Short-term synbiotic supplementation and intrahepatic triglyceride content assessed by proton MR spectroscopy: a pilot study

Abstract

Background

Non-alcoholic fatty liver disease (NAFLD), recently redefined as metabolic dysfunction-associated steatotic liver disease (MASLD), is strongly linked to obesity, insulin resistance, and metabolic syndrome. Alterations in gut microbiota have been implicated in its pathogenesis, and synbiotic therapies combining probiotics and prebiotics have gained interest as potential treatment strategies.

Objective

To investigate the effects of 3-month synbiotic supplementation on intrahepatic triglyceride (IHTG) content as the primary outcome, along with biochemical parameters, in patients with NAFLD, assessed by proton magnetic resonance spectroscopy (¹H-MRS).

Methods

Nineteen patients diagnosed with NAFLD based on ultrasonography or biopsy were recruited; after exclusions, 18 patients completed the study. All participants received a daily synbiotic capsule containing Lactobacillus acidophilus (LA-5), Bifidobacterium animalis subsp. lactis (BB-12), and prebiotics (inulin, oligofructose, potato starch) for 12 weeks. IHTG was quantified by ¹H-MRS before and after treatment. Routine biochemical and hematological parameters were also evaluated.

Results

After 12 weeks, IHTG content decreased significantly (13.02 ± 6.74% vs. 9.34 ± 4.44%, p = 0.021). Serum ALT (p = 0.023), AST (p = 0.013), and LDL cholesterol (p = 0.032) levels decreased significantly, while platelet count increased (p = 0.009). Fibrosis-4 Index (FIB-4) values also showed a significant reduction following treatment (p = 0.002). Body weight and BMI demonstrated a trend toward reduction without reaching statistical significance. Other biochemical parameters remained unchanged. Logistic regression analysis identified baseline triglyceride levels as the strongest predictor of steatosis progression despite therapy.

Conclusion

Short-term synbiotic supplementation significantly reduced hepatic steatosis and improved selected biochemical parameters in MASLD patients. These findings support the potential role of microbiota-targeted therapies as adjunctive treatment in NAFLD/MASLD. Larger randomized controlled trials are warranted.

Graphical Abstract

Introduction

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease worldwide, affecting approximately one-quarter of the global population and strongly associated with obesity, insulin resistance, and metabolic syndrome [1–4]. In 2023, an international expert consensus recommended the adoption of a new nomenclature, metabolic dysfunction-associated fatty liver disease (MASLD), to better reflect the underlying pathophysiology [5].

The pathogenesis of MASLD is multifactorial, involving insulin resistance, lipid metabolism dysregulation, oxidative stress, and inflammatory signaling [3, 6]. Increasing evidence suggests that alterations in the gut microbiota—through mechanisms such as increased intestinal permeability, endotoxin-mediated inflammation, and altered bile acid metabolism—play a critical role in disease initiation and progression [7, 8]. The gut–liver axis has therefore become a major target of therapeutic strategies [9].

Probiotics and prebiotics, and more recently synbiotics (combinations of both), are of particular interest. Probiotics may modulate gut microbiota composition, enhance intestinal barrier function, reduce systemic endotoxemia, and improve insulin sensitivity [10, 11]. Prebiotics such as inulin and oligofructose provide substrates for beneficial bacterial growth and short-chain fatty acid production, further promoting metabolic and anti-inflammatory effects [12, 13]. Synbiotic formulations have shown additive or synergistic benefits by combining these mechanisms [12, 14].

Several meta-analyses have suggested that microbiota-targeted therapies may reduce liver fat content, improve liver enzymes, and modulate metabolic risk factors in patients with NAFLD/MASLD [12, 15–20]. However, findings remain inconsistent due to heterogeneity in study design, patient characteristics, and probiotic strains used. Advanced imaging modalities such as proton magnetic resonance spectroscopy (¹H-MRS) provide reliable non-invasive quantification of intrahepatic triglyceride (IHTG) content, offering an objective means to assess treatment response [21, 22].

In this pilot study, we aimed to evaluate the effects of a 12-week synbiotic supplementation on intrahepatic triglyceride content in patients with MASLD, quantified by ¹H-MRS. We also investigated changes in routine biochemical and hematological parameters to provide insights into potential clinical benefits and predictors of treatment response. Although the study was conducted during a period when NAFLD terminology was in use, the patient population also fulfilled the updated diagnostic criteria for MASLD. Therefore, to ensure consistency and alignment with current nomenclature, the term MASLD is used throughout the manuscript.

Methods

Study design and ethical approval

This was a prospective, single-center pilot study conducted between December 2013 and June 2014 at the Gastroenterology outpatient clinic of Cumhuriyet University Hospital. The study followed a pre-test/post-test design, in which all patients received the same intervention and served as their own controls. The study protocol was approved by the Clinical Research Ethics Committee of Cumhuriyet University (decision no. 2013-12/07, 10.12.2013) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.

Participants

Patients aged 18–70 years with evidence of hepatic steatosis (Grade I–III) on liver biopsy and/or abdominal ultrasonography were screened for eligibility. Exclusion criteria included: alcohol consumption > 30 g/day for men and > 20 g/day for women, positive hepatitis B surface antigen or hepatitis C serology, autoimmune hepatitis, cirrhosis, or toxic hepatitis, alanine aminotransferase (ALT) > 10× the upper limit of normal, decompensated liver disease or malignancy, systemic corticosteroid or methotrexate use in the previous 6 months.

A total of 46 patients were initially assessed. After exclusions due to absence of steatosis on magnetic resonance spectroscopy (MRS), non-compliance, concomitant diagnoses, or technical reasons, 18 patients completed the study and were included in the analysis. The primary endpoint was the within-person change in intrahepatic triglyceride (IHTG) content measured by ¹H-MRS. As this study was designed as an exploratory pilot study, an a priori sample size calculation was not performed. Instead, a post hoc power analysis was conducted using G*Power software (version 3.1.9.7) based on the observed change in IHTG. Using a two-tailed paired t-test with an alpha level of 0.05 and an effect size (Cohen’s dz) of 0.60, the achieved statistical power with 18 participants was calculated as approximately 80.6%. These findings indicate that the present sample size was sufficient to detect a moderate effect in this exploratory setting.

Intervention

All participants received one capsule of a commercially available synbiotic supplement (Bakso capsule^®, Sandoz, Novartis) daily for 12 weeks. Each capsule contained Lactobacillus acidophilus (LA-5, 1 × 10⁹ CFU), Bifidobacterium animalis subsp. lactis (BB-12, 1 × 10⁹ CFU), inulin, oligofructose, and potato starch. In addition, patients were advised by dietitians to follow a low-calorie, low-fat diet consistent with clinical practice guidelines.

Magnetic Resonance Spectroscopy (¹H-MRS)

Hepatic triglyceride content was assessed using proton magnetic resonance spectroscopy (¹H-MRS) at baseline and after 12 weeks of treatment. Examinations were performed on a 1.5 Tesla MR scanner (Magnetom Aera, Siemens, Erlangen, Germany) using an 18-channel phased-array body coil. The PRESS (point-resolved spectroscopy) technique was employed. A single voxel of 8 cm³ (2 × 2 × 2 cm) was positioned in the right hepatic lobe, avoiding major vascular and biliary structures.

Spectral data were obtained using TE: 30 ms, TR: 1500 ms, and flip angle: 90°. No water or fat suppression was applied. Liver fat fraction (%) was calculated using the following formula:

A fat fraction ≥ 5.56% was considered diagnostic for hepatic steatosis, in line with established thresholds [21].

Biochemical and hematological parameters

Fasting blood samples were obtained at baseline and after 12 weeks. The following parameters were measured: fasting plasma glucose (FPG), total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), complete blood count (CBC) including hemoglobin, leukocyte, and platelet counts. Body weight and body mass index (BMI) were recorded at both visits.

Statistical analysis

The data are presented as mean±standard deviation. The difference between groups was compared by Student’s paired t test. Univariant and subsequent multivariant (backward selection) binary logistic regression analysis was used to test a possible independent association of measured parameters with steatosis grade so as with weight change after probiotic therapy.

The principal component analysis (PCA) with varimax-normalized rotation was performed to extract the factors which are further tested in binary logistic regression analysis. PCA analysis was considered adequate if Kaiser-Meyer-Olkin measure of sampling adequacy is above 0.500 and Bartlett’s test of sphericity produced P < 0.050. In particular, predefined number of 3 factors was used for factors’ selection, while only variables with factor loadings > 0.5 were considered for the interpretation of selected factors in PCA analysis. PASW^® Statistic v.18 (Chicago, IL, USA) software was used for data analysis. P values < 0.05 for all analyses was considered statistically significant.

Results

Comparison of all measured parameters before and after the synbiotic treatment (Table 1) showed decrease in weight and BMI, but with marginal significance (P = 0.063 and 0.054, respectively). IHTG percent significantly dropped after the therapy which is the main goal of this current study. Additionally, LDL-cholesterol concentration, ALT and AST activities decreased significantly as a result of therapy implementation, while number of platelets increase significantly after the synbiotic usage. In patients with MASLD, Fibrosis-4 Index (FIB-4) values significantly decreased following synbiotic treatment [0.565 (0.380–0.740) pre-treatment vs. 0.480 (0.400–0.550) post-treatment, p = 0.002]. Other measured parameters did not change as a result of synbiotic therapy.

Table 1.

Comparison of pre-treatment and post-treatment measurements

Variables	Pre-treatment	Post-treatment	P
Weight (kg)	91.61 ± 11.44	89.94 ± 11.58	0.063
BMI (kg/m2)	30.77 ± 3.92	30.19 ± 3.70	0.054
IHTG*(%)	13.02 ± 6.74	9.34 ± 4.44	0.021
PBG (mg/dL)	97.50 ± 19.60	98.11 ± 25.17	0.876
Triglycerides (mg/dL)	248.16 ± 158.63	222.22 ± 132.39	0.647
Total Cholesterol (mg/dL)	213.77 ± 41.70	206.88 ± 39.56	0.234
LDL-C* (mg/dL)	152.55 ± 34.88	138.22 ± 32.40	0.032
HDL-C (mg/dL)	38.0 ± 6.57	37.61 ± 7.32	0.771
ALT* (U/L)	62.44 ± 33.95	53.00 ± 25.07	0.023
AST* (U/L)	33.83 ± 14.11	28.27 ± 8.59	0.013
GGT (U/L)	64.72 ± 55.12	54.16 ± 35.20	0.527
ALP (U/L)	78.44 ± 31.30	75.22 ± 18.85	0.535
LDH (U/L)	215.44 ± 36.89	213.22 ± 42.89	0.744
WBC (103/UL)	7.45 ± 1.90	7.76 ± 1.65	0.547
Hemoglobin (g/dl)	16.14 ± 1.02	16.23 ± 1.03	0.393
Platelets (×10³/µL)*3	250.27 ± 40.42	273.83 ± 51.81	0.009
FIB-4	0.565 (0.380–0.740)	0.480 (0.400–0.550)	0.002

IHTG Intrahepatic triglycerides content, PBG Pre-prandial blood glucose, LDL Low-density lipoprotein, HDL High-density lipoprotein, ALT Alanin transaminase, AST Aspartate transaminase, GGT Gamma glutamyl transferase, ALP Alkaline phosphatase, LDH Lactate dehydrogenase, WBC White blood cell, BMI Body mass index, FIB-4 Fibrosis-4 Index

*Statistically significant (P < 0.05), paired t test

According to MRI measured liver steatosis before and after synbiotics’ use we indicated decrease in steatosis grade in 13, while increase was detected in 5 patients. This enabled formation of two distinct groups, i.e. “decreased steatosis” and “increased steatosis” subgroups. Binary logistic regression analysis of all measured biochemical parameters showed that 7 parameters had P values below 0.100 in univariant analysis, which is the basic criterion for the subsequent multivariant analysis. This analysis (backward selection) showed baseline triglycerides’ content is the most powerful predictor of steatosis increase even after implemented synbiotic therapy (Table 2).

Table 2.

Binary logistic regression analysis of steatosis increases predictors

Univariant analysis	B (SE)	Wald coefficient	OR (95% CI)	P
Triglycerides, baseline	0.024 (0.012)	4.151	0.976 (0.953–0.999)	0.042
Total cholesterol, baseline	0.025 (0.015)	2.702	1.025 (0.995–1.055)	0.100
ALT, baseline	0.058 (0.033)	3.112	1.060 (0.994–1.130)	0.078
WBC, baseline	-0.600 (0.348)	2.975	0.549 (0.277–1.085)	0.085
Platelet, baseline	-0.044 (0.023)	3.723	0.957 (0.916–1.001)	0.054
Platelet, time 2	-0.026 (0.015)	3.049	0.974 (0.946–1.003)	0.081
Weight difference	0.353 (0.207)	2.905	1.424 (0.948–2.137)	0.088
Multivariant analysis
Triglycerides, baseline	0.024 (0.012)	4.151	1.025 (1.001–1.049)	0.042

SE Standard error, OR Odds ratio (95th CI- confidence interval), P from the binary logistic regression analysis

In order to reduce the number of measured parameters and integrated them into a smaller number of factors we have implemented two separate factorial (PCA) analysis, one for the baseline measured parameters and second for the parameters measured at the time 2. Kaiser-Meyer-Olkin measure of sampling adequacy (0.500 for both PCA analysis) and Bartlett’s test of sphericity (P < 0.001 for both PCA analysis) proofed analysis competence.

PCA selected baseline parameters explained 72% of total variability in following order: 27%, 25% and 20% explained by the first, second and third factors, respectively. The first factor (F1) consisted of FIB-4, BMI and Hgb. The second factor (F2) included lipid status parameters, while the third factor F3 enclosed enzyme biomarkers (ALT, AST and LDH). The results of PCA are presented in Table 3.

Table 3.

Factorial analysis in a group of MASLD patients

Factors	Baseline			Time 2
	Parameters extracted in factors	Loadings	Variability (%) Total: 72	Parameters extracted in factors	Loadings	Variability (%) Total: 91
F1	FIB-4	0.842	27	ALP	0.905	31
	BMI	0.796		TG	0.896
	Hgb	-0.741		GGT	0.675
F2	TG	0.883	25	LDL-C	0.988	30
	HDL-C	-0.848
	TC	0.656		TC	0.949
	LDL-C	0.645
F3	ALT	0.902	20	AST	0.969	30
	AST	0.844		ALT	0.943
	LDH	0.661

Factor loadings > 0.50 are presented. Variability (%) indicates the proportion of total variance explained by each factor

Abbreviations: FIB-4 Fibrosis-4 Index, BMI Body mass index, Hgb Hemoglobin, ALP Alkaline phosphatase, TG Triglycerides, GGT Gamma-glutamyl transferase, LDL-C Low-density lipoprotein cholesterol, HDL-C High-density lipoprotein cholesterol, TC Total cholesterol, ALT Alanine aminotransferase, AST Aspartate aminotransferase, LDH Lactate dehydrogenase

PCA selected time 2 parameters explained almost 20% of variability more (91%), distributed at three extracted factors as follows: 31%, 30%, 30%, respectively. The first factor (F1) included ALP, TG and GGT, second factor (F2) included lipid status parameters LDL-c and TC, and third factor (F3) involved liver connected enzymes AST and ALT (Table 3).

Factorial analysis yielded three extracted factors at baseline and three at time 2 (six factors in total). These factor scores were subsequently entered into binary logistic regression models to evaluate their ability to predict steatosis increase. However, none of the extracted factors demonstrated a significant predictive value.

Discussion

In this pilot study, we demonstrated that 12 weeks of synbiotic supplementation significantly reduced IHTG content, as measured by ¹H-MRS, in patients with MASLD. In addition to the reduction in liver fat, FIB-4, serum ALT, AST, and LDL-cholesterol levels improved significantly, while platelet counts increased after treatment. Body weight and BMI showed a trend toward reduction, although these changes did not reach statistical significance. Taken together, these findings suggest that microbiota-targeted therapy may beneficially influence both hepatic steatosis and selected biochemical markers of liver injury and cardiometabolic risk.

Our results are in line with previous reports that highlighted the potential of probiotic and synbiotic interventions in NAFLD. Several meta-analyses have demonstrated improvements in hepatic fat content and liver enzymes following probiotic or synbiotic supplementation [12, 15–20]. For instance, Chaiyasut et al. reported that synbiotic therapy improved metabolic health markers in Thai Obese Adults [23]. The magnitude of improvement in our cohort, particularly in IHTG reduction, is comparable with these studies, underscoring the therapeutic relevance of gut microbiota modulation.

The mechanisms through which probiotics and synbiotics exert beneficial effects in MASLD are likely multifactorial. Modulation of the gut microbiota composition may reduce intestinal permeability and systemic endotoxemia, thereby attenuating hepatic inflammation [7, 24]. Production of short-chain fatty acids by prebiotic fermentation can enhance insulin sensitivity, reduce hepatic lipogenesis, and increase fatty acid oxidation [7, 9]. Furthermore, specific strains such as Lactobacillus acidophilus and Bifidobacterium animalis have been shown to improve lipid metabolism and reduce serum cholesterol levels, which may explain the LDL-lowering effect observed in this study [9].

An intriguing finding of our study was the significant increase in platelet count following therapy. While the clinical significance of this observation is unclear, it may reflect improved hepatic synthetic function or modulation of inflammatory and hematopoietic pathways by synbiotics. This warrants further investigation in larger cohorts.

It should also be acknowledged that this study was conducted under the diagnostic framework of NAFLD. In 2023, the term MASLD was introduced to better capture the underlying metabolic abnormalities [25, 26]. Our participants would meet MASLD criteria under the current nomenclature. This highlights the continued relevance of our findings within the broader context of metabolic liver disease.

A strength of our study is the use of ¹H-MRS for the quantitative assessment of intrahepatic triglyceride content. While liver biopsy remains the reference standard, it is invasive and limited by sampling variability. Conventional ultrasonography, although widely available, lacks sensitivity in detecting mild steatosis. In contrast, ¹H-MRS provides a validated, non-invasive, and highly reproducible measurement of hepatic fat fraction, with a diagnostic threshold of 5.56% widely accepted in clinical research [21, 22]. The application of this technique in our study adds methodological robustness and aligns with contemporary trends in NAFLD/MASLD clinical trials.

Despite encouraging results, several limitations should be noted. First, the sample size was small, limiting the generalizability of findings. Second, the study lacked a placebo or control group, which prevents definitive attribution of effects solely to the synbiotic intervention. Third, the treatment duration was relatively short (12 weeks), and long-term efficacy and sustainability of benefits remain unknown. Finally, dietary adherence was advised but not strictly monitored, which may represent a confounding factor.

A notable limitation of the present study is the lack of direct assessment of gut microbiota composition or intestinal contents following prebiotic supplementation. Given the well-established role of the gut–liver axis in the pathogenesis of metabolic dysfunction and hepatic steatosis, alterations in gut microbiota are likely to represent a key mechanistic pathway underlying the observed metabolic and hepatic improvements. However, due to the pilot nature of the study and resource constraints, microbiota analyses (e.g., 16 S rRNA sequencing or metagenomic profiling) were not performed. Future studies integrating detailed microbiota characterization are warranted to elucidate the mechanistic links between prebiotic supplementation and hepatic outcomes.

Finally, although a priori sample size calculation was not performed due to the exploratory nature of this pilot study, post hoc power analysis demonstrated that the study achieved a statistical power of approximately 80%, suggesting that the sample size was adequate to detect a moderate effect.

In conclusion, our study supports the potential utility of synbiotic supplementation as an adjunctive strategy in the management of NAFLD/MASLD. By reducing hepatic fat content and improving selected biochemical markers, synbiotics may complement lifestyle modification and pharmacological interventions. Larger, randomized controlled trials with longer follow-up are warranted to confirm these preliminary findings and to define optimal probiotic strains, dosages, and treatment durations.

Conclusion

In this pilot study, 12 weeks of synbiotic supplementation significantly reduced intrahepatic triglyceride content, as assessed by ¹H-MRS, and improved selected biochemical markers including ALT, AST, and LDL-cholesterol in patients with MASLD. These findings support the growing evidence that microbiota-targeted therapies may beneficially influence both hepatic and metabolic pathways in fatty liver disease.

An additional strength of this study was the use of ¹H-MRS, a validated and non-invasive imaging modality that provides accurate quantification of hepatic fat content, thus enhancing the reliability of treatment response assessment.

Given the small sample size, absence of a control group, and short follow-up period, these findings should be interpreted cautiously. Larger, randomized controlled trials are warranted to confirm the efficacy of synbiotic supplementation, to define the most effective bacterial strains and dosages, and to assess long-term outcomes.

Abbreviations

ALP

Alkaline phosphatase

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

BB–12

Bifidobacterium animalis subsp. lactis BB–12

BMI

Body mass index

CBC

Complete blood count

CFU

Colony–forming unit

CI

Confidence interval

FIB–4

Fibrosis–4 Index

FPG

Fasting plasma glucose

GGT

Gamma–glutamyl transferase

HDL–C

High–density lipoprotein cholesterol

IHTG

Intrahepatic triglyceride content

LA–5

Lactobacillus acidophilus LA–5

LDH

Lactate dehydrogenase

LDL–C

Low–density lipoprotein cholesterol

MASLD

Metabolic dysfunction–associated steatotic liver disease

MRI

Magnetic resonance imaging

MRS

Magnetic resonance spectroscopy

¹H–MRS

Proton magnetic resonance spectroscopy

NAFLD

Non–alcoholic fatty liver disease

OR

Odds ratio

PBG

Pre–prandial blood glucose

PCA

Principal component analysis

PLT

Platelet count

PRESS

Point–resolved spectroscopy

SE

Standard error

TE

Echo time

TR

Repetition time

WBC

White blood cell

Authors' contributions

O.G., H.A., B.O., and S.G. conceived and designed the study. O.G., S.G., and H.A. collected the data and performed clinical and laboratory assessments. B.O. performed and analyzed the magnetic resonance spectroscopy measurements. J.K.S. and A.K. conducted the statistical analyses. F.M. contributed to data visualization and graphical abstract design. O.G. and F.M. drafted the manuscript. All authors critically reviewed the manuscript, approved the final version, and agreed to be accountable for all aspects of the work.

Funding

This study was supported by Cumhuriyet University Scientific Research Projects Commission (CÜBAP), project number T-584. The funding body had no role in the design of the study, data collection, analysis, interpretation of data, or writing of the manuscript.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding authors on reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the Clinical Research Ethics Committee of Cumhuriyet University (decision number 2013-12/07, dated 10 December 2013) and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants prior to inclusion in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.