June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Research Paper

Sex-speciﬁc eﬀect of ethanol on colonic short-chain fatty acids andderivatives in the mouse model using targeted LC/MS
1,2#

Siddabasave Gowda B. Gowda*, Lipsa Rani Nath1#, Yonghan Li3,

Jayashankar Jayaprakash , Divyavani Gowda , Shu-Ping Hui *
1

1

2

2

Graduate School of Global Food Resources, Hokkaido University, Kita-9, Nishi-9, Kita-Ku, Sapporo 060‒0809, Japan
2
Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo 060‒0812, Japan
3
Graduate School of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo 060‒0812, Japan

Abstract

Ethanol consumption aﬀects the human body, particularly the digestive system, leading to gut dysbiosis anddysregulation of metabolites. Although previous studies have focused on the eﬀects of ethanol on liver metabolism, colon-speciﬁc metabolites have not been well explored. In this study, we investigated the eﬀects of ethanol on colon-speciﬁcshort-chain fatty acids (SCFAs) and their derivatives in a mouse model using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Colonic ﬂush samples obtained from ethanol-fed (EF) and pair-fed (PF) mice of each sex were
subjected to targeted analysis of SCFAs and SCFA esters of hydroxy fatty acids (SFAHFA) using LC-MS/MS. The levelsof acetic acid (AA), propanoic acid (PA), and butyric acid (BA) were signiﬁcantly increased in the male mouse EF groupcompared to those in the PF group. In contrast, the levels of AA and PA were decreased in female mice in the EF groupcompared to those in the PF group. Furthermore, the levels of AA-derived SFAHFAs were decreased in the male EF groupbut increased in the female EF group compared to those in the control group. Compared to the control groups, hydroxy
fatty acid levels were signiﬁcantly decreased in the EF groups of both sexes. Overall, ethanol exerted diverse eﬀects oncolon-speciﬁc metabolites in both male and female mice. This study provides new insights into sex-dependent metabolicchanges associated with alcohol-induced gut microbiota dysfunction and their potential health impacts.
Key words: ethanol, short-chain fatty acids, SFAHFA, liquid chromatography, mass spectrometry, gut-microbial metabolites

1.

Introduction

health risks. Alcohol was ranked as the seventh leading

Alcohol is a globally prevalent psychoactive substancecause of death globally and contributed to 2.8 million

deeply rooted in various cultures that poses signiﬁcantdeaths in 2016 . The harmful eﬀects of alcohol on thedigestive system are closely related to the gut‒liver axis, in1)#

which the enterohepatic circulation is essential for trans-

Equally contributing authors*Corresponding author

12)

porting products from the gut to the liver . Alcohol disrupts
the gut‒liver axis, altering the microbial composition andSiddabasave Gowda B. Gowda

Faculty of Health Sciences, Hokkaido University, Kita-12,
Nishi-5, Kita-ku, Sapporo 060‒0812, Japan3)

gut barrier integrity , which leads to impaired nutrient
absorption and contributes to conditions such as alcoholicE-mail: gowda@gfr.hokudai.ac.jp
2
Shu-Ping Huihepatitis and cirrhosis. Dysbiosis disrupts tight junction
proteins, allowing the translocation of particles into portal

Faculty of Health Sciences, Hokkaido University, Kita-12,
Nishi-5, Kita-ku, Sapporo 060‒0812, Japan4)

circulation . Tight junction maintenance is facilitated byshort-chain fatty acids (SCFAs), which are byproducts ofTel: ＋81‒11‒706‒3693
E-mail: keino@hs.hokudai.ac.jp

anaerobic fermentation by the food ﬁbers . Fecal metabo5)lome analysis of patients with alcohol use disorders showeda decrease in SCFAs, which could be explained by dysbio-

Received: February 4, 2024. Accepted: February 28, 2024.
Epub April 16, 2024.

sis-impacting bacteria that produce SCFAs, including Fae-

DOI: 10.24508/mms.2024.06.002
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June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Chemical (Ann Arbor, MI, USA). Solid-phase extraction
cartridges (MonoSpin C18-AX) were provided by GL Sci-

6)

calibacterium .
Recent ﬁndings revealed a novel class of derivative lipids
of SCFAs, namely SCFA esters of hydroxy fatty acids
7)(SFAHFAs), which are abundant in the gut . Gut microbi-

ence (Tokyo, Japan). 2-Chloro-1-methylpyridinium iodide

ota is signiﬁcant in the formation of these lipids, which arevaleric acid (VA), and caproic acid (CA), were sourced(CMPI), along with propionic acid (PA), butyric acid (BA),8)

present in the colon and cecum . Understanding the eﬀectof alcohol on colonic-speciﬁc SCFAs and their derivatives

from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
The SFAHFA standards were synthesized in-house as per

was signiﬁcantly advanced when chemically generated

the protocols described in our previous study .

9)

SFAHFAs were measured in rat intestinal contents and
9)

2.2.

fecal samples .

Animal samples

Male and female C57BL6 mice (8‒10 weeks old) were

Although existing research has explored lipid metabolism in alcohol-induced gut dysbiosis, notable gaps exist in
our understanding of how ethanol speciﬁcally aﬀects

bred at the institutional core facility after obtaining
approval from The Institutional Animal Care and Use Com-

colonic SCFAs and SFAHFAs. A previous study used gas
chromatography‒mass spectrometry (MS) for analyzing

mittee at the University of Tennessee Health Science Center. Bothe male and female mice were fed a Lieber‒DiCarli

human stool samples and demonstrated that excessive alco-liquid diet (D710260, Dyets Inc., Bethlehem, PA, USA) for

hol consumption disrupts the intestinal microbiota andfour weeks with ethanol (0% for two days, 1% for twocauses dysbiosis, thus highlighting importance of fatty
acids (FAs) in intestinal health and linking microbiota dys-days, 2% for two days, 4% for one week, 5% for one week,
and 6% for one week) or pair-fed (PF) isocaloric maltodex-

10)function to alcohol-related pathologies . Furthermore,using liquid chromatography coupled with triple quadru-

trin (BioServ, Flemington, NJ, USA). At the end of the
four-week feeding period, both groups of mice were eutha-

pole and quadrupole time-of-ﬂight MS, previous studies

nized, and the colonic contents were ﬂushed with 3 mL of
0.9% saline. Flushed samples were frozen until lipid analy-

revealed that ethanol alters lipid metabolism by inﬂuencing
11,12)

the gut microbiota.

sis. All experiments were performed in compliance with the

However, the existing literature on lipid metabolism
in ethanol-induced gut dysbiosis is limited, and thesex-speciﬁc eﬀects of ethanol on gut lipid metabolism remain

relevant laws and/or institutional guidelines.

2.3.

unclear. Therefore, using liquid chromatography-tandem MS(LC-MS/MS), we investigated the sex-speciﬁc eﬀects ofExtraction and targeted analysis of SCFA by
LC-MS/MSThe extraction and analysis of SCFA were performed

ethanol on gut lipid metabolism in a mouse model. Unveil-using the method developed in our previous studies . Ini-

ing the intricate connections between alcohol consumption
and SCFA/SFAHFA variations, our ﬁndings contribute to atially, 80 μL of acetonitrile was added to 20 μL of colonic
ﬂush, followed by the addition of 100 μL of 20 μM AA-d4

nuanced understanding of the mechanisms, oﬀering thepotential for sex-speciﬁc therapeutic strategies against alco-

and 100 μL of 20 μM VA-d9 as internal standards. The mixture was then vortexed for 3 min at 3500 rpm and then cen-hol-related pathologies.

trifuged for 10 min at 15000 rpm and 4°
C. The supernatant
was carefully transferred into a new microtube, and derivat-

2.

Materials and Methods

2.1.

Materials

13)

ization was initiated by adding 20 μL of TEA and 10 μL of
CMPI of 2 mM each. The mixture was vortexed for 3 min at

LC/MS-grade solvents acetonitrile, methanol, and isopro-

3500 rpm, following which 20 μL of 2 mM DMED was

panol, 1 M aqueous ammonium acetate, and acetic acidadded to the microtube; the sample was further vortexed for(AA) were purchased from Wako Pure Chemical Industries,

30 min at 3500 rpm and centrifuged (10 min, 15000 rpm, 4°Ltd. (Osaka, Japan). The reagents triethylamine (TEA),
N,N-Dimethylethylenediamine (DMED), and acetic acid-d4

C). The centrifugate was transferred to an LC vial, and 5 μLof this solution was injected into the LC-MS system.

(AA-d4) and HPLC-grade chloroform were acquired from
Sigma-Aldrich (St. Louis, MO, USA). Pentanoic acid-d9

Targeted analysis was performed using a Prominence
ultrafast liquid chromatography (UFLC; Shimadzu, Kyoto,(PA-d9) and palmitic acid-d31 were obtained from Cayman

Japan) connected to a TSQ Quantum Mass Spectrometer
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Medical Mass Spectrometry Vol. 8 No. 1

9)

System (Thermo Fisher Scientiﬁc Inc., San Jose, CA, USA)
operated in the positive-ion mode. The chromatographic

in our previous research . The elution solvents consisted of

separation was performed on a Hypersil GOLD C8 column(50 mm × 2.1 mm), which is a reversed-phase-type columnB), and methanol (pump C). An Atlantis T3 C18 column

with particle and pore sizes of 1.9 μm and 175 Å, respectively. The column oven was maintained at 40°
C through-for the chromatographic separation, which was maintained

10 mM ammonium acetate (pump A), isopropanol (pump(50 mm × 2.1 mm, 3 μm, Waters, Milford, USA) was used
at 40°
C with a ﬂow rate of 300 μL/min. The elution gradient
was set as follows: 40% B and 20% C (0‒7 min), 80% B

out the analysis. The mobile phase comprised a dual-solvent system: solvent A was Milli-Q water containing

and 10% C (7‒14 min), 30% B and 35% C (14‒18 min),

20 mM ammonium acetate, and solvent B was a mixture ofand returned to the initial conditions. For each injection, themethanol and acetonitrile (50:50, v/v). The ﬂow rate was

sample volume was set to 10 μL.

set to 0.3 mL/min. The gradient program was initiated with

2.5.

solvent B at 100% for the ﬁrst 1.5 min and held constant for

Statistical analysis

2 min. At 2.1 min, the proportion of solvent B was adjusted

Data were visualized using Microsoft Excel 2021 andto 50% and maintained for 7 min. Subsequently, solvent B

GraphPad Prism version 8.0.1. Statistical analysis involved
Studentʼs t-test with p＜0.05, which was considered signiﬁ-

was returned to 100% after 7.5 min and maintained for
10 min. The electrospray ionization (ESI) source parame-cant.

ters were as follows: the spray voltage was 3,500 V; the3.

sheath gas was 40 arbitrary units; the auxiliary gas was 25

Results
Fig. 1 shows the outcomes of SCFA analysis of the colon

arbitrary units; the capillary temperature was 250°
C; andthe HESI vaporizer temperature was 150°
C. The SCFA con-contents of EF and PF mice. The concentration of eachcentrations were calculated from the linearity curves constructed using Xcalibur 2.2 software (Thermo Fisher Scien-acquired SCFA level is shown in the bar graphs. Male PF

tiﬁc., San Jose, CA, USA).

mice. In particular, AA and PA levels signiﬁcantly

and EF mice exhibited lower SCFA levels than female
decreased in the EF group of females, whereas the trend

2.4.

Extraction and targeted analysis of SFAHFAs by
LC-MS/MS

was reversed in males. Conversely, BA and VA levels
increased in male and female mice in the EF group, respec-

The extraction and analysis of SFAHFAs were performed

tively. However, no signiﬁcant diﬀerences were observed in9)using a method developed in our previous studies .
Approximately 50 μL of colonic ﬂush from ethanol-fed (EF)CA levels between sexes.

mice samples were transferred to Eppendorf tubes. To this,samples are shown in Fig. 2 (A‒C). The overall SFAHFA

100 μL of an internal standard mixture in methanol wasconcentrations were higher in males than in females. In
particular, the levels of the AA ester of 2-hydroxy palmitic

The concentrations of SFAHFAs detected in colonic ﬂush

added, which comprised 5 μM deuterated SFAHFA standard
mixture (PAHPA-d30, BAHPA-d30, VAHPA-d30, andacid (2-AAHPA), AA ester of 2-hydroxy arachidic
acid (2-AAHAA), AA ester of 2-hydroxy behenic

HAHPA-d30). In addition, 50 μL of methanol and 400 μL of
Milli-Q water were added to the mixture. The resulting mix-

acid (2-AAHBA), AA ester of 2-hydroxy tricosylic
acid (2-AAHTA), AA ester of 2-hydroxy lignoceric acidture was vortexed (3500 rpm, 1 min) and centrifuged(15,000 rpm, 1 min, 4°C) to collect the supernatant for sol-

(2-AAHLA), and AA ester of 2-hydroxy pentacosylic acid
(2-AAHPSA) exhibited signiﬁcant increases in the EF

id-phase extraction using the MonoSpin C18-AX cartridges.
The detailed workﬂow for the solid-phase extraction forgroup of females compared to those in the PF group, whereas

9)

SFAHFAs is described in our previous study .

a reversed trend was observed in males. Furthermore, the AA
ester of 2-hydroxy heneicosylic acid (2-AAHHA), BA ester

Lipidomic analysis was conducted using a TSQ Quantum(Thermo Fisher Scientiﬁc, Waltham, MA, USA) coupledof 2-hydroxy behenic acid (2-BAHBA), PA ester of
2-hydroxy cerotic acid (2-PAHCA), BA ester of 2-hydroxywith the Prominence UFLC system (Shimadzu, Kyoto,
Japan) operating under ESI in negative mode. The LC-MS

cerotic acid (2-BAHCA), CA ester of 2-hydroxy cerotic
acid (2-CAHCA), BA ester of 2-hydroxy lignoceric acidconditions closely adhered to the methodology established(2-BAHLA), and PA ester of 2-hydroxy pentacosylic acid

Access MAX Triple Quadrupole Mass Spectrometer

13

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Medical Mass Spectrometry Vol. 8 No. 1

Fig. 1.

Amount of SCFAs in colon contents of PF and EF groups of male and female mice.

(M_PF: Male pair-fed, M_EF: Male ethanol-fed, F_PF: female pair-fed, F_EF: female ethanol-fed, n＝4 for each group). Studentʼs t-test: p＜
0.001 (a), p＜0.01 (b), p＜0.05 (c), and ns: non-signiﬁcant.

Fig. 2.

Amount of SFAHFAs detected in colon contents of the PF and EF groups of male and female mice.

The concentrations of SFAHFAs at the molecular level are shown in A, B, and C, and the total SFAHFAs of each SCFA-derived lipids areshown in D. (M_PF: Male pair-fed, M_EF: Male ethanol-fed, F_PF: female pair-fed, F_EF: female ethanol-fed, n＝4 for each group). Studentʼs t-test: p＜0.001 (a), p＜0.01 (b), p＜0.05 (c), and ns: non-signiﬁcant.

(2-PAHPSA) did not show any signiﬁcance between the EF

whereas no such diﬀerences were observed in male EF
mice. However, the levels of the VA ester of 2-hydroxy

and PF groups of sexes.
The levels of the AA ester of 2-hydroxy cerotic acid
(2 AAHCA) and PA ester 2-hydroxy lignoceric acid(2-PAHLA) were signiﬁcantly increased in female EF micecerotic acid (2-VAHCA) and VA ester of 2-hydroxy pentacosylic acid (2-VAHPSA) were signiﬁcantly decreased infemale EF mice, but no signiﬁcant diﬀerence was observed
14June 2024

Medical Mass Spectrometry Vol. 8 No. 1

in male mice. Fig. 2(D) shows the total amount of SFAH-

16)

lism . Therefore, metabolites (such as SCFAs) produced
by beneﬁcial bacteria in the gut aid absorption and utiliza-

FAs based on their SCFA acyl chains in the PF and EF miceof both sexes. In female mice, the levels of AA-derived

tion. These metabolites also support mucosal immunity,

SFAHFAs were signiﬁcantly increased in the EF group,

preserve intestinal homeostasis, and shield the intestinal

whereas the opposite trend was observed for the male mice.

mucosal barrier.

However, BA showed no signiﬁcant diﬀerences between
sexes. The levels of PA-derived SFAHFAs were signiﬁ-

As shown in Fig. 1, a signiﬁcant decrease in the SCFA
concentration was observed for the female EF mice com-

cantly increased in the female EF group, whereas those of
VA-derived SFAHFAs decreased. The concentrations of

pared to female PF mice. The relevance of these ﬁndings ishydroxy FAs (HFAs) in PF and EF mice of both sexes are

humans with alcohol use disorders. This independent study

shown in Fig. 3. Notably, higher concentrations of HFAs
were observed for females than males except for 2-hydroxy

revealed a reduction in SCFA levels, which is consistentpalmitic acid (2-HPA) in both the PF and EF groups. A sig-

attributable, in part, to dysbiosis, which negatively aﬀects
6,10)
SCFA-producing bacteria, namely Faecalibacterium .

further supported by a study on the fecal metabolome inwith our results. The observed decrease in SCFAs is likely

niﬁcant decrease in HFA concentration was observed in

The observed increase in SCFA levels in male EF mice may
be attributed to inherent sex-speciﬁc hormonal and physio-

female mice with EF. However, no signiﬁcant variations
were observed for HFA concentrations in male EF mice,
except for 2-HPA and 2-hydroxy behenic acid (2-HBA),

logical diﬀerences. This is in agreement with a recent study
on Chinese bamboo rats, which showed signiﬁcant diﬀer-

which were signiﬁcantly decreased in the EF group. The
concentrations of all the SCFA, SFAHFA, and HFA ana-

ences in microbial composition and genetic/metabolic path-

provided in Table 1. The data shows a large deviation in theways between female and male rats despite shared captive
17)
and feeding conditions . These sex-speciﬁc variations may

concentration levels among each animal, suggesting that

contribute to unique responses to alcohol exposure, inﬂu-

additional experiments with a large animal number would

encing SCFA levels. The congruence between our mousebe essential to draw any further conclusions.

model results and the insights from the bamboo rat study
highlights a robust association between ethanol consump-lyzed in the colonic ﬂush samples of PF and EF mice were

4.

Discussions

tion, gut dysbiosis, and alterations in SCFA levels in a
sex-speciﬁc manner. Furthermore, parallel ﬁndings across

Alcohol-induced dysbiosis has a detrimental eﬀect on gut

integrity, leading to the development of acute (alcoholic
hepatitis) and chronic (alcohol-related cirrhosis) liver disor-

diﬀerent species enhance our understanding of the intricate

ders. Many metabolites such as SCFAs are produced by theand downstream physiological eﬀects.

relationships between alcohol consumption, gut microbiota,

anaerobic gut microbiota as byproducts of the fermentation

The modulation of SFAHFAs in the PF and EF groups of

14,15)and have been demonstrated to havemultiple favorable eﬀects on mammalian energy metaboof dietary ﬁbers

Fig. 3.

both sexes is detailed in Fig. 2. Previous investigations into
SFAHFAs have indicated a decline in their levels in theAmount of hydroxy fatty acids detected in the PF and EF groups of male and female mice.

(M_PF: Male pair-fed, M_EF: Male ethanol-fed, F_PF: female pair-fed, F_EF: female ethanol-fed). Studentʼs t-test: p＜0.001 (a) and p＜0.01 (b).

15

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Medical Mass Spectrometry Vol. 8 No. 1

Table 1. The concentrations of short-chain fatty acids, SFAHFAs, and hydroxy fatty acids analyzed in the colonic ﬂush samples
Male mice
PF1

PF2

PF3

PF4

EF1

Female mice
EF2

EF3

EF4

PF1

PF2

PF3

PF4

EF1

EF2

EF3

EF4

SCFA
Acetic acid
Propanoic acid
Butyric acid
Valeric acid
Caproic acid170.27
12.49
4.52
12.41
0.32

179.21
13.89
4.27
15.56
0.83

141.17
9.47
4.21
9.97
0.59

72.10
7.98
1.15
3.67
0.78

347.47
22.05
28.96
12.65
1.24

359.45
23.56
35.08
14.94
0.28

Concentration (μmol/L)
303.28 361.51 1938.33 1781.13 2181.98 2822.14 1205.66 1526.45 1266.54 1112.41
21.44
18.05 113.18 118.68 123.34 180.34
72.94
69.97
63.74
65.13
26.96
32.18 381.42 375.36 428.11 440.38 388.65 410.53 384.06 309.70
10.60
13.81
16.82
18.53
21.19
17.47
22.38
23.92
26.68
17.78
2.33
0.33
0.51
1.48
0.95
1.73
1.87
1.75
2.05
0.68

SFAHFA
2-AAHPA
2-AAHAA
2-AAHHA
2-AAHBA
2-BAHBA
2-AAHTA
2-AAHCA
2-PAHCA
2-BAHCA
2-VAHCA
2-CAHCA
2-AAHLA
2-PAHLA
2-BAHLA
2-AAHPSA
2-PAHPSA
2-VAHPSA
AA_SFAHFA
PA_SFAHFA
BA_SFAHFAVA_SFAHFA

13.14
9.86
4.69
82.97
14.76
53.74
24.07
4.93
59.86
11.10
200.01
86.57
9.73
8.80
4.69
7.44
10.54
279.72
22.10
83.43
21.64

16.03
13.74
3.27
125.93
19.52
40.22
24.41
15.74
40.31
15.50
190.97
58.90
4.99
1.34
10.92
0.54
13.67
293.42
21.26
61.17
29.17

18.49
33.51
14.75
206.27
8.33
56.92
27.96
8.32
94.74
20.02
207.92
75.62
2.63
5.80
6.39
9.40
11.44
439.9120.35
108.87
31.46

24.00
56.24
14.28
210.27
8.45
71.75
26.50
7.66
108.01
20.39
165.63
95.98
6.17
11.45
15.34
4.28
10.61
514.3518.11
127.91
31.00

0.78
4.31
5.58
18.07
0.00
8.25
23.91
4.33
77.16
20.21
200.45
20.05
13.49
0.00
2.21
2.17
13.28
83.16
20.00
77.16
33.49

4.00
0.59
2.59
5.62
0.00
3.45
21.59
22.30
56.25
20.60
208.75
13.44
13.10
0.88
5.40
2.28
12.37
56.67
37.68
57.13
32.97

Concentration (pmol/L)
3.62
1.99
0.87
0.00
4.91
2.68
1.90
1.20
0.62
4.80
2.48
3.41
12.66
14.81
4.11
8.23
6.20
17.58
23.43
7.32
11.69
20.53
5.33
6.45
25.06
27.32
17.04
17.26
8.36
6.18
1.74
1.76
83.23
67.73
67.42
78.72
23.68
13.51
17.03
16.63
191.88 158.62 135.42 112.72
22.17
13.00
11.16
13.41
3.13
5.24
0.50
5.43
17.23
0.00
1.37
0.00
4.91
2.64
3.23
2.64
7.06
1.90
12.05
6.17
17.55
11.49
7.23
11.35
85.63
87.77
46.12
52.59
18.55
13.33
14.29
13.35
106.66
85.31
92.22
86.05
41.23
25.00
24.25
27.98

0.48
0.07
0.74
0.43
0.96
0.08
0.11

0.40
0.04
0.58
0.30
0.66
0.06
0.09

0.48
0.04
0.50
0.23
0.54
0.05
0.08

0.98
0.08
1.27
0.69
1.60
0.12
0.11

0.23
0.07
0.60
0.28
0.68
0.08
0.10

0.15
0.04
0.30
0.13
0.28
0.05
0.07

HFA
2-HPA
2-HAA
2-HHA
2-HBA
2-HTA
2-HLA
2-HPSA

Concentration (nmol/L)
0.20
0.28
0.29
0.05
0.06
0.20
0.36
0.42
1.90
0.19
0.20
1.04
0.44
0.49
2.33
0.05
0.07
0.21
0.09
0.10
0.33

0.49
0.41
6.79
2.13
4.87
0.45
0.66

0.00
0.87
0.00
6.56
0.00
1.70
22.09
4.32
47.28
18.31
109.77
11.19
2.12
3.27
2.22
4.45
10.07
44.63
10.89
50.55
28.38

0.56
2.33
4.09
8.57
2.62
3.43
23.29
1.80
104.3520.12
128.03
7.28
0.94
0.69
4.01
2.55
11.41
53.55
5.29
107.66
31.53

3.94
3.69
0.93
31.37
2.39
19.71
26.82
3.10
76.47
18.54
109.25
36.32
31.76
0.00
7.38
3.13
5.81
130.16
37.99
78.86
24.35

5.77
4.58
2.16
18.15
13.73
7.34
38.15
0.00
78.12
14.51
150.49
30.99
7.71
0.00
6.74
4.61
7.49
113.89
12.32
91.85
22.00

7.77
10.88
1.08
70.79
0.00
29.85
27.33
4.94
50.83
13.28
68.24
115.21
66.58
4.29
15.12
5.24
7.00
278.04
76.76
55.13
20.28

4.59
14.77
1.81
74.44
1.29
28.01
21.54
2.89
60.93
11.31
63.15
119.97
28.26
1.95
14.14
11.52
7.06
279.26
42.67
64.17
18.37

0.52
0.44
5.78
2.19
5.11
0.47
0.71

0.38
0.31
2.95
1.50
3.41
0.32
0.53

0.21
0.06
0.66
0.31
0.80
0.09
0.14

0.18
0.04
0.59
0.21
0.58
0.07
0.10

0.41
0.10
1.39
0.47
1.20
0.11
0.14

0.39
0.12
1.38
0.51
1.29
0.11
0.12

colons of rats subjected to a high-fat diet, in contrast to an

observed. As the generation of HFAs is linked with theincrease observed in mice infected with the inﬂuenza
7,18)
virus . The observed downregulation of speciﬁc SFAH-

abundance of free FAs (FFAs), these HFA results are inagreement with previous research that highlighted the piv-FAs derived from PA and BA in antibiotic-treated mouseotal role of FFA metabolism in the progression of alcoholic
22)
liver disease . The implications of FFAs in alcohol-in-

fecal samples underscores their speciﬁcity for the gut
12)
microbiota . Previous studies have consistently demon-

duced gut dysbiosis, characterized by microbiota imbalances and potential contribution to gastrointestinal disor-

strated that supplementation with SCFAs, achieved throughinterventions such as a high-ﬁber diet or probiotics,

23)

ders, have been accentuated in numerous studies . An
animal study has suggested that maintaining optimal intesti-enhanced gut epithelial integrity and mitigated liver injury
19,20,21). Considering the apparent associin alcoholic models

nal FFA levels, particularly saturated fatty acids, canenhance and preserve the integrity of the intestinal gut bar-

ation between SFAHFAs and SCFAs, these two biomole-rier . The observed decline in HFA concentration after eth24)

cules are reasonably correlated. This consistency strongly
supports the continued exploration of interventions involv-

anol consumption in our study adds to this understandingby emphasizing the intricate interplay between alcohol con-ing SCFAs and SFAHFAs as promising strategies foraddressing pathologies associated with alcohol consump-

sumption, FA metabolism, and gut health. These ﬁndings
reinforce the importance of targeted interventions to modu-

tion.

late HFAs, particularly in maintaining intestinal barrier
integrity, as a potential strategy for addressing alcohol-re-

A notable decrease in the HFA concentration in female
EF mice is shown in Fig. 3. In male EF mice, except for
2-HPA and 2-HBA, no signiﬁcant alterations werelated pathologies. The ﬁndings of the study are summarized16June 2024

Fig. 4.

Medical Mass Spectrometry Vol. 8 No. 1

Summary of changes in the precursor (FA 22:0(2OH), AA) and product (2-AAHBA) SFAHFA metabolism.

English editing expenses.

in Fig. 4 using a representative FA and its metabolites. The
concentration of precursor FA 22:0(2OH) was decreased in

Conﬂict of interest

the EF group of both sexes, and that of AA was increased in
the male EF mice but decreased in female EF mice. How-

The authors declare no competing ﬁnancial interest.

ever, an opposite trend was observed for 2-AAHBA in the
EF groups of both sexes. This study has some limitations,

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