Journal of. High Hemoglobin Level Predicts Non-alcoholic Fatty Liver Disease in Japanese Women

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1 Journal of Gastroenterology and Heatology Research Online Submissions: htt:// doi:0.605/j.issn Journal of GHR 203 June 2 2(6): ISSN (rint) ISSN (online) ORIGINAL ARTICLE High Hemoglobin Level Predicts Non-alcoholic Fatty Liver Disease in Jaanese Women Takemi Akahane, Kennichi Fukui, Yasuyo Shirai, Hitoshi Yoshiji, Masahito Uemura, Hiroshi Fukui Takemi Akahane, Kennichi Fukui, Yasuyo Shirai, Deartment of Internal Medicine, Nara Health Promotion Center, Miyako 404-7, Tawaramoto-cho, Shiki-gun, Nara , Jaan Hitoshi Yoshiji, Masahito Uemura, Hiroshi Fukui, Third Deartment of Internal Medicine, Nara Medical University, Shijocho 840, Kashihara, Nara , Jaan Corresondence to: Takemi Akahane, MD, Deartment of Internal Medicine, Nara Health Promotion Center, Miyako 404-7, Tawaramoto-cho, Shiki-gun, Nara , Jaan. stakemi@naramed-u.ac.j Telehone: Fax: Received: Aril 2, 203 Revised: May, 203 Acceted: May 3, 203 Published online: June 2, 203 ABSTRACT AIM: To identify whether hemoglobin () level is a redictor of non-alcoholic fatty liver disease (NAFLD) in Jaanese women. METHODS: The subjects were 673 Jaanese women (mean age: 52.±8.7 years, age range: years) who underwent a hysical checku during Women with heatitis B or C virus infection, other heatobiliary disease or alcohol consumtion>20 g/day were excluded from the study. NAFLD was diagnosed by ultrasonograhy. Alanine aminotransferase (ALT) >30 U/L was defined as elevated ALT. Ferritin level was measured in 00 selected subjects to examine the relationshi between and ferritin levels. RESULTS: NAFLD was diagnosed in 245 subjects and NAFLD with elevated ALT were detected in 73 subjects. level was significantly higher in subjects with NAFLD than those without NAFLD. BMI, menoausal status and, triglycerides and glucose levels were indeendently associated with ALT level. Logistic regression analysis showed that BMI [odds ratios (OR) =.54, 95% CI ], menoausal status (OR=.98, 95% CI ) and Q4 (to 25th ercentile) level (OR=2.8, 95% CI ) were associated with NAFLD. Q3 (5 st -75 th ercentiles) level (OR=2.7, 95% CI ) and Q4 level (OR=2.69, 95% CI ) were associated with NAFLD with elevated ALT. Multivariate analysis showed that level was indeendently associated with ferritin level and insulin resistance. CONCLUSION: High level is a redictor of NAFLD. level is associated with iron stores and insulin resistance in Jaanese women. Key words: NAFLD; Hemoglobin; Menoause; Ferritin; Insulin resistance Akahane T, Fukui K, Shirai Y, Yoshiji H, Uemura M, Hiroshi Fukui. High Hemoglobin Level Predicts Non-alcoholic Fatty Liver Disease in Jaanese Women. Journal of Gastroenterology and Heatology Research 203; 2(6): Available from: URL: htt://www. ghrnet.org/index.h/joghr/article/view/40 INTRODUCTION Obesity-related liver disease, articularly non-alcoholic fatty liver disease (NAFLD), is increasingly common in develoed countries, and reresents the heatic manifestation of metabolic syndrome. NAFLD includes a sectrum of liver tissue alterations ranging from simle heatic steatosis to heatitis and rogressive fibrosis, and is associated with a high risk of tye 2 diabetes and cardiovascular disease [,2]. In Jaan, the revalence of NAFLD is higher in men than women in the oulation aged<50 years. The revalence of NAFLD increases raidly in older women to equal or surass that in men [3]. This suggests that menoause is associated with an increase in the risk of NAFLD, and that ovarian hormones hel to rotect remenoausal women. The revalence of obesity increases after menoause. The low estrogen level in ostmenoausal women is associated with loss of subcutaneous fat and gain of visceral fat [4-6]. Increased visceral fat is an indeendent redictor of NAFLD, and is much more harmful than subcutaneous fat [7,8]. Weight gain after menoause therefore increases the risk of NAFLD, and body mass index (BMI) is a useful marker for redicting NAFLD. However, not all obese women develo NAFLD. The frequent association between heatic steatosis and body iron overload is known as insulin resistance-associated heatic iron overload [9]. Hyerferritinemia associated with mild heatic iron accumulation is frequently observed in NAFLD [0-2]. Insulin stimulates ferritin synthesis and facilitates iron utake [3], and conversely, iron influences insulin signaling [4], reduces heatic extraction and metabolism of insulin which leads to eriheral hyerinsulinemia [5], and may increase cellular oxidative stress, which inhibits the internalization and actions of insulin [6]. 623

2 Premenoausal women have lower heatic iron stores because of iron loss during menstruation and increased requirements during regnancy. The body iron stores increase after menoause [7], which may contribute to the increased risk of NAFLD in ostmenoausal women. Liver enzymes are not considered to be sensitive or secific for diagnosing A NAFLD, and a significant B number of NAFLD atients may have normal enzymes. Although serum ferritin level is a reliable marker for heatic iron stores, it is not measured as a screening test during hysical checkus. If there are useful markers besides BMI which can redict NAFLD, it would be ossible to detect atients with NAFLD more accurately. We focused on hemoglobin () level instead of ferritin level, because level is often measured as a screening test. is a redominant comonent in erythrocytes and functions as an oxygen transorter in blood. Recently exression was reorted in heatocytes and was increased in NASH [8]. The urose of this study was to investigate whether level is a redictor of NAFLD in Jaanese women. METHODS Our cross-sectional study used data from 825 women (age range: years), who had undergone hysical checkus at Nara Health Promotion Center during Of these, 52 were excluded from the study for the following reasons: heatitis B surface antigen or antiheatitis C virus antibody ositive, other heatobiliary disease or alcohol consumtion of >20 g/day. The remaining 673 articiants (934 ostmenoausal women and 739 remenoausal women, mean age: 52.±8.7 years, age range: years) were included in this study. One hundred women (mean age: 52.6±7.8 years, age range: 3-74 years) were selected from this grou for measurement of ferritin levels. This study was aroved by the Ethics Committee of Nara Health Promotion Center. All subjects gave informed consent. Each subject underwent a comlete hysical examination including anthroometric measurements. Height and body weight were measured using a digital scale, with the subject wearing a light gown. BMI was calculated as weight (kg)/height squared (m 2 ). Subjects were asked not to ingest any food for 2 h or water for 2 h rior to the examination. Resting blood ressure was measured using an automatic shygmomanometer. All biochemical analyses were erformed within 2 h after obtaining blood samles, in the same laboratory, by standard methods using commercially available kits and an automated analyzer. Alanine aminotransferase (ALT)>30 U/L was defined as elevated ALT. The level was classified into 4 quartiles: Q (0 th -25 th ercentiles), Q2 (26 th -50 th ercentiles), Q3 (5 st - 75 th ercentiles), Q4 (76 th -00 th ercentiles). Insulin resistance was assessed using the homeostasis model assessment (HOMA) of insulin resistance=fasting insulin (μu/ml) fasting glucose (mg/dl)/405. NAFLD was defined by ultrasonograhy when there was increased liver echogenicity ( bright liver ) and contrast between the heatic and renal arenchymal tissues [9]. Statistical analysis Data are exressed as the mean±standard deviation. The Student s t-test was used to comare quantitative variables with a normal distribution. The Chi-square test was used to comare qualitative variables. Odds ratios and 95% confidence intervals for NAFLD risk factors were calculated using the standard logistic regression model. Multivariate analysis was erformed based on the results of univariate analyses. A -value of <0.05 was considered to be statistically significant. All calculations were erformed using SPSS software (Version 8.0, Inc., Chicago, IL). RESULTS Clinical characteristics NAFLD was diagnosed in 245 subjects, of which 80 were ostmenoausal. The mean age was higher in subjects with NAFLD than those without. Waist circumference, BMI, blood ressure, insulin resistance, and triglyceride (TG), glucose, insulin and CRP levels were significantly higher in subjects with NAFLD than those without. HDL cholesterol was significantly lower in subjects with NAFLD than those without. and hematocrit (Ht) levels were significantly higher in subjects with NAFLD than those without (Table and Figure ). Table Anthroometric, clinical and laboratory data of the study oulation. Age (year) Menoause [n (%)] Waist circumference (cm) BMI (Kg/m 2 ) SBP (mmhg) DBP (mmhg) AST (U/L) ALT (U/L) GGT (U/L) Triglycerides (mg/dl) HDL (mg/dl) Glucose (mg/dl) Insulin (µu/ml) HOMA CRP Red blood cells (0 4 /µl) Hematocrit (%) Platelet (0 4 /µl) Current smokers [n (%)] With NAFLD n= ±7. 80 (73.5%) 89.0± ± ± ± ± ± ± ± ± ± ± ±2. 0.± ±36 3.4±.2 4.4± ±5.8 8 (3.3%) Without NAFLD n= ± (52.8%) 78.5± ±2.8 20± ± ± ± ± ± ± ± ±2.3.± ± ±32 2.9± ± ± (4.%) SBP: Systolic blood ressure; DBP: Diastolic blood ressure; AST: Asartate aminotransferase; ALT: Alanine aminotransferase; GGT: γ-glutamyltransferase; HDL: High-density liorotein-cholesterol; HOMA: Homeostasis model assessment of insulin resistance., 2 <0.05 between subjects with NAFLD and those without. g/dl a 0 with NAFLD without NAFLD Figure Comarison of hemoglobin () level between subjects with NAFLD and those without. a between subjects with NAFLD and those without. Comarison between re- and ostmenoausal women The revalence of NAFLD was significantly higher in ostmenoausal than remenoausal women. Risk factors for metabolic syndrome such as increased waist circumference, BMI, blood ressure, insulin resistance, and TG and glucose levels were significantly more frequent in ostmenoausal than remenoausal women. AST, ALT, GGT, and Ht levels were also significantly higher in ostmenoausal than remenoausal women (Table 2 and Figure 2). 624

3 Table 2 Comarisons of anthroometric, clinical and laboratory data between re- and ostmenoausal women. NAFLD [n (%)] Waist circumference (cm) BMI (Kg/m 2 ) SBP (mmhg) DBP (mmhg) AST (U/L) ALT (U/L) GGT (U/L) Triglycerides (mg/dl) Glucose (mg/dl) HOMA Red blood cells (0 4 /µl) Hematocrit (%) Platelet (0 4 /µl) Current smokers [n (%)] Table 3 Results of multivariate analysis of relationshis between alanine aminotransferase level and clinical characteristics. BMI SBP Triglycerides Glucose Menoause Premenoausal women n= (8.8%) 3 78.± ± ± ± ± ± ± ± ± ± ±33 2.5± ± ± (4.7%) β Postmenoausal women n= (9.3%) 8.6± ± ± ± ± ± ± ± ±5..4±.3 447±33 3.3±0.9 4.± ± (3.4%) SBP: Systolic blood ressure; DBP: Diastolic blood ressure; AST: Asartate aminotransferase; ALT: Alanine aminotransferase; GGT: γ-glutamyltransferase; HOMA: Homeostasis model assessment of insulin resistance. 3 < 0.00, 4 < 0.05 between re- and ostmenoausal grous. g/dl a 0 Premenoausal Postmenoausal Figure 2 Comarison of hemoglobin () level between re- and ostmenoausal women. a between subjects with re- and ostmenoausal women. Factors correlated with ALT level We used multivariate analysis to analyze factors correlated with ALT level. High BMI, ostmenoausal status, and high TG, glucose and levels were identified as indeendent redictors of high ALT level (Table 3). < 0.00 NS NS: Nonsignificant; SBP: Systolic blood ressure; : Hemoglobin. Factors associated with NAFLD Logistic regression analysis with NAFLD as the deendent variable showed that ostmenoausal status, high BMI and Q4 level were significantly associated with an increased risk of NAFLD (Table 4). Furthermore, the statistically significant risk factors for NAFLD with elevated ALT were high BMI, ostmenoausal status and Q3 and Q4 levels (Table 5). Table 4 Logistic regression analysis with NAFLD as a deendent variable. Menoausal status Pre menoause Post menoause Q Q2 Q3 Q4 CI: Confidence interval. NAFLD [n (%)] 65 (8.8%) 80 (9.3%) 38 (8.5%) 37 (9.0%) 67 (6.2%) 03 (25.8%) Odds ratio (95%CI).54 ( ).98 ( ) 0.79 ( ).34 ( ) 2.8 ( ) Table 5 Results of Logistic regression analysis with NAFLD with elevated alanine aminotransferase (ALT) as a deendent variable. Menoausal status Pre menoause Post menoause Q Q2 Q3 Q4 NAFLD with ALT elevation [n (%)] 5 (2.0%) 58 (6.2%) 7 (.6%) 7 (.7%) 26 (6.3%) 33 (8.3%) Odds ratio (95%CI).47 ( ) 2.34 ( ) 0.9 ( ) 2.7 ( ) 2.69 ( ) ALT: alanine aminotransferase; CI: Confidence interval Relationshi between insulin resistance assessed by HOMA and level We investigated the correlation between level and insulin resistance. Multivariate analysis showed that BMI and level were indeendent redictors of insulin resistance (Table 6). Table 6 Results of multivariate analysis of relationshis between insulin resistance assessed by HOMA and age, BMI and hemoglobin level. Age β HOMA: Homeostasis model assessment of insulin resistance Relationshi between ferritin and levels in 00 selected subjects Thirty-eight of the 00 subjects selected for measurement of ferritin level were remenoausal. NAFLD was diagnosed in 52 subjects, of which 37 (7.%) were ostmenoausal. Ferritin level was significantly higher in ostmenoausal than remenoausal women (68.7±52. versus 28.9±33.6 ng/ml, ). Univariate analysis showed that level correlated with ferritin level, but BMI did not correlate with ferritin level. Multivariate analysis showed that level was an indeendent redictor of ferritin level (Table 7). Table 7 Results of univariate and multivariate analysis of relationshis between ferritin level and age, BMI and level. Age (g/dl) : Hemoglobin. DISCUSSION Univariate analysis β Multivariate analysis β The major finding of this study is that a high level is an indeendent redictor of NAFLD in Jaanese women. We found that level was higher in subjects with NAFLD than those without. Multivariate analysis showed that level correlated with both 625

4 ferritin level and insulin resistance assessed by HOMA, indicating a high robability that level is associated with iron stores and insulin resistance. We analyzed levels of 94 men, who had undergone hysical checkus at our center during 2009 (Individuals with heatitis B surface antigen or anti-heatitis C virus antibody ositive, other heatobiliary disease or alcohol consumtion of >20 g/day were excluded). Univariate analysis showed that level was negatively associated with age (β=-0.9, ). Age did not differ significantly between subjects with NAFLD (n=79) and those without, while level was significantly higher in subjects with NAFLD than those without (5.4±0.9 versus 4.8±.0 g/dl, ) (unublished data). Therefore, age did not correlate with NAFLD or level in Jaanese men and high level may be redictive of NAFLD in both Jaanese women and men. Patients with NAFLD often have a high serum ferritin level. Zelber-Sagi et al [20] demonstrated that NAFLD is a major determinant of increased serum ferritin level. They also showed that the association between serum ferritin and insulin levels is much more evident in atients with NAFLD than those without. Recent studies have suggested that serum ferritin level is a marker for insulin resistance [2-23]. Although serum ferritin level is a useful marker for NAFLD, it is not measured in screening tests at the time of hysical checkus. However, level is often measured in screening tests, and our results showed that level was indeendently correlated with ferritin level in 00 subjects, and with insulin resistance assessed by HOMA in all subjects. Our results suggest that level can be as useful a marker for NAFLD as ferritin level. A recent roteomic study has shown that free α and β subunits in serum were significantly increased from normal controls to steatosis and to NASH, suggesting free subunits in serum could be a biomarker for liver lesions [24]. In another oulation study, higher free serum has been associated with higher revalence rates of NAFLD [25]. The source of free in serum was not identified, but resumed to result from oxidative stress-induced hemolysis. On the other hand, Liu et al [8] reorted that was exressed in heatocytes and was increased in NASH. They suggested that elevated oxidative stress in NASH could induce exression and suression of oxidative stress by could be a mechanism to rotect heatocytes from oxidative damage. Obstructive slee anea is a ossible comorbidity in atients with morbid obesity, and is also associated with a high level [26]. Slee anea is rare in Jaanese women, and none of the subjects in this study suffered from slee anea. Slee anea is therefore not a confounder in this study. Smoking might also be associated with an increased level. A national survey in 2009 reorted that the smoking rate in Jaanese women was 0.9% [27]. Univariate analysis in this study did not show an association between smoking and level (data not shown), and there were no significant differences in the roortions of smokers between subjects with NAFLD and those without, or between re- and ostmenoausal women (Tables and 2). The likelihood of increased levels in this study having been caused by smoking is therefore robably small. In conclusion, high level is an indeendent redictor of NAFLD in Jaanese women. Our findings suggests that level can be a useful marker for NAFLD. REFERENCES Tziomalos K, Athyros VG, Karagiannis A. Non-Alcoholic Fatty Liver Disease in Tye 2 Diabetes: Pathogenesis and Treatment Otions. 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