in
liver disease: Pathophysiological basis for therapy. J Hepatol 2020,
72(3):558-577.

{[}9{]} Canfora EE, Meex RCR, Venema K, et al. Gut microbial metabolites
in obesity, NAFLD and T2DM. Nat Rev Endocrinol 2019, 15(5):261-273.

{[}10{]} Aron-Wisnewsky J, Vigliotti C, Witjes J, et al. Gut microbiota
and human NAFLD: disentangling microbial signatures from metabolic
disorders. Nature reviews Gastroenterology \& hepatology 2020,
17(5):279-297.

{[}11{]} Chen M, Hui S, Lang H, et al. SIRT3 deficiency promotes
high-fat diet-induced nonalcoholic fatty liver disease in correlation
with impaired intestinal permeability through gut microbial dysbiosis.
Mol Nutr Food Res 2019, 63(4):e1800612.

{[}12{]} Giorgio V, Miele L, Principessa L, et al. Intestinal
permeability is increased in children with non-alcoholic fatty liver
disease, and correlates with liver disease severity. Digestive and liver
disease : official journal of the Italian Society of Gastroenterology
and the Italian Association for the Study of the Liver 2014,
46(6):556-560.

{[}13{]} Luther J, Garber JJ, Khalili H, et al. Hepatic injury in
nonalcoholic steatohepatitis contributes to altered intestinal
permeability. Cell Mol Gastroenterol Hepatol 2015, 1(2):222-232.

{[}14{]} Mouries J, Brescia P, Silvestri A, et al. Microbiota-driven gut
vascular barrier disruption is a prerequisite for non-alcoholic
steatohepatitis development. J Hepatol 2019, 71(6):1216-1228.

{[}15{]} Miele L, Valenza V, La Torre G, et al. Increased intestinal
permeability and tight junction alterations in nonalcoholic fatty liver
disease. Hepatology 2009, 49(6):1877-1887.

{[}16{]} Zhou D, Pan Q, Shen F, et al. Total fecal microbiota
transplantation alleviates high-fat diet-induced steatohepatitis in mice
via beneficial regulation of gut microbiota. Sci Rep 2017, 7(1):1529.

{[}17{]} Zhao Z, Chen L, Zhao Y, et al. Lactobacillus plantarum NA136
ameliorates nonalcoholic fatty liver disease by modulating gut
microbiota, improving intestinal barrier integrity, and attenuating
inflammation. Appl Microbiol Biotechnol 2020, 104(12):5273-5282.

{[}18{]} De Munck TJI, Xu P, Verwijs HJA, et al. Intestinal permeability
in human nonalcoholic fatty liver disease: A systematic review and
meta-analysis. ~Liver Int 2020, 40(12):2906-2916.~{}

{[}19{]} Xu R, Tan C, He Y, et al. Dysbiosis of gut microbiota and
short-chain fatty acids in encephalitis: a Chinese pilot study. Front
Immunol 2020, 11:1994.

{[}20{]} Verbeke KA, Boobis AR, Chiodini A, et al. Towards microbial
fermentation metabolites as markers for health benefits of prebiotics.
Nutr Res Rev 2015, 28(1):42-66.

{[}21{]} National Workshop on Fatty L, Alcoholic Liver Disease CSoHCMA,
Fatty Liver Expert Committee CMDA. Guidelines of prevention and
treatment for nonalcoholic fatty liver disease: a 2018 update. Zhonghua
Gan Zang Bing Za Zhi 2018, 26(3):195-203.

{[}22{]} Matthews DR, Hosker JP, Rudenski AS, et al. Homeostasis model
assessment: insulin resistance and beta-cell function from fasting
plasma glucose and insulin concentrations in man. Diabetologia 1985,
28(7):412-419.

{[}23{]} Ling Z, Liu X, Guo S, et al. Role of probiotics in mycoplasma
pneumoniae pneumonia in children: a short-term pilot project. Front
Microbiol 2018, 9:3261.

{[}24{]} Liu X, Cheng Y, Shao L, et al. Alterations of the predominant
fecal microbiota and disruption of the gut mucosal barrier in patients
with early-stage colorectal cancer. Biomed Res Int 2020, 2020:2948282.

{[}25{]} Hendy OM, Elsabaawy MM, Aref MM, et al. Evaluation of
circulating zonulin as a potential marker in the pathogenesis of
nonalcoholic fatty liver disease. APMIS 2017, 125(7):607-613.

{[}26{]} Gabele E, Dostert K, Hofmann C, et al. DSS induced colitis
increases portal LPS levels and enhances hepatic inflammation and
fibrogenesis in experimental NASH. J Hepatol 2011, 55(6):1391-1399.

{[}27{]} Di Ciaula A, Baj J, Garruti G, et al. Liver Steatosis,
Gut-Liver Axis, Microbiome and Environmental Factors. A Never-Ending
Bidirectional Cross-Talk. J Clin Med 2020, 9(8):2648.

{[}28{]} Svegliati-Baroni G, Patricio B, Lioci G, et al.
Gut-pancreas-liver axis as a target for treatment of NAFLD/NASH. Int J
Mol Sci 2020, 21(16):5820.

{[}29{]} Verdam FJ, Rensen SS, Driessen A, et al. Novel evidence for
chronic exposure to endotoxin in human nonalcoholic steatohepatitis. J
Clin Gastroenterol 2011, 45(2):149-152.

\textbf{Table 1:} Patient characteristics\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{Normal intestinal permeability} \textbf{(n=111)}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{Elevated intestinal permeability} \textbf{(n=79)}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{P-value}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Age (years)}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
56.00 (48.00--64.00)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
59.00 (50.00--65.00)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.283\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Male sex, n (\%)}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
63 (56.76)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
38 (48.10)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.239\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Body mass index (kg/m\textsuperscript{2})}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
25.71\selectlanguage{ngerman}±2.94\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
25.70\selectlanguage{ngerman}±3.53\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.990\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Smoking, n (\%)}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
28 (25.23)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
19 (24.05)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.853\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Hypertension, n (\%)}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
34 (30.63)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
30 (37.97)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.291\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Diabetes, n (\%)}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
18 (16.22)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
18 (22.78)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.255\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Normal intestinal permeability was defined as D-lactate\textless{}15
U/L, and elevated intestinal permeability was defined as D-lactate[?]15
U/L.

\textbf{Table 2:} Comparison of clinical parameters in NAFLD patients
with normal or elevated intestinal permeability\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& \textbf{Normal intestinal permeability} \textbf{(N=111)} &
\textbf{Elevated intestinal permeability} \textbf{(N=79)} &
\textbf{P-value}\tabularnewline
\textbf{Liver test parameters} & \textbf{Liver test parameters} &
\textbf{Liver test parameters} & \textbf{Liver test
parameters}\tabularnewline
\textbf{ALT (U/L)} & 18.00 (13.00--24.50) (n=109) & 24.00 (18.00--36.00)
(n=75) & \textbf{<0.001}\selectlanguage{english}\tabularnewline
\textbf{AST (U/L)} & 19.00 (16.00--23.00) (n=109) & 21.00 (19.00--29.00)
(n=75) & \textbf{<0.001}\selectlanguage{english}\tabularnewline
\textbf{GGT (U/L)} & 24.50 (18.00--32.53) (n=92) & 28.05 (20.95--45.50)
(n=66) & \textbf{0.022}\tabularnewline
\selectlanguage{greek}\textbf{TBIL (μmol/L)}\selectlanguage{english} & 11.00 (9.15--14.30) (n=109) & 12.90
(10.40--15.50) (n=75) & \textbf{0.010}\tabularnewline
\selectlanguage{greek}\textbf{DBIL (μmol/L)}\selectlanguage{english} & 3.35 (2.70--4.30) (n=108) & 3.70 (2.80--4.80)
(n=74) & 0.218\tabularnewline
\selectlanguage{greek}\textbf{IBIL (μmol/L)}\selectlanguage{english} & 7.80 (6.50--9.80) (n=108) & 9.20 (7.50--11.40)
(n=75) & \textbf{0.003}\tabularnewline
\textbf{Liver ultrasonographic parameters} & \textbf{Liver
ultrasonographic parameters} & \textbf{Liver ultrasonographic
parameters} & \textbf{Liver ultrasonographic parameters}\tabularnewline
\textbf{LSM (kPa)} & 6.70 (5.58--8.80) (n=106) & 6.80 (5.80--8.30)
(n=76) & 0.924\tabularnewline
\textbf{FAP (dB/m)} & 259.50 (247.00--285.00) (n=106) & 276.00
(255.00--295.50) (n=77) & \textbf{0.001}\tabularnewline
\textbf{Metabolic parameters} & \textbf{Metabolic parameters} &
\textbf{Metabolic parameters} & \textbf{Metabolic
parameters}\tabularnewline
\textbf{TG (mmol/L)} & 1.37 (0.99--2.20) (n=104) & 1.68 (1.15--2.44)
(n=73) & \textbf{0.023}\tabularnewline
\textbf{TC (mmol/L)} & 4.70 (4.27--5.78) (n=104) & 4.98 (4.52--5.79)
(n=73) & 0.096\tabularnewline
\textbf{HDL (mmol/L)} & 1.10 (0.96--1.30) (n=104) & 1.14 (1.00--1.25)
(n=73) & 0.814\tabularnewline
\textbf{LDL (mmol/L)} & 2.82 (2.23--3.62) (n=104) & 3.04 (2.50--3.60)
(n=73) & 0.258\tabularnewline
\textbf{HOMA-IR value} & 1.83 (0.74--2.62) (n=110) & 2.71 (1.14--3.91)
(n=79) & \textbf{0.020}\tabularnewline
\textbf{Intestinal parameters} & \textbf{Intestinal parameters} &
\textbf{Intestinal parameters} & \textbf{Intestinal
parameters}\tabularnewline
\textbf{DAO (U/L)} & 4.54 (2.66--9.80) & 6.15 (3.20--12.45) &
\textbf{0.025}\tabularnewline
\textbf{LPS (U/L)} & 9.20 (4.39-10.51) & 9.30 (4.18--11.22) &
0.355\tabularnewline
\bottomrule
\end{longtable}

ALT: alanine transaminase; AST: aspartate transaminase; DAO: diamine
oxidase; DBIL: direct bilirubin; FAP: fat attenuation parameter; GGT:
gamma-glutamyl transpeptidase; HDL: high-density lipoprotein; HOMA-IR:
homeostasis model assessment of insulin resistance; IBIL: indirect
bilirubin; LDL: low-density lipoprotein; LPS: lipopolysaccharide; LSM:
liver stiffness measurement; NAFLD: nonalcoholic fatty liver disease;
TBIL: total bilirubin; TC: total cholesterol; TG: triglyceride. Normal
intestinal permeability was defined as D-lactate\textless{}15 U/L, and
elevated intestinal permeability was defined as D-lactate[?]15 U/L.
Significant P values are indicated in bold font.

\textbf{Table 3:} Correlations between D-lactate and clinical parameters
in patients with NAFLD\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& \textbf{D-lactate (U/L)} & \textbf{D-lactate (U/L)}\tabularnewline
& r & P-value\tabularnewline
\textbf{Liver test parameters} & \textbf{Liver test parameters} &
\textbf{Liver test parameters}\tabularnewline
\textbf{ALT (U/L)} & 0.312 & \textbf{<0.001}\selectlanguage{english}\tabularnewline
\textbf{AST (U/L)} & 0.303 & \textbf{<0.001}\selectlanguage{english}\tabularnewline
\textbf{GGT (U/L)} & 0.190 & \textbf{0.017}\tabularnewline
\selectlanguage{greek}\textbf{TBIL (μmol/L)}\selectlanguage{english} & 0.214 & \textbf{0.004}\tabularnewline
\selectlanguage{greek}\textbf{DBIL (μmol/L)}\selectlanguage{english} & 0.130 & 0.081\tabularnewline
\selectlanguage{greek}\textbf{IBIL (μmol/L)}\selectlanguage{english} & 0.247 & \textbf{0.001}\tabularnewline
\textbf{Liver ultrasonographic parameters} & \textbf{Liver
ultrasonographic parameters} & \textbf{Liver ultrasonographic
parameters}\tabularnewline
\textbf{LSM (kPa)} & 0.049 & 0.515\tabularnewline
\textbf{FAP (dB/m)} & 0.252 & \textbf{0.001}\tabularnewline
\textbf{Metabolic parameters} & \textbf{Metabolic parameters} &
\textbf{Metabolic parameters}\tabularnewline
\textbf{TG (mmol/L)} & 0.173 & \textbf{0.021}\tabularnewline
\textbf{TC (mmol/L)} & 0.117 & 0.121\tabularnewline
\textbf{HDL (mmol/L)} & 0.066 & 0.384\tabularnewline
\textbf{LDL (mmol/L)} & 0.096 & 0.203\tabularnewline
\textbf{HOMA-IR value} & 0.100 & 0.173\tabularnewline
\textbf{Intestinal parameters} & \textbf{Intestinal parameters} &
\textbf{Intestinal parameters}\tabularnewline
\textbf{DAO (U/L)} & 0.218 & \textbf{0.002}\tabularnewline
\textbf{LPS (U/L)} & 0.132 & 0.069\tabularnewline
\bottomrule
\end{longtable}

ALT: alanine transaminase; AST: aspartate transaminase; DAO: diamine
oxidase; DBIL: direct bilirubin; FAP: fat attenuation parameter; GGT:
gamma-glutamyl transpeptidase; HDL: high-density lipoprotein; HOMA-IR:
homeostasis model assessment of insulin resistance; IBIL: indirect
bilirubin; LDL: low-density lipoprotein; LPS: lipopolysaccharide; LSM:
liver stiffness measurement; NAFLD: nonalcoholic fatty liver disease;
TBIL: total bilirubin; TC: total cholesterol; TG: triglyceride.
Significant P values are indicated in bold font.

\textbf{Table 4:} Effects of metabolic therapy in NAFLD patients with
normal or elevated intestinal permeability\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{Normal intestinal permeability} \textbf{(N=7)}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{Elevated intestinal permeability} \textbf{(N=23)}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{P-value}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{ΔTG (mmol/L)}\selectlanguage{english}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1.00 (0.90--1.30)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
-0.10 (-0.39--0.39)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{0.014}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{ΔTC (mmol/L)}\selectlanguage{english}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0.09 (-0.12--0.55)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
-0.20 (-0.81--0.49) (n=22)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0.469\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{ΔHDL (mmol/L)}\selectlanguage{english}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
-0.02\selectlanguage{ngerman}±0.17\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
-0.03\selectlanguage{ngerman}±0.17\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.848\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{ΔLDL (mmol/L)}\selectlanguage{english}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
-0.04 (-0.37--0.00)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.25 (-0.67--0.48)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.598\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{greek}Δ: \selectlanguage{english}baseline results minus results at one month after metabolic therapy;
HDL: high-density lipoprotein; LDL: low-density lipoprotein; TC: total
cholesterol; TG: triglyceride. Normal intestinal permeability was
defined as D-lactate\textless{}15 U/L, and elevated intestinal
permeability was defined as D-lactate[?]15 U/L. Significant P-value is
indicated in bold font.

\textbf{Figure legends}

\textbf{Figure 1:} Selection process of patients with nonalcoholic fatty
liver disease\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

