Fatty acid composition but not quantity is an important indicator of non-alcoholic fatty liver disease: a systematic review

There is still paucity on the effects of dietary and supplemental fatty acid on non-alcoholic fatty liver disease (NAFLD). The aim of this review is to systematically review and summarise the effect of fatty acids intake on liver-related outcomes in adult patients with NAFLD. The review was conducted using Cochrane CENTRAL Library, Scopus, Embase, MEDLINE, PubMed, and Web of Science. A total of 2786 records were identified, and of these, 36 studies (31 were randomised control trials (RCTs), and 5 were case-control studies) were included. Quality assessment was conducted using the Revised Cochrane Risk of Bias tool and Joanna Briggs Institute checklists. Of 36 articles, 79% of RCTs and 66% of case-control studies had a low risk of bias. Potential heterogeneity has been observed in assessment of liver-related outcomes. According to the RCTs, there was moderate evidence (3/6 studies) that a diet characterised by a high MUFA, PUFA and low SFA showed reduced liver fat and stiffness. The using of culinary fats that are high in MUFA (4/6 studies) reduces liver steatosis. n-3 PUFA supplementation in combination with a hypocaloric or heart healthy diet with a low SFA improved liver enzyme level (5/14 studies) and steatosis score (3/14 studies). Effects on NAFLD parameters, including liver fat content (assessed via magnetic resonance imaging/spectroscopy), stiffness and steatosis score (assessed by ultrasonography), were primarily related to fatty acid composition independent of energy intake. Further investigation is needed to determine the mechanism of specific fatty acid on the accumulation of liver fat.


INTRODUCTION
Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide, and it currently affects ~25% of the adult population [1].In the Western world, including the United States, Europe and Brazil, NAFLD appears to be the most common cause of chronic liver disease in the adult population [2][3][4].Since the global estimated prevalence of NAFLD is expected to increase alarmingly (ranging from 6 to 35%) [5], action is urgently needed to reduce its global health and economic burden.While multiple interventions have been evaluated in clinical studies for the management of NAFLD, there is no consensus on the assessment of these patients or optimal pharmacological therapy [6].Hence, current treatment options are limited to lifestyle changes, including diet and exercise, which are cost-effective and efficacious for improving the prognosis of patients with NAFLD [6].
The liver is exposed to different types of fats (namely fatty acids, cholesterol, and triacylglycerol) derived from the diet, as well as visceral adipose tissues through the hepatic portal vein, which may contribute to fatty liver disease and hepatic insulin resistance [7].Dietary fatty acids are associated with hepatic lipogenesis and may play a dual role in the pathogenesis of fatty liver disease as they are implicated in their development and conversely, the prevention or reversal of liver fat accumulation [8].Further, the dietary fatty acid composition is an important factor in NAFLD development since 15% of liver triacylglycerol originates from the diet [9].Thus, it is important to know the specific type of fatty acids in the diet that causes fat accumulation in liver cells and, consequently, NAFLD development.Saturated fatty acid-rich diets increase liver fat due to increased lipolysis, mediated by inflammatory pathways in adipose tissue [10].Several studies revealed that increased intake of saturated fats, trans fat and cholesterol are associated with a deterioration of clinical features of NAFLD [10,11].
Conversely, unsaturated fat intake is associated with decreased lipolysis and preventing fat accumulation in the liver [12].Adequate consumption of polyunsaturated fatty acid (PUFA) has been associated with the improvement of NAFLD [13,14].Previous systematic reviews and meta-analyses have shown that supplementation with n-3 PUFA significantly improved liver fat content (assessed by magnetic resonance imaging, MRI/spectroscopy, MRS) [12,13,15], steatosis score (assessed by ultrasonography), and liver enzyme levels in patients with NAFLD [12][13][14][15].However, the effect of fatty acid intake on liver histological outcomes (i.e., steatosis, lobular inflammation, hepatocellular ballooning, fibrosis) are still inconclusive [14].Additionally, above mentioned systematic reviews, in general, have focused on fatty acids modification, particularly, n-3 PUFA supplementation.However, in a recent systematic review and meta-analysis that evaluated the effect of dietary interventions with different, quantitative, macronutrient compositions on hepatic steatosis attenuation, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lipid profile, glucose metabolism markers, and anthropometric parameters among adults and the elderly (60 years and over) with NAFLD [1].In relation to the effect of dietary fatty acid pattern on NAFLD, the results showed that hypocaloric diet that contains lower proportion of fat or higher monounsaturated fat rich hypocaloric diet significantly reduce the degree of hepatic steatosis (assessed by ultrasonography) [1].However, the meta-analysis of 6 included studies showed no significant changes in degree of hepatic steatosis [1].Given that cooking oil is considered an important source of fatty acids, it is essential to evaluate the effects of various cooking oil fatty acids on NAFLD [16][17][18][19][20]. Thus, considering the diversity of dietary fatty acids for NAFLD in this current review, we attempt to include all studies assessing the effects of dietary and supplemental fatty acids on liver-related outcomes (i.e., histological outcomes, liver fats and liver enzyme levels) in adults with NAFLD.

METHOD
The current systematic review was registered at the PROSPERO International Prospective Register of Systematic Reviews (Registration number: CRD42021243799).Additionally, the study was reported following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines (Table S1).

Screening and selection criteria
At the first screening stage, the titles and abstracts were assessed based on inclusion and exclusion criteria for each record.Selection criteria are described in detail in Table 1.Next, all titles and abstracts of electronically identified studies were screened by two reviewers (AD and YT) independently.Finally, these two reviewers performed the second screening step for each selected article based on the eligibility criteria.Selected journals included all studies published until November 2021.

Data extraction
After selecting eligible studies, different types of data were extracted, including author, year of publication, country, sample size, participants' age, study design, intervention, outcomes, outcome measures, statistical analysis, and results.In the case of incomplete data, the authors of the articles were contacted via email.Given that the results of the biochemical variables were presented using different units of measurement, only data on patients who completed the studies were used for analysis.Liver histological outcomes (i.e., steatosis, lobular inflammation, hepatocellular ballooning, fibrosis), liver fat content assessed by imaging studies (i.e., ultrasonography, magnetic resonance imaging, or computed tomography) and levels of the liver enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were determined.

Quality assessment
The quality of included studies was critically assessed in duplicates by the two reviewers (AD and YT) independently.The risk of bias was evaluated using the revised Cochrane risk of bias tool for randomised trials (RoB 2.0).Each journal article was assessed individually at the outcome level and scores as "low risk of bias", "high risk of bias" or classified as "some concerns".In addition, the Joanna Briggs Institute (JBI) Critical Appraisal Checklists were used to assess the risk of bias assessment for studies other than case-control studies [21].The JBI tools are comprised of 9 to11 questions with options "yes" indicating higher quality, "no" indicating poor quality or "unclear".The overall appraisal allowed authors to "Include" or "Exclude" studies.Studies with ≥3 "no" or "unclear" categories were excluded (see Fig. 1).

Study selection
A total of 2786 records were obtained from the initial database search.After removing duplicates (n = 1913), 873 studies were screened by titles and abstracts based on inclusion and exclusion criteria.From these studies, 58 studies were retrieved for full-text screening.After careful review of these studies, a total of 36 studies (31 RCTs and 5 case-control studies) were included for quality assessment and data extraction.The study selection is detailed in the PRISMA flow diagram (Fig. 1).

Outcomes
Effects of dietary fat and fatty acids on NAFLD Case control studies: The effects of dietary fatty acid intake on NAFLD were evaluated in five case-control studies [50][51][52][53][54] (Table 3).Of the five studies, one study showed a positive association between total fat intake and the risk of NAFLD [50].This study did not report the effects of saturated, monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) on NAFLD [50].The authors were contacted to obtain the data, but the necessary data was not provided [50].Another study by Cheng et al. showed a positive association between total fat and saturated fatty acid intake and hepatic fat content [51].While no associations were observed between MUFA or PUFA and NAFLD [51].Entezari et al. observed that the highest MUFA/SAFA ratio tertiles (>0.57) have lower odds for NAFLD in crude (OR: 0.47, 95% CI: 0.24-0.95) and multivariable adjustment (OR: 0.42, 95% CI: 0.20-0.87)[52].Furthermore, a recent study by Xie et al. showed a positive association between total PUFA intake (18.8-29.3g/day) and the risk of NAFLD among Chinese adults [54].Further analysis (total PUFA intake to energy intake ratio) also showed that the risk of NAFLD increased as total PUFA intake to energy intake ratio increased (OR: 1.14, 95% CI: 1.08-1.20)[54].In contrast, Han et al. showed that lower intake of n-3 PUFA was significantly associated with an increased risk of NAFLD in men [53].
Effects of dietary fat on histologic parameters and liver fat content RCTs.The effect of hypocaloric diets with modified dietary fat assessed in 4 RCTs [34,39,40,44] (Table 4), however, no effects were observed in liver histology.The effect of the Mediterranean dietary pattern that is low SFA and high PUFA and MUFA on NAFLD was evaluated in six RCTs [29,35,37,[41][42][43].Of 6 studies, 2 studies showed a significant reduction in liver stiffness [35,41], 2 studies reported a significant reduction in liver fat [29,41], 1 study reported a significant reduction in liver enzyme level [37] Table 1.PICOS criteria for inclusion and exclusion of studies.

Population
• Individuals aged ≥18 years with NAFLD confirmed and 1 study reported a significant reduction in NAFLD score assessed by ultrasonography [42].Interestingly, the effects intervention varies from 12 weeks to 104 weeks [29,35,41].In addition, liver stiffness was measured using ultrasonography [35] and fibro scan [41].Similarly liver fat was measured by magnetic resonance imaging [29,41].Furthermore, of the 6 studies that evaluated the effects of culinary fats, 4 studies reported that the consumption of culinary fats which are high in MUFA, n-3 and n-6 PUFA had positive effects in reducing liver steatosis as measured by ultrasound [16,[18][19][20].These studies observed a significant reduction in liver steatosis after using 20 ml or less MUFA and PUFA rich oil for 12-24 weeks [16,[18][19][20].
Effects of dietary fat on liver enzymes.The effect of low-fat diets on liver enzyme was inconclusive (Table 4).A study by Jang et al. reported a significant reduction of serum ALT in control group compared to low fat diet group after 8-week of intervention [34].Only 1 study that used the Mediterranean diet, reported a significant reduction in AST level [37].Another study by Razavi et al. observed a significant reduction in ALT level with a Dietary Approaches to Stop Hypertension (DASH) diet [36].Maciejewska et al. observed that a hypocaloric diet with modified fat for 24weeks resulted a significant reduction of serum ALT and AST [40].
Similarly, 2 studies found significant reductions in ALT and AST using cooking oil rich in MUFA and n-6 PUFA [16,45].These studies used nigella sativa oil and olive oil for 12 weeks [16,45].
Effects of n-3PUFA supplementation on histologic parameters.The effect of n-3 PUFA supplementation on NAFLD on liver fibrosis score, hepatocellular ballooning score, steatosis score, lobular inflammation score, or the NAFLD activity score using histologic assessment was examined in 6 studies [22-25, 30, 46] (Table 4).There were inconclusive effects on histological parameters with a very low certainty of evidence.Only Li et al. showed a significant improvement in steatosis score, necro-inflammatory score, fibrosis score and hepatocellular ballooning score with 50 ml/day n-3 PUFA supplementation for 24 weeks [24].While in the study by Dasarathy et al. reported a significant improvement in hepatic steatosis and lobular inflammation aggravated in placebo group, but no improvement observed in intervention group [23].Other studies found no improvements in any histological parameters [22,25,30,46].Effects of n-3 PUFA supplementation on liver fat content.The effects of n-3 PUFA supplementation on liver fat content, using MRI or MRS, were evaluated in 4 studies [22,26,30,47] (Table 4).Only Argo et al. reported a significant reduction of hepatic fat content with 3000 mg/day n-3 PUFA supplementation at 52 weeks [22].However, this study did not report post-intervention liver fat content values in the article [22].The remaining 3 studies did not reveal any significant changes in liver fat levels after the PUFA n-3 intervention [26,30,47].There was no effect of n-3 PUFA supplementation on liver fat content.The certainty of evidence of this outcome at different timepoint was very low.Effects of n-3 PUFA supplementation on steatosis score assessed by ultrasonography.The effect of n-3 PUFA supplementation on steatosis score was evaluated in 5 studies [28,32,33,38,47] (Table 4).Most studies reported an improvement of steatosis score with n-3 PUFA supplementation compared to control group.Two of these studies assessed the effect of the supplement at 24 weeks [38,47] and one study investigated the effect of the intervention at 52 weeks [32].Whereas two studies found no effect of n-3 PUFA supplementation on steatosis at 12 weeks [28,33].The consumption n-3 PUFA supplementation had a beneficial effect on steatosis evaluated by ultrasonography.The certainty of evidence was high.

DISCUSSION
The current systematic review presents a relatively comprehensive summary of the existing evidence on the effects of fatty acids on liver-related outcomes through histologic, radiologic, ultrasonographic and biochemical assessments in adults with NAFLD.The screening stages resulted in 36 studies, which comprised 31 RCTs [22-28, 30-32, 38, 46, 47] and 5 case-control studies [50][51][52][53][54].The results of this study are heterogenous and showed some similarities and differences with previous systematic reviews and meta-analyses, indicating the need for further study.

Effects of total dietary fat and fatty acids on NAFLD
There was inconclusive evidence for the effects of dietary fat on NAFLD.In this review, we evaluated the effects of almost all fats, diets with modified amount of fats and culinary fats.All the included studies were not directly comparable due to the variation of dietary intake measures, outcome measures, duration of intervention, quantity of fatty acids and ethnicity.In line with our review, a previous systematic review and meta-analysis also showed that there was no effect of fat modified diets on NAFLD [1].Findings from included studies indicated that the consumption of Mediterranean diet rich in MUFA, PUFA and low in SFA may have a beneficial effect on hepatic fat and stiffness.However, the duration of intervention was heterogenous.Likewise, a recent systematic review of RCTs indicated that the Mediterranean diet may be beneficial for reducing hepatic fat content, while the duration of the intervention ranged between 6 to 24 weeks [55].While, a systematic review by Saeed et al. described a significant reduction of liver steatosis with a Mediterranean diet that is rich in PUFA, however, the duration of the intervention varied [56].Mediterranean diet is mainly supplemented with extra virgin olive oil, avocados, nuts, and oily fish including salmon and sardines, which are high in n-3 PUFA.Olive oil is the primary source of recommended fat, replacing other oils and fats.The major fatty acid in olive oil is MUFA (i.e., oleic acid, 65-79%).However, the Mediterranean diet contains bioactive compounds, such as polyphenols and phytosterols, which makes it difficult to isolate the effects of MUFA and n-3 PUFA in Mediterranean diet on NAFLD.
Further, using of cooking oil namely olive, canola or flax seed oil has been shown to reduce liver steatosis [16,[18][19][20].Flaxseed oil is rich in n-3 PUFA, while olive oil is rich in MUFA and canola oil is rich in n-6 PUFA [57,58].Since olive oil can reduce the accumulation of liver fat independently of metabolic pathways, likely through increased oxidation of fatty acids [59].To date, there is no study has compared the effects of n-3 and n-6 PUFA on NAFLD.However, it would be difficult to isolate the nutritional effects of n-3 and n-6 PUFA on NAFLD.In regards to the effect of olive oil on inflammatory status, evidence from animal model, suggested that olive oil, significantly improves glucose homoeostasis and inflammatory status through regulation of TNF-α IL-1, IL-10, and adipokines [59].On the other hand, olive oil also reduces the oxidative and nitrosative stress induced by a high fat diet in the liver and a high oxidation of liver fatty acids [59].Olive oil reduces NF-κB activation and improves insulin resistance [59].Another animal study by Zhang et al. showed that flaxseed oil significantly reduced liver inflammation by reducing plasma and liver tissue levels of TNF-α, IL-1β and IL-6 in alcoholic fatty liver disease [60].There is limited evidence that there appears to be a gap in the oxidation of unsaturated and saturated fats.However, irrespective of the discrepancy, some unsaturated fatty acids may stimulate general fat oxidation by activating transcription factors as Peroxisome proliferator-activated receptors (PPARα) [61].
Animal studies and a limited number of early human clinical trials have suggested that PPARα has a protective effect on NASH through certain important mechanisms, such as inducing expression of genes involved in beta oxidation, decreasing inflammation, decreasing oxidative stress, and increasing the secretion of beneficial adipokines like adiponectin [62].

Effects of n-3 PUFA supplementation on liver histological parameters
In line with the previous systematic review and meta-analysis [14], there is no effect of n-3 PUFA supplementation on fibrosis score, hepatocellular ballooning score, lobular inflammation score, and NAS.Of the 5 studies that reported liver histology outcomes, 2 studies showed a significant improvement in liver histology outcomes with n-3 PUFA supplementation in combination with a hypocaloric or heart healthy diet for 24 weeks [23,24].However, it was suggested that supplementation with n-3 PUFA may be beneficial at the early stage of NAFLD.The incorporation of a combination of dietary interventions can address the multiple impacts occurring during the onset and evolution of NAFLD [63].
Effects of n-3 LC-PUFA supplementation on liver fat content, steatosis score and enzymes Liver fat.The inconclusive effects of n-3 PUFA supplementation on liver fat content, assessed by MRI/MRS in patients with NAFLD was consistent with the results of previous systematic review and meta-analysis [14].Only two out of five studies showed a significant decrease in liver fat in patients with NAFLD [14].In the current systematic review, only one study found a significant reduction in liver fat content, assessed by MRI, with n-3 PUFA supplementation along with a hypocaloric diet for 52 weeks [22].
In contrast, previous systematic reviews and meta-analyses reported a beneficial effect of n-3 PUFA supplementation on liver fat [13,15].These studies also found an effect size favouring n-3 PUFA treatment.Although these meta-analyses used a random effect model due to significant heterogeneity among included studies [13,15].
Steatosis score.The effects of n-3 PUFA supplementation on steatosis score assessed by ultrasonography and CT scan were similar to previous systematic reviews [13][14][15].In the present review, of the 5 studies, 3 studies indicated a significant reduction in steatosis scores with the n-3 PUFA supplementation [32,38,47].Two studies have used American Health Foundation diet and n-3 PUFA supplementation.However, the effects of dosage and duration of the intervention on the changes in the steatosis score were inconclusive because a higher dose (6 g/day), longer duration (12 months) and a lower dose (0.83 g/day), short duration (6 months) of n-3 PUFA supplementation produced similar results.
Liver enzymes.Contrary to previous systematic reviews and meta-analyses [13][14][15]64], the effects of the n-3 PUFA intervention on ALT and AST levels were inconclusive in our current systematic review.Using n-3 PUFA supplementation 4 studies showed a significant reduction in ALT and AST levels [26,27,38,46], 3 studies showed a significant decrease only in serum ALT levels [27,38,46], and only one study demonstrated a significant reduction in serum AST levels in intervention group [26].There were 2 studies that reported significant reduction in serum ALT and AST levels in the control group but not in the intervention group [30,31].For control group, both studies used olive oil [30,31].Additionally, there was also a significant reduction in ALT and AST with a macronutrient modified diet and n-3 PUFA supplementation [38,47].These studies used the American Heart Foundation diet, which consisted of 50% carbohydrate, 20% protein and 30% fat [38,47].Similarly, some studies that used dietary approaches, showed a significant reduction in serum ALT and AST levels [40,41].
Although the majority of RCTs examined the effect of n-3 PUFA supplementation in combination of hypocaloric or hearth healthy diet on liver-related outcomes.Only 35% of studies indicated that n-3 PUFA supplementation may attenuate liver enzyme levels and a small number of studies (20%) found a reduced steatosis score.As the duration of the intervention ranges from 6 weeks to 78 weeks, along with the wide range of n-3 PUFA doses.As a result, it is difficult to determine the optimal dose of n-3 PUFA supplementation for improvement of liver-related outcomes.
The present systematic review has several strengths.First, we selected primary outcomes of liver-related outcomes i.e., liver histological outcomes, liver fat content, steatosis score and liver enzyme levels.Because it gave a more comprehensive indication of the effect of fatty acids on liver-related outcomes.Second, a broad definition of the fatty acids was evaluated, which ranged from specific fatty acids, such as total fat, SFA, MUFA and PUFAs, fat modified diets and cooking oil.The inclusion of both dietary and supplemental fatty acid intake allowed for a relatively comprehensive review of the association with NAFLD.Third, the search strategy was extensive and comprehensive, using six electronic databases.Fourth, 31 RCTs and 5 case-control studies with large sample sizes and sample diversity across 16 countries were included, thus enabling a full evaluation of fatty acid intake on NAFLD in adult populations worldwide.Fifth, the quality assessment tools, ROB 2.0 for RCTs and the JBI checklist for casecontrol studies, allowed for risk of bias assessment at the study level using.Majority of RCTs (79%) and case-control studies (66%) had a low risk of bias.Finally, most studies in this systematic review investigated both male and female participants.
Of course, it also comes with its limitations, mainly the heterogeneity of the duration of interventions, a broad range of liver-related outcome as well as outcome measures, and that pooled results from meta-analysis were not conducted.Additionally, the participant inclusion criteria included all adults with NAFLD, except those with any acute or chronic disease and that inherently affects dietary intake.Further, we excluded the lower quality case-control studies due to the non-report of information regarding confounding factors and strategies to deal with confounding factors.Also, given the inconsistencies of types of dietary and supplemental fat, dosages and the duration of intervention, the systematic review could not provide any recommendation related to optimal dosage and duration of fatty acid supplementation for the treatment of NAFLD.Finally, the heterogeneity of the studies (i.e., age, geographic location, study quality, severity of NAFLD, and adjustment for main confounders) prevented the synthesis of a meta-analysis.

CONCLUSION
This systematic review provides an update on the effect of dietary and supplemental fatty acid intake on NAFLD.Results from the current review revealed that dietary fatty acid composition plays an important role in liver-related outcomes in NAFLD patients.Overall n-3 PUFA supplementation in combination with a hypocaloric or heart healthy diet may improve liver enzymes levels, however, further research is required to examine the effect of n-3 PUFA supplementation with a diet rich in healthy fat and low SFA.The use of cooking oils such as flax or olive oil or canola oil or a combination of three oils can be beneficial for the

Fig. 1
Fig. 1 PRISMA diagram.Flow diagram of study selection process for the systematic review.

Table 3 .
Summary of the case-control studies included in the systematic review (n = 5).

Table 4 .
Summary of the clinical trials included in the systematic review (n reduction of liver fat content.Existing evidence of the mechanism of dietary fatty acid composition or ratio on NAFLD is limited.Additionally, effect of individual fatty acid on NAFLD is limited because it is difficult to isolate certain fatty acids from foods.More studies with large samples, an adequate methodological design is recommended to understand the mechanism of specific fatty acids to modify hepatic outcomes in patients with NAFLD.