Low Serum Iron Status Is Associated With Low Incidence of Coronary Artery Disease In Women

Background: Disorders of iron metabolism has been implicated in cardiovascular disease. However, the association of serum ferritin and coronary artery disease (CAD) remains inconsistent. Here, we investigated the associations of serum iron metabolism with the incidence of CAD, the severity of coronary artery stenosis, metabolic biomarkers, and 1-year restenosis after coronary artery revascularization. Methods: A total of 643 CAD patients and 643 healthy controls were enrolled to assess the associations of serum iron status with the presence of CAD, the severity of CAD, and 1-year rehospitalization after revasculation. Serum iron metabolism and other metabolic markers were measured in all subjects. All statistical analyses were analyzed using SPSS22.0 software and STATA statistical package. Results: Serum level of iron metabolism markers, including serum iron, ferritin, unsaturated transferrin iron binding capacity (UIBC), Total iron binding capacity (TIBC) levels, in CAD groups was signicantly higher than the control group (P<0.001). UIBC and TIBC were negatively correlated with ferritin in both sexes. Serum level of iron (OR=0.806, 95% CI (0.687-0.944), UIBC (OR=0.919, 95% CI (0.852-0.992), and TIBC (OR=0.864, 95% CI (0.787-0.95) were found to have a protective role for CAD in women (P<0.05, Table 3). The OR for ferritin was signicant in the both sexes (OR=1.029, 95% CI (1.002-1.058) in men, OR=1.02, 95% CI (1.005-1.034) in women, P<0.05). Conclusion: Low Serum level of iron, UIBC, TIBC and ferritin levels were found to have a protective role for CAD in women, but not in men. Elevated serum ferritin is independently and positively associated with CAD in men and women.


Introduction
Iron plays a role in several fundamental biological processes such as erythropoiesis and cell metabolism. Iron status is a modi able feature associated with cardiovascular disease. Iron metabolism disorders, either de ciency or overload, were associated with increased cardiovascular morbidity and mortality [1,2]. The increase of iron stores was also positively correlated with the risk factors of cardiovascular disease, such as the risk of metabolic syndrome [3], insulin resistance (IR) [4,5] and new-onset type 2 diabetes mellitus [6]. The role of body iron indices has been reviewed in the pathogenesis of coronary artery disease (CAD) [7][8][9][10][11]. It has been suggested iron plays an important role in the pathogenesis of atherosclerosis by catalyzing the atherogenic modi cation of low density lipoprotein (LDL) [10,[12][13][14][15][16]. Some studies supported the iron hypothesis [4,[17][18][19][20][21], some found no supporting evidence, and the results of others contradicted the hypothesis [22][23][24][25][26].
Although iron status was implicated in cardiovascular disease, the relationship between iron states and CAD has long been a controversial topic in the literature. Because of the inconsistent results of the association between body iron stores and CAD risk, we conducted a case-control study enrolled a total of 643 CAD patients and 643 healthy controls to analyze the associations of serum iron levels with the presence of CAD, the severity of coronary artery stenosis, and 1-year rehospitalization after revasculation.

Study population
The control group consisted of 643 (male/female 381/262) healthy persons without cardiovascular disease via physical examination and electrocardiogram. The controls were frequency-matched to the cases on age and sex. The CAD group consisted of 643 patients, 349 men and 294 women. All CAD patients underwent physical examination and review of their medical history. Patients were excluded if they 1) had heart failure, acute myocardial infarction, coronary bypass surgery or angioplasty, coronary spasm, or myocardial bridge; 2) had cardiac diseases such as cardiomyopathy, valvular or congenital heart disease, arrhythmia; 3) patients who had malignant tumors, acute or chronic infection, iron de ciency anemia, digestive system disease, fever, infection, connective tissue disease, autoimmune disease; 4) patients whose Blood pressure ≥180/110 mmHg after taking standard antihypertensive drugs, severe hepatic or renal dysfunction; 5) A history of major surgical trauma, pregnancy, mental illness, or any other cause of active blood loss within 2 weeks. The Zhengzhou University ethics Committee approved this study (The approval number 2020-KY-172). All the investigations were performed in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all participants or from a parent or legal guardian if participants are under 16.

Methods
All subjects underwent anthropometrical evaluation with measurements of weight, height, and body mass index (BMI). Laboratory determinations: All blood samples were obtained after overnight fasting. Fasting plasma glucose (FPG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), triglycerides (TG), high sensitive C-reactive protein (hs-CRP), iron metabolism biomarkers, liver function and renal function markers were measured on a Cobas 8000 Analyzer (Roche Diagnostics, Germany) using the Original Roche Reagent following the manufacturer's manual.
Plasma glucose was measured by the hexokinase method; TG, TC, LDL-C and HDL-C, Alanine aminotransferase (ALT) and aspartate aminotransferase (AST), creatinine, urea nitrogen (UREA), uric acid (UA) concentrations were evaluated by enzymatic methods. Hemoglobin A1c (HbA1c) was assayed by high-performance liquid chromatography on a Primer-Premier Hemoglobin Testing System (reagents were supplied by Primus). hs-CRP, homocysteine (Hcy), Apolipoprotein A (ApoA), Apolipoprotein B (ApoB) and serum ferritin levels were measured using an immune-turbid metric assay. The serum iron was measured with a colorimetric method; while the levels of unsaturated transferrin iron binding capacity (UIBC) was measured using a colorimetric test. Total iron binding capacity (TIBC) equaled the serum iron levels plus serum UIBC levels. Finally, estimated glomerular ltration rate (eGFR) was computed by using the chronic kidney disease epidemiology (CKD-EPI) collaboration equation [27].

Statistical analysis
Continuous variables were expressed as mean ± standard deviation (normally distributed data). Categorical variables are expressed as the frequency and its percentage. Continuous variables were analyzed using Student's ttest in normally distributed data, and Mann-Whitney test in non-normally distributed data. Chi-squared test was utilized for categorical variables. The association between continuous variables was assessed by Pearson correlation. The association of iron metabolism biomarkers with CAD was analyzed by logistic regression models with three progressive degrees of adjustment. Model 1 was a crude model without any confounders; model 2 was adjusted for age and cardiovascular risk factors including smoking habit, alcohol drinking habit, body mass index (BMI), hypertension, and dyslipidemia; model 3 was additionally adjusted for laboratory tests including HbA1C, ALT, AST, TC, TG, hs-CRP, eGFR. To avoid collinearity, we did not include iron and transferrin saturation into the multiple liner regression models because these two variables lied into the same causal pathway between ferritin and CAD. All the statistical analyses were executed using Statistical Package for Social Science (SPSS, version 22.0) and STATA statistical package (version 13, Texas, USA).

Results
Clinical characteristics Table 1 showed the general characteristics of the study groups according to the sexual groups. There was no signi cant difference in age and diastolic blood pressure (DBP) between CAD group and controls. Overall, CAD patients were more likely to be smokers, alcohol drinkers, obese, hypertensive, dyslipidemia and hyperglycemia. Comparing to the controls, individuals in the male CAD group showed signi cantly lower plasma concentrations of Total proteins (TP), Albumin (ALB), and ApoB, Tfs serum iron levels, systolic blood pressure (SBP) and body mass index (BMI). The plasma concentrations of ferritin, UIBC, HbA1c, FBP, hs-CRP, liver function and renal function were signi cantly higher in the CAD group compared to the control in men (P<0.05) (Table 1, Figure 1). Similar difference was found in the female group (Table 1, Figure 1). Additionally, signi cant differences were found in the diabetes management styles between male and female patients with CAD (Table 1). Correlation between ferritin and other metabolic variables in CAD patients We analyzed the correlation between ferritin and other metabolic variables in CAD by Pearson correlation. We totally tested the correlation between serum ferritin and 17 biomarkers, i.e. blood lipids (LDL-C, HDL-C, TC, TG, ApoA, ApoB and Lp(a)), blood glucose (FPG, HbA1c), blood pressure (systolic blood pressure, SBP and diastolic blood pressure, DBP), eGFR, proin ammatory measures (hs-CRP), adiposity measure (BMI) and other iron metabolism markers. TC, TG, LDL-C and Tfs were positively correlated with ferritin in men, HDL-C, ApoA, and eGFR were negatively correlated with ferritin in women, while BMI was inversely associated with ferritin in women ( Table  2). UIBC and TIBC were negatively correlated with ferritin in both sexes. Interestingly, HbA1C level was positively correlated with ferritin in men, while inversely associated with ferritin in women. Logistic regression analysis of iron metabolic markers and the risk of CAD and 1-year rehospitalization To illustrate the continuous relationships between parameters of iron metabolism and the risk of CAD, we assessed the concentration-risk relationship between serum UIBC, ferritin levels and CAD risk by multivariate random-effects meta-regression based on the restricted cubic spline model with four knots (Figure 2). Visual inspection revealed Jshaped relationships between UIBC, ferritin and the ORs for the risk of CAD. The association between ferritin and high CAD was further explored by categorizing ferritin levels into low, normal and high groups, and using the normal ferritin level as the reference.
From an unadjusted multivariable logistic regression analysis, each unit increase in ferritin was associated with a 1.003-fold (95% con dence interval (CI), 1.002-1.004) and 1.01-fold (95% CI, 1.000-1.01) increased Odds Ratio (OR) of CAD in male and female groups respectively (   Table 3). When analyzed with the normal ferritin group as the reference, the low level of serum ferritin showed a decreased OR for CAD in women (OR=0.087, 95% CI (0.01-0.724) P<0.05, Table 3).
Regrettably, there was no statistical signi cance between the correlation of iron metabolism markers and 1-year rehospitalization after calibrating for confounders (model 3, P>0.05, Table 4). Association of body iron status with the severity of CAD The severity of CAD was quanti ed the modi ed Gensini scores based on the number and the extent of lesions in coronary arteries [32]. The detail calculation of modi ed Gensini scores was described in our previous study [33].
Serum iron status markers exhibited no signi cant association with the severity of CAD (Supplement Table 1).

Discussion
There are limited data concerning serum iron and iron saturation in CAD patients and the results are also inconsistent. This study was conducted to assess the association between serum iron metabolism markers and CAD. For physiological reasons, the reference interval for iron metabolism markers in women are different from men, we analyzed the association of iron status and CAD strati ed by gender. In a cohort of 643 CAD patients and 643 controls, iron imbalance, as characterized by either high serum ferritin or low iron levels, was associated with an increased risk of CAD.
Overall, our ndings suggested that low Serum level of iron, UIBC, TIBC and ferritin levels showed a protective role for CAD in women, but not in men. Increased serum ferritin was independently associated with CAD in men and women. Our data add to previous reports that showed a protective role of low iron level on the risk of CAD. Sullivan proposed that reduced iron stores can protect against ischemic heart disease and for the rst time to explain the sex difference in CAD risk [34]. Salonen, J.T. et al. observed that a higher level of iron is a risk factor for myocardial infarction in Finnish men [35]. Later, it has been reported that reducing iron stores through phlebotomy could decelerate the progression of atherosclerotic plaque [12,22]. However, we did not nd iron de ciency was associated with increased risk for CAD, which was inconsistent with some previous report. Iron de ciency was associated with increased risk for CAD and had detrimental effects in patients with CAD [1,26]. The patients' status and comorbidities might explain the inconsistence. In our study, we enrolled low percentage (less than 30%) of iron de ciency in the patient group, and we exclude the CAD patients with clinical anemia. However, there was a high prevalence of iron de ciency in acute coronary syndrome and its association with poor outcome [36]. Moreover, the de nition of iron de ciency to be applied in heart disease remains controversial.
Iron is a trace element that exists in serum at low concentration of mg/dL. Iron values exhibit diurnal variation depending on dietary iron intake or patient condition [37].  [24,42]. These studies implied that ferritin might be an in ammatory marker for atherosclerosis. It is hard to tell whether high ferritin levels re ect in ammation caused by hyperglycemia or indicate iron overload, which can also lead to in ammation.
Iron is an essential mineral, which participates in different functions of the organism under physiological conditions. Numerous biological processes, such as oxygen and lipid metabolism, protein production, cellular respiration, and DNA synthesis, require the presence of iron [43]. Maintaining iron metabolism is important for cardiovascular health as its high energy consumption and high mitochondrial activity [44]. Intravenous iron administration in acute myocardial infarction (MI) exerted bene cial effects in MI patients [45]. However, excessive iron accumulation accelerates the formation of atherosclerosis through several putative mechanisms. Firstly, iron catalyzes Fenton reaction to produce reactive oxygen species (ROS) which promote LDL peroxidation and induce endothelial dysfunction by reducing the bioavailability of nitric oxide [46]. Secondly, excess iron may make vascular endothelium vulnerable to other pathogenic factors. Ultimately, atherosclerosis is accelerated as a result of increased platelet activity and leucocyte adhesion [47]. Thirdly, ROS increased the expression of LOX-1 receptor on endothelial cells, resulting in mitochondrial DNA damage and autophagy activation [48]. Fourthly, accumulated iron in adipocytes leads to adipocyte IR by increasing lipolysis and by decreasing insulin-stimulated glucose transport [2]. Adipocyte IR accelerates atherosclerosis [49].

Conclusion
There are limited data concerning serum iron and iron saturation in CAD patients and the results are also inconsistent. The discrepancies among the studies may be partly attributable to the differences in race, dietary habits, sample size and confounding factors. Low Serum level of iron, UIBC, TIBC and ferritin levels were found to have a protective role for CAD in women, but not in men. Increased serum ferritin is independently associated with CAD in men and women. The main limitation of the study is its cross-sectional design; therefore, a causal relationship could not be established. In addition, the measurements of hepcidin, a well-known regulator of body iron uxes, were not available. However, we evaluated transferrin saturation which is an important determinant of hepcidin release. Moreover, the daily iron intake needs to be estimated in the future study. Finally, there is a pragmatic need to identify circulating iron biomarkers reliably characterizing iron status within tissues. The serum levels of iron metabolism markers in CAD and Control groups strati ed by gender. Figure 2