Serum Iron:Ferritin Ratio Predicts Healthy Body Composition and Reduced Risk of Severe Fatty Liver in Young Adult Women

Dysregulated iron metabolism is associated with altered body composition and nonalcoholic fatty liver disease (NAFLD); however, mechanisms underlying this association remain undefined. We investigated this association in 117 women. Middle-aged women (≥45 years old (y)) were heavier and had lower serum iron, higher serum hepcidin, ferritin, and severe NAFLD incidence than young adult women (<45 y). Age-adjusted linear regression analysis revealed that young adult women with the highest serum iron:ferritin ratio (Tertile 3) had a 5.08-unit increased percentage of muscle mass [β = 5.08 (1.48–8.68), p < 0.001] and a 1.21-unit decreased percentage visceral fat mass [β = −1.21 (−2.03 to −0.39), p < 0.001] compared with those with the lowest serum iron:ferritin ratio (Tertile 1; reference). The iron:ferritin dietary pattern, characterized by high consumption of beef, lamb, dairy products, fruits, and whole grains, and low consumption of refined carbohydrates (rice, noodles, and bread and pastries), and deep- and stir-fried foods, predicted a 90% [odds ratio: 0.10, 95% confidence interval: 0.02–0.47, p < 0.001] reduced risk of mild vs. moderate and severe NAFLD in young adult women. Our findings suggest that the serum iron:ferritin ratio more accurately predicts body composition and reduced risk of severe fatty liver progression in young adult women compared to middle-aged women.


Introduction
Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide [1]. However, mechanisms underlying the etiology and progression of NAFLD remain unclear. NAFLD can progress from fat accumulation in the liver (steatosis) to nonalcoholic steatohepatitis (NASH) or from NASH to fibrosis or cirrhosis. Recently, NAFLD is considered to be the hepatic manifestation of metabolic syndrome (MetS) because NAFLD is strongly associated with obesity and the clustering of metabolic risk factors, including dyslipidemia and insulin resistance (IR) [2,3]. Emerging evidence suggests that iron contributes to NAFLD progression [3]. Although

Evaluation of Body Composition
The body mass index (BMI) was calculated as mass (kg)/[height (m)] 2 . Overweight and obesity were defined based on the criteria of the World Health Organization for Asia, which defines overweight as BMI ≥24 kg/m 2 and obesity as BMI ≥27 kg/m 2 [27]. Central obesity was defined as waist circumference ≥80 cm. The body composition was measured by bioelectrical impedance analysis (BIA) by using a direct segmental multifrequency BIA meter (X-SCAN Plus-II analyzer; Jawon, Korea). Body compositions were divided by body weight and expressed as percent skeletal muscle, percent body fat, percent visceral fat, and percent subcutaneous fat.

Blood Biochemistry Examination
Fasting blood samples were collected from overnight fasting participants, and serum and plasma were stored at −80 • C until analysis. Heparinized whole blood was collected for Hb measurement. Serum iron and total iron-binding capacity (TIBC) were measured using a ferrozine-based colorimetric method. %TS was determined as (serum iron/TIBC) × 100. Serum ferritin was measured by electrochemiluminescence immunoassay and was quantitated with a Roche Modular P800 analyzer (Mannheim, Germany). Furthermore, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using a colorimetric method. Serum-free Hb (free Hb; Immunology Consultants Laboratory, Inc., Portland, OR, USA), serum hepcidin (DRG International Inc; Springfield, NJ, USA), and serum Nε-(carboxymethyl)lysine (CML; Cell Biolabs, San Diego, CA, USA) were analyzed by enzyme-linked immunosorbent assay, according to the manufacturer's instructions.

Dietary Assessment
A self-reported food frequency questionnaire (FFQ) was used to determine the dietary pattern of the participants. Dietary data were assessed using a modified Chinese version of the FFQ for the Taiwanese population [28]. The modified questionnaire comprises three components: the intake frequency of 66 food items, frequency of eating away from home, and cooking methods used. The food groups were as follows: bread and pastries, noodles, rice, whole grains, root starch, brightly-colored vegetables, seaweed, white and light green vegetables, dark green vegetables, fruits, desserts, western dishes, fried desserts, organs, processed meat, animal fats, dairy products, coffee, sugary beverages, beef and lamb, chicken and pork, duck and goose, seafood, soy products, eggs, homemade food, eating away from home, deep-fried food, grilled or barbecued food, stir-fried food, stewed food, and steamed/boiled/raw food. The FFQ was divided into eight levels: (1) 0-1 times/week; (2) 2-3 times/week; (3) 4-5 times/week; (4) 6-7 times/week; (5) 8-10 times/week; (6) 11-13 times/week; (7) 14-16 times/week; and (8) ≥17 times/weeks. No portion sizes were available.

Statistical Analyses
Statistical analyses were conducted using SPSS 19 (IBM Corp., Armonk, NY, USA) and SAS Version 9.4 (SAS Institute Inc., Cary, NC, USA). Categorical and continuous variables are presented as numbers (percentages) and mean ± standard deviation, respectively. Middle-aged women were defined as those aged ≥45 y, and young adult women were those younger than 45 y. For comparing baseline characteristics between young adult and middle-aged women, the Mann-Whitney U and chi-squared tests were used for analyzing continuous and categorical variables, respectively. Multivariate linear regression was performed to evaluate the associations between the dependent variables (percent visceral fat or percent muscle mass) and potential variables, including iron biomarkers and age. Odds ratio (OR) and 95% confidence intervals (CIs) were calculated to estimate fatty liver progression. The trend test was performed using simple linear regression after age adjustment. p < 0.05 was considered statistically significant.
The iron:ferritin-specific dietary patterns were determined using reduced rank regression (RRR). This technique was described in detail by Hoffmann et al. [29] and has been applied in studies of dietary patterns and disease prediction [30][31][32]. In RRR, the food groups and biomarkers were considered as predictor and response variables, respectively. In this analysis, we used 32 food groups as the predictor variables. Five food groups with the highest factor loadings (≥0.20) and five with the lowest factor loadings (≤0.20) were used to describe the serum iron:ferritin dietary pattern. The response variables were as follows: percent skeleton muscle mass, percent visceral fat mass, serum ALT, hepcidin, LDL-C, and CML. The response variables were selected based on a significant correlation between serum iron:ferritin ratio and response variables by partial correlation after adjustment for age (r = 0.312 to −0.422; all p < 0.05). The directed acyclic graph in Figure 1 explains the RRR conceptual framework.

Statistical Analyses
Statistical analyses were conducted using SPSS 19 (IBM Corp., Armonk, NY, USA) and SAS Version 9.4 (SAS Institute Inc., Cary, NC, USA). Categorical and continuous variables are presented as numbers (percentages) and mean ± standard deviation, respectively. Middle-aged women were defined as those aged ≥45 y, and young adult women were those younger than 45 y. For comparing baseline characteristics between young adult and middle-aged women, the Mann-Whitney U and chi-squared tests were used for analyzing continuous and categorical variables, respectively. Multivariate linear regression was performed to evaluate the associations between the dependent variables (percent visceral fat or percent muscle mass) and potential variables, including iron biomarkers and age. Odds ratio (OR) and 95% confidence intervals (CIs) were calculated to estimate fatty liver progression. The trend test was performed using simple linear regression after age adjustment. p < 0.05 was considered statistically significant.
The iron:ferritin-specific dietary patterns were determined using reduced rank regression (RRR). This technique was described in detail by Hoffmann et al. [29] and has been applied in studies of dietary patterns and disease prediction [30][31][32]. In RRR, the food groups and biomarkers were considered as predictor and response variables, respectively. In this analysis, we used 32 food groups as the predictor variables. Five food groups with the highest factor loadings 0.20) and five with the lowest factor loadings (≤0.20) were used to describe the serum iron:ferritin dietary pattern. The response variables were as follows: percent skeleton muscle mass, percent visceral fat mass, serum ALT, hepcidin, LDL-C, and CML. The response variables were selected based on a significant correlation between serum iron:ferritin ratio and response variables by partial correlation after adjustment for age (r = 0.312 to −0.422; all p < 0.05). The directed acyclic graph in Figure 1 explains the RRR conceptual framework.

Participant Characteristics
In total, 117 adult women (19 controls and 98 patients with NAFLD) were recruited for analyses. The mean age was 43.02 ± 13.32 y (controls: 38.99 ± 14.35 y and patients with NAFLD: 43.80 ± 13.04 y). A total of 33 middle-aged women were self-reported to be post-menopause (63.5%) and
A total of 33 middle-aged women were self-reported to be post-menopause (63.5%) and all of the young adult women had not entered post-menopausal stage. The overall prevalence rates of dyslipidemia, MetS, anemia, and iron overload were 37.2%, 22.3%, 13.3%, and 9.7%, respectively. We then stratified the participants by age. Table 1 shows that middle-aged women (≥45 y) were heavier and had higher prevalence of dyslipidemia, MetS, and severe fatty liver than did young adult women (<45 y; all p < 0.05; Table 1). Contrastingly, young adult women had a higher percent skeletal muscle mass and lower percent visceral fat mass and body fat mass than did middle-aged women (all p = 0.001; Table 1). Furthermore, young adult women had higher levels of serum iron, but lower levels of ferritin, hepcidin, HbA1c, and free Hb than did middle-aged women (all p < 0.001; Table 1).

Associations among Serum Iron:Ferritin Ratio, Body Composition, and Fatty Liver Severity
We investigated the association between serum iron:ferritin ratio and body composition in women. Figure 2 shows that young adult women with the highest serum iron:ferritin ratio tertile (Tertile 3; T3) had a 5.08-unit increased percent muscle mass [β = 5.08 (1.48-8.68), p < 0.001; Figure 2A] and a 1.21-unit decreased percent visceral fat mass [β = −1.21 (−2.03 to −0.39), p < 0.001; Figure 2B] compared with those with the lowest serum iron:ferritin ratio (T1) (Reference; Ref). No significant associations were observed between body compositions and the iron:ferritin ratio in middle-aged women.  Age-adjusted multivariate logistic regression analysis revealed that the serum iron:ferritin ratio in young adult women was associated with a 47% reduced [OR = 0.53, 95% CI: 0.30-0.95, p < 0.05] risk of progression from mild to moderate and severe fatty liver (Model 1, Table 2). However, no such relationship was observed in middle-aged women [OR = 1.01, 95% CI: 0.91-1.11; Model 1, Table 2].  Age-adjusted multivariate logistic regression analysis revealed that the serum iron:ferritin ratio in young adult women was associated with a 47% reduced [OR = 0.53, 95% CI: 0.30-0.95, p < 0.05] risk of progression from mild to moderate and severe fatty liver (Model 1, Table 2). However, no such relationship was observed in middle-aged women [OR = 1.01, 95% CI: 0.91-1.11; Model 1, Table 2].

Iron:Ferritin-Specific Dietary Pattern Is Associated with Reduced Risk of Fatty Liver Progression in Young Adult Women
We investigated the dietary pattern associated with the iron:ferritin ratio and determined whether this pattern predicted fatty liver severity. Table 3 shows items that such as beef and lamb, dairy products, fruits, whole grain, and eggs, were positively correlated with dietary pattern scores (factor loadings ≥0.20). Contrastingly, rice, noodles, bread and pastries, and stir-and deep-fried foods were negatively correlated with dietary pattern scores (factor loadings ≤0.20). Age-adjusted multivariate logistic regression analysis revealed that in young adult women, the iron:ferritin-specific dietary pattern was associated with a 90% reduced [OR = 0.10, 95% CI: 0.02-0.47, p < 0.01] risk of progression from mild to moderate and severe fatty liver (Figure 3). No such relationship was observed in middle-aged women.

Discussion
Our study revealed that the serum iron:ferritin ratio was associated with a healthy body composition, indicated as increased muscle mass and decreased visceral fat mass, and a reduced risk of moderate and severe fatty liver progression in young adult women, but not in middle-aged women. The study findings suggest that age or age-related factors (e.g., obesity) affect the body iron status and influence fatty liver progression in women. The serum iron:ferritin-specific dietary pattern (high intake of beef and lamb, dairy products, eggs, fruits, and whole grains and low intake of refined carbohydrates and stir-and deep-fried foods) can supply adequate iron to young adult women. This pattern was also associated with a 90% reduced risk of mild vs. moderate and severe fatty liver progression. Hence, it is likely that the serum iron:ferritin ratio reflects an adequate supply of iron to maintain a healthy body composition and, consequently, protect the liver against iron overload-related liver injury.
Approximately 5-10% of the body's iron is stored in myoglobin. Iron is an essential nutrient for muscle growth and performance. For example, muscles require iron-containing proteins for oxygen transportation and oxidation. An early study reported that serum iron exerts lipolytic effects in isolated adipocytes [33]. Elevated serum ferritin, considered as an acute-phase reactant, is associated with visceral fat mass and NAFLD, suggesting that serum iron or ferritin has direct or indirect effects on adiposity and liver function. However, correlations between the serum iron:ferritin ratio and body composition may be disrupted by obesity-related factors in middle-aged women. Our study revealed that middle-aged women were heavier and had higher levels of serum hepcidin and ferritin and lower levels of serum iron than did young adult women. Increased hepcidin is well-established to gradually cause hypoferremia and tissue iron overload. Moreover, decreased serum iron levels may affect muscle growth in middle-aged women. Hence, elevated serum hepcidin and ferritin are likely to reflect a degree of chronic inflammation and tissue iron retention in middle-aged women. Overall, the serum iron:ferritin ratio may not only reflect the bioavailability of iron for the body, but also a healthy body composition. Disruptions in tissue iron sequestration because of elevated serum hepcidin may result in an unhealthy body composition and fatty liver progression.
Our study showed that the high consumption of beef and lamb, dairy products, eggs, fruits, and whole grains and low consumption of refined carbohydrates (rice, noodles, and bread and pastries) as well as deep-and stir-fried foods protect young adult women against fatty liver progression. These food groups explained approximately 90% of the variation in the dietary pattern scores. Beef and lamb consumption explained approximately 17.8% of the variation in the serum iron:ferritin dietary pattern scores. Consistent with our result, Hodgson et al. reported an association between lean red meat and increased serum iron levels, but not serum ferritin levels [34]. Doyle et al. examined 1268 elderly patients and reported that a varied diet (meat, poultry and fish, and vegetables and fruits, with a moderate alcohol consumption) was positively correlated with serum

Discussion
Our study revealed that the serum iron:ferritin ratio was associated with a healthy body composition, indicated as increased muscle mass and decreased visceral fat mass, and a reduced risk of moderate and severe fatty liver progression in young adult women, but not in middle-aged women. The study findings suggest that age or age-related factors (e.g., obesity) affect the body iron status and influence fatty liver progression in women. The serum iron:ferritin-specific dietary pattern (high intake of beef and lamb, dairy products, eggs, fruits, and whole grains and low intake of refined carbohydrates and stir-and deep-fried foods) can supply adequate iron to young adult women. This pattern was also associated with a 90% reduced risk of mild vs. moderate and severe fatty liver progression. Hence, it is likely that the serum iron:ferritin ratio reflects an adequate supply of iron to maintain a healthy body composition and, consequently, protect the liver against iron overload-related liver injury.
Approximately 5-10% of the body's iron is stored in myoglobin. Iron is an essential nutrient for muscle growth and performance. For example, muscles require iron-containing proteins for oxygen transportation and oxidation. An early study reported that serum iron exerts lipolytic effects in isolated adipocytes [33]. Elevated serum ferritin, considered as an acute-phase reactant, is associated with visceral fat mass and NAFLD, suggesting that serum iron or ferritin has direct or indirect effects on adiposity and liver function. However, correlations between the serum iron:ferritin ratio and body composition may be disrupted by obesity-related factors in middle-aged women. Our study revealed that middle-aged women were heavier and had higher levels of serum hepcidin and ferritin and lower levels of serum iron than did young adult women. Increased hepcidin is well-established to gradually cause hypoferremia and tissue iron overload. Moreover, decreased serum iron levels may affect muscle growth in middle-aged women. Hence, elevated serum hepcidin and ferritin are likely to reflect a degree of chronic inflammation and tissue iron retention in middle-aged women. Overall, the serum iron:ferritin ratio may not only reflect the bioavailability of iron for the body, but also a healthy body composition. Disruptions in tissue iron sequestration because of elevated serum hepcidin may result in an unhealthy body composition and fatty liver progression.
Our study showed that the high consumption of beef and lamb, dairy products, eggs, fruits, and whole grains and low consumption of refined carbohydrates (rice, noodles, and bread and pastries) as well as deep-and stir-fried foods protect young adult women against fatty liver progression. These food groups explained approximately 90% of the variation in the dietary pattern scores. Beef and lamb consumption explained approximately 17.8% of the variation in the serum iron:ferritin dietary pattern scores. Consistent with our result, Hodgson et al. reported an association between lean red meat and increased serum iron levels, but not serum ferritin levels [34]. Doyle et al. examined 1268 elderly patients and reported that a varied diet (meat, poultry and fish, and vegetables and fruits, with a moderate alcohol consumption) was positively correlated with serum iron status in elderly patients [35]. Approximately 40% of iron in red meat is heme iron, which contains ferrous iron and has a higher absorption rate than does non-heme iron [36]. However, red meat is a risk factor for NAFLD [37]. In Taiwan, pork is the most consumed red meat (31.3 kg per capita per year) compared with beef and lamb (3.9 and 2.9 kg per capita per year, respectively) [38]. Adequate meat consumption is important for increasing muscle mass. A study suggested that 25-30 g of protein per meal can stimulate muscle protein synthesis [39]. Hence, it seems reasonable to advise young adult women to moderately consume beef and lamb to improve their iron and muscle mass statuses. In the present study, eggs accounted for approximately 9.1% of the variation in the serum iron:ferritin dietary pattern scores. Eggs are rich in protein and cholesterol. Mokhtari et al. reported that the consumption of more than four eggs per week had no effect on NAFLD [40]. Our study also reported that a high consumption of whole grains and fruits (accounting for 11.5% of the variation in the dietary pattern scores) and low consumption of deep-and stir-fried foods and refined carbohydrates (accounting for 44.4% of the variation in the dietary pattern scores) protect against fatty liver progression. Whole grains are rich in ferric iron. Fruits contain vitamin C, which can facilitate ferric iron absorption in the small intestine. Georgoulis et al. showed that switching from refined carbohydrates to whole grains can reduce abdominal fat and prevent NAFLD [41]. Dry-heat cooking methods, such as deep-frying, may increase the levels of exogenous advanced glycation end products (AGEs) by the Maillard reaction during dry-heat processing. Recent evidence suggested that serum CML, a major immunogen of AGEs, was accumulated in the liver and positively correlated with NAFLD severity [42].
Our resultsuggested that middle-aged women were more likely to have central obesity (30% vs. 59%, p = 0.002) and obesity-related comorbidities, such as MetS (13% vs. 35%, p = 0.006), dyslipidemia (28% vs. 49%, p = 0.02), and moderate and severe FLD (15% vs. 40%, p = 0.006), than were young adult women. Despite low serum iron levels in middle-aged women (99.7 vs. 92.1 µg/L, p < 0.001), the prevalence rates of anemia (14% vs. 12%), iron deficiency (8% vs. 6%, p = 0.73), and IDA (8% vs. 6%, p = 0.73) did not vary between young adult and middle-aged women in our study. Normally, reproductive-age women are more likely to have iron deficiency than are middle-aged women because of menstrual blood loss or increased iron demand during pregnancy and lactation. Our data suggest that, despite the low prevalence rate of iron deficiency, middle-aged women are more likely to develop altered iron metabolism because of obesity-related comorbidities and elevated serum hepcidin. Altered iron metabolism may affect the body composition, which consequently, may accelerate NAFLD progression.
The study has several limitations. First, the relatively small sample size and cross-sectional design limited the study findings. The relative low number of moderate (n = 25) and severe FLD (n = 6) among women is another limitation. The prevalence of hepatitis virus infection in Taiwan is high. Therefore, the number of moderate and sever FLD are low after the exclusion of patients with hepatitis virus infection (e.g., hepatitis virus A, B, or C). Second, NAFLD was diagnosed based on abdominal ultrasonography and not liver biopsy. Abdominal ultrasonography is commonly used for diagnosing NAFLD in Taiwan because it offers relatively low cost, non-invasiveness, and fewer side effects than liver biopsy. Third, the body composition was evaluated using a bioelectrical impedance analysis device, and not dual-energy X-ray absorptiometry, because of budget constraints. Fourth, we used RRR to generate iron:ferritin-specific dietary pattern scores; this method requires in-depth knowledge of the diet-disease relationship to select intermediate variables. Our study selected six response variables as the intermediate variables, namely skeletal muscle mass, visceral fat mass, ALT, hepcidin, LDL-C, and CML. These were based on the literature on the association between iron and NAFLD, as well as the correlation between the serum iron:ferritin ratio and response variables. However, selecting response variables can be subjective and personal and may result in different dietary patterns in different age groups or studies. For example, the intermediate variables may be influenced by age, sex, or disease status. In our study, the association between the serum iron:ferritin ratio and response variables varied between young and middle-aged women, suggesting that age has direct effects on the intermediate variables and influences the diet-disease relationship. Future studies are required to validate our findings in a large cohort study. Moreover, it is important to identify dietary factors that may contribute to altered iron metabolism in middle-aged women because this population has a high risk of anemia of inflammation and severe fatty liver injury.

Conclusions
Our findings suggest that the serum iron:ferritin ratio predicts a healthy body composition and reduced risk of severe fatty liver in young adult women. The increased consumption of beef and lamb, dairy products, fruits, whole grains, and eggs, and decreased consumption of refined carbohydrates (rice, noodles, bread and pastries), as well as deep-and stir-fried foods may supply adequate iron to young adult women to maintain a healthy body composition and prevent fatty liver progression. Additional studies are required to determine dietary risk factors for iron metabolism alterations and fatty liver progression in middle-aged women.