INTRODUCTION
In South Korea, the proportion of older adults aged ≥ 65 is 18.4% and is projected to reach 20.6% by 2025, becoming a super-aged society [1]. The rapid rise in obesity prevalence among older adults not only increases the risk of metabolic disease but also causes various social problems. According to the National Health Survey, the obesity prevalence increased from 31.1% in 2012 to 33.5% in 2021, with older women having a higher obesity prevalence rate of 36.6% compared with 29.2% in older men [2]. Older women experience lower physical activity levels [1] and significant hormonal changes before and after menopause, leading to a decreased lean body mass and increased body fat mass [3], increasing the risk of obesity. Excessive fat accumulation due to obesity increases the plasma free fatty acid (FFA) levels [4]. Elevated serum FFA levels promote lipid accumulation in the liver, resulting in lipotoxic damage to hepatocytes [5,6]. Previous studies have reported that increased serum FFA levels are closely associated with liver metabolism [7]. Furthermore, obesity is an independent factor related to non-alcoholic fatty liver disease (NAFLD) [8] and is associated with liver dysfunction and disease [9].
Four plasma enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and γ-glutamyl transferase (GGT), are primarily used as biomarkers to evaluate liver function [10]. Elevated levels of these enzymes in the plasma indicate liver damage [11]. Marchesini et al. (2008) reported higher serum levels of ALT, AST, ALP, and GGT in individuals with obesity [12].
There is increasing interest in natural substances for the prevention and improvement of obesity-related liver diseases. Therefore, the development of functional materials using natural products with minimal side effects on the human body is needed. Oysters, which are widely farmed in coastal countries such as Korea, are nutritionally balanced and are among the top ten foods globally. Oysters are rich in essential minerals such as glycogen, protein, iodine, iron, and taurine (4.2%/dry base) [13]. Previous studies using oyster extracts in animal models have reported the recovery of damaged liver cell [14]. The antioxidant activity of taurine found in oysters is known to play an important role in liver protection [15,16,17] and in regulating lipid metabolism [18,19]. Moreover, fermented oysters have been reported to have a greater effect on improving liver damage compared with non-fermented oysters [18].
Studies that have investigated the effects of fermented oyster intake on the liver function of older individuals with obesity are few. Therefore, this study aimed to investigate the effects of fermented oyster extract on FFA and liver enzymes in older women with obesity, and to provide basic data for improving liver function in older individuals with obesity.
METHODS
Participants
The study participants included older women with obesity, with a body mass index (BMI) ≥ 25 kg/m2 [19], residing in B Metropolitan City. After explaining the purpose and details of the study to the participants, only those who voluntarily expressed their intention to participate were included after completing a consent form. Forty participants were randomly assigned to the fermented oyster intake group (n = 20) or the control group (n = 20). The physical characteristics of the participants are presented in Table 1.
Study design
A randomized, double-blind, placebo-controlled clinical trial method aimed to confirm the effects of fermented oyster extract on FFA levels and liver function in older women with obesity. The participants were allocated to fermented oyster extract (FSO) and control (CON) groups at a 1:1 allocation ratio using the block randomization method. Randomization was performed using the SPSS statistical software for Windows (version 22.0; IBM Corp., Armonk, NY, USA). The block size and number were determined by an independent third-party statistician, and this information was not disclosed to any of the investigators. The randomization schedule was sealed in opaque envelopes to ensure that all participants, researchers, and assessors remained blinded to the treatment allocation until the conclusion of the study. All participants were requested to attend four visits during the study period (visit 1: screening; visit 2: random supplement distribution; visit 3: six weeks post-intervention; and visit 4: 12 weeks post-intervention for final observation).
Fermented oyster preparation and consumption
The FSO and placebo were purchased from Marine Bioprocess Co., Ltd. (Busan, Korea). Oysters (Crassostrea gigas) sourced from Tongyeong were thawed, desalted, and preprocessed using wet grinding (Han Sung Pulverizing Machinery Co. Ltd., Gyeonggi, South Korea). They were then hydrolyzed with alcalase 2.4 L FG (Novozyme, Brenntag Korea) at 60±5℃ for 4 h, followed by filtration (120 mesh) and centrifugation (Disc Separator, Alpalaval, Swiss) to separate soluble components. The resultant solution was concentrated using a rotary evaporator (BÜCHI, Essen, Germany) to a brix of 14. After concentration and fermentation, the final product was spray dried to produce FSO containing GABA (11.2%), lactic acid (5.0%), moisture (5.5%), protein (31.8%), and carbohydrates (56.1%).
Participants in the FSO group received 1,000 mg of FSO daily, administered orally in the form of four 250 mg capsules, taken 30 min after meals for 12 weeks. The placebo capsules, identical in appearance to the FSO capsules, were filled with dextrin. Previous animal studies demonstrated no toxicity at FSO dosages of 100 and 200 mg/kg over 28 days20. Therefore, a safe and effective 200 mg/kg dose, converted to 960 mg/day based on the human surface area for a 60 kg adult, was established. A preliminary clinical study set a safe FSO extract dosage of 1,000 mg/day [21], which was selected for convenience.
Measurement items and methods
In this study, the anthropometric measurements of all the participants were consistently conducted under the same standardized conditions and procedures. These measurements were taken on an empty stomach, before and after substance intake, and were specifically scheduled between 8 A.M. and 10 A.M. to ensure uniformity.
Anthropometry
Body weight (kg) and height (cm) were measured using digital height and weight meters (BSM370; InBody, Seoul, Korea). Body fat (kg), body fat percentage, and muscle mass (kg) were assessed using a bioelectrical impedance analysis device (InBody 270; InBody, Seoul, Korea). BMI was calculated from height and weight measurements (kg/m²).
Blood sampling
Biochemical analyses of serum FFA, AST, ALT, ALP, and GGT levels were performed. A registered nurse collected 10 mL of blood from the antecubital vein of each participant. Before analysis, the samples were centrifuged at 3,000 rpm for 10 min using a Combi-514R centrifuge (Hanil, Korea). FFA concentrations were measured using an enzymatic method with a specific reagent (Sicdia NEFAZYME; Eiken Co., Japan). AST, ALT, GGT, and ALP levels were measured using an automated chemical analyzer (Olympus AU 5400, Japan).
Statistical analysis
The ideal sample size was calculated using G*Power software (version 3.1; University of Kiel, Germany), with parameters set to an effect size of 0.25 (default), a significance level (α) of 0.05, and a power (1-β) of 0.86. All statistical analyses were performed using SPSS for Windows (version 27.0; IBM Corp., Armonk, NY). The means and standard deviations were calculated and recorded. Normality tests were initially performed to assess the effects of 12 weeks of FSO consumption on FFA levels and liver enzyme function. This was followed by a 2 × 2 factorial repeated-measures analysis of variance, with treatment and time as independent variables. The Scheffe’s method was applied for post-hoc tests, and the threshold for statistical significance was set at p<0.05.
RESULTS
Changes in FFA concentration following FSO supplementation
We performed statistical analyses to assess the effects of the 12-week FSO supplementation on FFA levels. The detailed findings of these analyses are presented in Table 2. The analyses revealed a statistically significant interaction between time and group for changes in FFA levels (p<0.05). Specifically, in the FSO supplementation group, FFA levels were significantly reduced from a baseline mean of 518.85 ± 125.69 μEq/L to 423.50 ± 150.97 μEq/L at the end of the intervention (p<0.05). Conversely, the CON group did not exhibit statistically significant changes in FFA levels, with initial measurements at 519.70 ± 94.56 μEq/L slightly rising to 524.25 ± 139.43 μEq/L, indicating no effective alteration due to the lack of treatment. The statistically significant reduction in FFA levels within the FSO group suggested that 12-week supplementation with FSO has a beneficial effect in lowering FFA levels in older women with obesity. The lack of a significant change in the CON group supported the conclusion that these effects can be attributed to FSO supplementation.
Changes in liver enzyme concentrations following FSO supplementation
We conducted statistical analyses to evaluate the effects of 12-week FSO supplementation on liver enzyme levels. The detailed results of these analyses are presented in Table 3. The analyses demonstrated a statistically significant time × group interaction for AST, ALT, and GGT levels (p<0.05).
AST
Post-hoc analysis revealed that AST levels in the FSO group significantly decreased from 26.80 ± 3.82 IU/L to 25.15 ± 3.50 IU/L (p<0.01). In contrast, the CON group showed no significant change, with AST levels moving from 26.25 ± 3.34 IU/L to 26.50 ± 4.19 IU/L.
ALT
ALT levels in the FSO group significantly reduced from 24.50 ± 7.74 IU/L to 20.90 ± 5.12 IU/L (p<0.05), whereas the CON group showed no significant variation, with levels changing from 21.45 ± 7.35 IU/L to 21.70 ± 7.98 IU/L.
GGT
For GGT, levels in the FSO group significantly decreased from 17.20 ± 6.98 U/L to 15.55 ± 6.68 U/L (p<0.05), while the CON group showed an insignificant change from 17.25 ± 6.89 U/L to 17.80 ± 7.95 U/L.
ALP
ALP levels in the FSO group changed from 58.85 ± 12.01 IU/L to 56.85 ± 10.58 IU/L, and in the CON group from 51.90 ± 11.59 IU/L to 51.90 ± 11.43 IU/L, with neither group exhibiting significant differences.
The statistically significant reductions in AST, ALT, and GGT levels in the FSO group suggested that 12-week FSO supplementation improved liver enzyme levels in older women with obesity. The lack of a significant change in the control group supported the conclusion that these effects can be attributed to FSO supplementation.
DISCUSSION
This study aimed to assess the effects of FSO on FFA and liver enzyme levels in older women with obesity. Both the FSO and control groups maintained FFA and liver enzyme levels within the normal ranges. The results demonstrated significant reductions in serum FFA levels, as well as significant decreases in AST, ALT, and GGT levels in the FSO group.
It is known fact that the increase in body fat due to obesity is associated with elevated serum FFA levels [22]. Elevated serum FFA levels contribute to the increased accumulation of triglycerides in liver cells, combining glycerol and FFA, which can lead to the development of NAFLD [23]. Depending on severity, fatty liver disease can progress to steatohepatitis, cirrhosis, and hepatocellular carcinoma [24]. However, oyster extract consumption has been reported to reduce fatty acid synthesis in the liver [25] and animal studies have shown a reduction in FFA levels following oyster extract consumption [26]. It has been suggested that the anti-inflammatory, antioxidant, and anti-apoptotic effects of taurine, which accounts for approximately 80% of the total amino acids in oysters, may help prevent liver lipid metabolism disorders [27,28]. In this study, the significant reduction in FFA in the FSO group suggested that fermented oyster intake may help reduce the accumulation of FFA in the liver due to obesity, potentially preventing NAFLD. Overweight and obesity are significant risk factors for liver function impairment [29]. The prevalence of NAFLD among individuals with obesity has increased from 57% to 74% [30], and a correlation has been reported between the severity of obesity and liver disease [31].
ALT, AST, GGT, and ALP are serum enzymes commonly used as biomarkers for liver damage [10]. ALT and AST are enzymes predominantly found in the cytoplasm of liver cells, and their serum levels increase upon liver cell damage [10]. GGT is mainly located on the cell surface [33], and elevated serum levels are closely related to fatty liver disease [34]. ALP is present in the liver, bones, and other tissues [34], and elevated serum ALP levels are associated with liver disease [35]. Increased plasma levels of these biomarkers increase the risk of metabolic diseases, such as NAFLD [36]. Elevated levels of these biomarkers are associated with obesity [37,38]. Previous studies have reported higher serum levels of ALT, AST, GGT, and ALP in individuals with obesity [37,39].
Oyster extracts have been reported to enhance antioxidant defense systems, aiding in the recovery from liver damage [40]. In animal experiments, significant reductions in ALT and AST levels were observed following a 9-week administration of oyster extract [41]. In a study involving patients with alcohol-induced liver disease, a 12-week oyster extract consumption resulted in improved GGT levels [42]. It has been suggested that a reduction in FFA, which is associated with markers of liver damage [41], along with the anti-inflammatory and anti-apoptotic pathways of taurine, and are abundantly found in oysters, contributes to the alleviation of liver tissue damage [43,44]. In this study, significant reductions in ALT, AST, and GGT levels were observed in the FSO group, indicating that fermented oyster intake may improve liver function and prevent NAFLD by improving liver damage biomarkers in older women with obesity.
The 12-week supplementation with fermented oyster extract significantly reduced FFA, ALT, AST, and GGT levels. These findings suggested that fermented oysters improve liver function, highlighting their potential as a natural food source for the prevention and treatment of liver damage. However, this study has several limitations. First, the study population was limited to elderly women with obesity. Second, the relatively short 12-week intake period and the small sample size may have limited the generalizability of our findings.
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