Mutual relationship between serum ferroxidase activity ... - Springer Link

Ann Hematol (2016) 95:1333–1339 DOI 10.1007/s00277-016-2709-8

ORIGINAL ARTICLE

Mutual relationship between serum ferroxidase activity and hemoglobin levels in elderly individuals Arianna Romani 1 & Alessandro Trentini 1 & Angelina Passaro 2 & Cristina Bosi 2 & Tiziana Bellini 1 & Carlo Ferrari 3 & Carlo Cervellati 1 & Giovanni Zuliani 2

Received: 5 April 2016 / Accepted: 23 May 2016 / Published online: 27 May 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract The identification of hemoglobin (Hb) biological determinants is of primary clinical interest, in particular in the elderly because of the well-documented relationship between anemia and cognitive and functional decline. Ceruloplasmin (Cp) and non-Cp ferroxidase activity might influence Hb production because of its role in modulating iron mobilization. This potential connection has never been explored so far. Therefore, in the present study, we evaluated the possible association between serum ferroxidase activity (sFeOx) and Hb in a sample of 136 apparently healthy older individuals. The results revealed that nonlinear (quadratic) regression explained the relationship between the two variables of interest better than did the linear one (R2 = 0.09 vs. R2 = 0.03). The same analysis highlighted a linear behavior for the relationship between Hb and sFeOx, for two separate subsamples stratified on the basis of the Hb value (141 g/L) corresponding to the parabola vertex. In the subset with higher Hb (high Hb), sFeOx was positively associated (r = 0.44, p = 0.003) while in the low Hb subset, the association was Electronic supplementary material The online version of this article (doi:10.1007/s00277-016-2709-8) contains supplementary material, which is available to authorized users. * Carlo Cervellati [email protected]

1

Department of Biomedical and Specialist Surgical Sciences, Section of Medical Biochemistry, Molecular Biology and Genetics, University of Ferrara, Via Luigi Borsari 46, 44121 Ferrara, Italy

2

Department of Medical Science, Section of Internal and Cardiopulmonary Medicine, University of Ferrara, Via Savonarola 9, 44100 Ferrara, Italy

3

Department of Clinical and Molecular Sciences, Faculty of Medicine, Le Marche Polytechnic University, Via Tronto 10/A, IT-60126 Ancona, Italy

negative (r = −0.26, p = 0.01). Notably, we found that the concentration of Cp was significantly higher in Low Hb compared to High Hb subsample (p < 0.05), with this multicopper oxidase selectively contributing to sFeOx in the former group (r = 0.348, p = 0.001). Collectively, this exploratory study suggests that ferroxidases might play a role in dispatching the body’s iron toward erythropoietic tissues, with Cp contribution that might become more important in stress-like conditions. Keywords Anemia . Ceruloplasmin . Elderly . Hemoglobin . Ferroxidase

Introduction It is well documented that the incidence and prevalence of anemia increase with advancing age, with the condition becoming a frequent health issue among the elderly [1, 2]. In this population, anemia is associated with a poor functional and cognitive status [3] as well as several unfavorable outcomes in a variety of diseases, including dementia [4] and cardiovascular diseases (CVDs) [5]. Data from the Third National Health and Nutrition Examination Survey (NHANES III) showed that anemias affecting older people are mostly linked to nutritional deficiency, inflammatory diseases, renal insufficiency, stem cell aging, androgen insufficiency, etc. [2, 6]. Regardless of the putative driving-factors, the decrease in hemoglobin (Hb) below the clinical threshold always reflects in an impairment of erythropoiesis and/or iron homeostasis [1]. There is ample experimental and clinical evidence suggesting that copper is one of the factors that can influence the metabolism of iron [7]. Indeed, it is now well established that anemia commonly occurs in humans and animals with copper

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deficiency [7]. However, the mechanism underlying this connection is still far to be fully elucidated [8, 9]. At present, one of the most widely accepted hypothesis considers the ability of plasma copper proteins, in primis ceruloplasmin (Cp), to function as ferroxidase enzyme, catalyzing the oxidation of Fe2+ to Fe3+ [10–12]. Ferroxidase activity promotes the binding to apotransferrin of Fe3+ thereby creating a negative ferrous iron gradient that, in turn, increases the efflux of iron from tissues such as gut and liver [10, 12]. Therefore, Cp stimulates iron transport from storage pools to erythroid tissue where Hb synthesis takes place. In support of this proposed mechanism, patients with aceruloplasminemia, a rare genetic disorder characterized by mutation in Cp gene and low levels of the protein, presents iron accumulation mostly in the liver and brain, along with, although mild, anemia [13]. Collectively, this body of evidence is suggestive of a possible role of Cp-related iron oxidase activity in maintaining the level of Hb within a Bhealthy^ range. This contribution might be more relevant in older age when the yield of Hb synthesis is low even in absence of a full-blown iron deficiency [2]. However, the definitive confirmation of this potential biological interaction has not been achieved yet because of the lack of clinical-epidemiological studies on this specific topic. In this light, we set out the present population-based study that aimed to explore the possible association between serum ferroxidase activity (sFeOx) and Hb in a cohort of older individuals.

Materials and methods Subjects One hundred thirty-six outpatients referring to the Day Service for the evaluation of Cognitive Decline (University of Ferrara, Italy) were included in this study from 2006 to 2014. This study conforms to The Code of Ethics of the World Medical Association (Declaration of Helsinki) and was conducted according to the guidelines for Good Clinical Practice (European Medicines Agency, http://www.ema. europa.eu). Signed informed consent, which was written in compliance with local and national ethical guidelines, was obtained from each patient prior to the inclusion into the study. Personal data and medical history were collected by a structured interview from participants. Individuals were included in the study protocol only if they were older than 65 years, had normal cognitive function as assessed by a standardized battery of cognitive tests [14]. and were independent in the activities of daily living (ADLs). Subjects with diagnosis of severe congestive heart failure (New York Heart Association class III-IV); severe liver or kidney disease; severe chronic obstructive pulmonary, chronic, or acute inflammatory disease (e.g., all acute known

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infections or rheumatologic conditions); cancer; or taking NSAIDS, antibiotics, or steroids were excluded. Subjects with serum levels of ferritin, folate, or vitamin B12 below the lower limit of normal range (10 μg/mL, 3.5 μmol/L, and 200 pmol/ L, respectively) were excluded from the analyses to avoid possible cases of evident iron deficiency or vitamin deficiency anemia. Biochemical assays Venous blood was collected from subjects upon an overnight fast, between 8.30 and 9.30 a.m., and blood was centrifuged at 3000 rpm for 10 min. The isolated serum/plasma was stored at −80 °C until analysis. The levels of sFeOx were measured in serum samples according to Erel’s method [15] with some minor modifications [16, 17]. Briefly, 5 μL of the sample was added to 195 μL of acetate buffer (0.45 M, pH = 5.8) in a 96-well microplate. After 1 min of incubation at 37 °C, 43 μL of 370 mM Fe(NH4)2SO4 was added, and the resulting mix was incubated for further 3.8 min at 37 °C. At the end of the incubation, 20 μL of chromogen (3-(2-Pyridyl)-5,6-bis-[2-(5furylsulfonic acid)]-1,2,4-triazin) was added. The rate of formation of colored complex (formed by the chromogen and ferrous ions) was recorded at 600 nm by a UV–Vis microplate reader (Tecan infinite M200 from Tecan group Ltd, Switzerland). Cp concentration was determined by competitive enzymelinked immunosorbent assay (ELISA) according to the manufacturer’s protocol (AssayMax Human Ceruloplasmin ELISA Kit EC4001–1, Assaypro LLC, St. Charles, MO, USA). Briefly, serum samples were diluted 1:100. The standards and serum samples were added to plates precoated with a polyclonal antibody specific for human Cp. The Cp protein present in the samples competed with biotinylated Cp, added to the wells in a constant amount, for the binding to the wells. After incubation and washing steps, a streptavidin–peroxidase conjugate was added to all wells and the color development was started by dispensing tetramethylbenzidine, a peroxidase substrate. The enzymatic reaction was stopped by the addition of 1 M sulfuric acid and the absorbance measured at 450 nm by Tecan infinite M200; inter- and intra-assay coefficients of variation were 8.4 and 1.3 %, respectively. Clinical-chemistry analyses including serum B-12 vitamin, serum folate, ferritin, copper, and Hb were performed by routine laboratory methods. Statistical analysis Statistical analyses were performed by SPSS 21.0 for windows (SPSS Inc., Chicago, Illinois, USA), with p < 0.05 as level of significance for all analyses. The normality distribution of the variables was checked by the Kolmogorov–

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Smirnov and Shapiro–Wilk tests. Correlations between normal variables were checked with Pearson’s analysis, while those between non-normal variables with Spearman’s rank test. When the relationship between variables of interest did not appear to be properly described by a linear function, we tried with other non-linear regression models (quadratic, cubic, logarithmic, and exponential). Model selection was based on the best fit of observed data, using R2 value as discriminatory factor. Finally, stepwise multiple linear regression analysis was performed among variables to examine the independence of an association and the contribution of each predictor in the variance of independent variable. Variables were included as covariate in the multivariate models if they were biologically plausible or significantly related based on simple correlation analysis. Criteria for variable inclusion in the stepwise regression analysis were entry if p ≤ 0.05 and exclusion if p > 0.10. A two-tailed probability value 200 pmol/L; folates = >4.5 nmol/L; copper = 10–22 μmol/L; Cp = 0.2–0.6 g/L Hb hemoglobin, Cp ceruloplasmin, sFeOx serum ferroxidase activity

We found that in the both Bsubsets^ obtained by using this Hb level as discriminating threshold, the relationship between Hb concentration and sFeOx was adequately described by a linear regression model (Fig. 1b, c). In particular, within the subgroup characterized by Hb lower than 141 g/L (named as low Hb) including 96 subjects, an inverse correlation between the two variables was present (Fig. 1a; r = −0.26, p = 0.01, R2 = 0.07). In contrast, in the high Hb subgroup (n = 42), the correlation between the two variables was positive and much stronger (Fig. 1 c; r = 0. 44, p = 0.003, R2 = 0.19).

Table 2 Correlation coefficients for the relationship between Hb/ sFeOx and age, gender, and markers of iron and copper metabolism Variables

Hb (r)

FeOx activity (r)

Hb Age Gender Ferritin Vitamin B12 Folates

– −0.28** 0.40*** 0.21* 0.05 −0.01

−0.23* 0.05 −0.15 −0.09 0.03 0.13

Copper Cp

−0.26* −0.10

−0.11 0.37***

Hb hemoglobin, Cp ceruloplasmin, sFeOx serum ferroxidase activity *p < 0.05; **p < 0.01;***p < 0.001

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Fig. 1 Graphical analysis of the relationship between Hb and sFeOx in whole sample (a), in low Hb (b), and high Hb (c) subsamples. a Linear (solid line) and quadratic (dashed line) regression plots for the relationship between Hb and sFeOx. Linear regression: y = −1.71x + 729 (R2 = 0.03, p = 0.02); quadratic regression: y = 25.6x + 0.09x2 + 2284 (R2 = 0.09, p = 0.003). The x coordinate of the parabola vertex was

found by solving the relative standard formula for quadratic function: −25:6 xðvertexÞ ¼ −b 2a ¼ 2x0:09 ¼ 141. b Linear regression plot obtained by fitting the data in low Hb subsample: r = −0.261, p = 0.014, R2 = 0.07). c Linear regression plot obtained by fitting the data in High Hb subsample (r = 0.438, p < 0.001, R2 = 0.19)

Evaluation of the possible difference in the sFeOx/Hb relationship between high and low Hb subsamples

the high subgroup. Since Cp represents the main, but not the sole, biological agent of serum with ferroxidase function, we checked in which extent the multicopper oxidase contributes to the variance of serum sFeOx in the two subsamples. Noteworthy, Cp was found to be significantly correlated with sFeOx (r = 0.348; p = 0.001) only in the low Hb subsample (Fig. 2a), in a way that is independent of age, gender, and copper levels (data not shown).

Multivariate analysis was performed to check whether the univariate associations between sFeOx and Hb observed in the two subsamples were independent of potential confounders such as age, gender, ferritin, vitamin B12, folates, Cp, and copper (Table 3). We chose to design sFeOx and Hb as explanatory and outcome variable, respectively, but not vice versa, in virtue of the proposed biological interaction that assume ferroxidase activity as, although indirectly, promoting the biosynthesis of the erythrocyte protein [9, 10]. From these analyses, it emerged that sFeOx remained inversely and positively related to Hb in low and high Hb subset, respectively, regardless of the covariates entered in the regression models. Notably, the relative contribution of sFeOx to Hb variance was larger among subjects belonging to the high compared to the low Hb, both in the non-adjusted (model 1 19.2 vs. 6.8%) and the fully adjusted models (model 3 21.2 vs. 11.2 %). Differences between the two subsamples were also evidenced by the comparison of the mean, or median, levels of the examined markers, as displayed in online resources 1. Within the aim of this study, the most interesting results of this analysis were the significantly higher levels of Cp and sFeOx (p0.001 >0.001

19.2 (17.1) 46.4 (29.8)

Regression model 1: sFeOx. Regression model 2: sFeOx, age, gender, ferritin, copper, cp, folates, vitamin B12 Hb hemoglobin, Cp ceruloplasmin, sFeOx serum ferroxidase activity a

The squared semipartial correlation coefficient accounts for the proportion of variance in the dependent variable that is explained by the covariate

role in promoting iron egress from cells through two mechanisms: (1) by stabilizing the specific transmembrane iron carrier, i.e., pore ferroportin [12] and (2) by enabling the incorporation of iron into transferrin [21]. The latter function is directly linked to the ability of Cp to utilize copper for oxidizing the outgoing Fe2+ to Fe3+, the only form that can be bound by transferrin. Thus far, the in vivo studies on humans have solely explored whether the concentration of circulatory Cp, but not the sFeOx, may influence iron mobilization and Hb levels [8, 22, 23]. Owing to these premises, the aim of the present study was to investigate for the first time the possible association between sFeOx and Hb levels in a sample of elderly individuals. At first sight, our findings appeared, at least partially, in contrast with the hypothesis of sFeOx as a factor that can contribute to Hb production. Indeed, the fitting of the data points with a linear regression model resulted in a weak and negative correlation coefficient (Table 2 and Fig. 1a). On the contrary, a quadratic plot was found to be a more suitable mathematical model for the relationship between the Hb and sFeOx. By allowing quadratic association between the two variables, we were able to separate two opposite behaviors

of the sFeOx in the function of Hb. More specifically, only when Hb was higher than 141 g/L, sFeOx positively correlated with the concentration of the oxygen carrier. Conversely, after considering subjects with lower Hb, the association between the two factors was still significant, but with an opposite direction. The picture emerging from our data is that other ferroxidases besides Cp might contribute in iron mobilization and, thus, conceivably, in the enhancement of Hb production more likely by increasing the charge of iron on transferrin. However, this might occur in a condition of a relatively high abundance of Hb. Indeed, 141 g/L is considerably higher than the WHO recommended cutoffs for Hb (120 and 130 g/L for men and women, respectively), especially considering the advanced age of the sample subjects. This hypothesis is supported by the lack of correlation between serum Cp concentration and sFeOx observed in the subset characterized by high Hb concentration. Thus, in this condition of Bgood^ oxygenation, the contribution of Cp to iron efflux from cells and loading into transferrin might be negligible and this role could be sufficiently covered by other biological players [24]. This result is not surprising, since Cp

Fig. 2 Scatter plots for the relationship between sFeOxs and Cp in the Low Hb* subgroup (n = 94, a) and High Hb* subgroup (n = 42, b). *cutoff value = 141 g/L

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is the main, but not the sole, agent with ferroxidase function present in the circulatory system [8, 25]. According to this statement, humans and rodents with aceruloplasminemia showed iron overload into the hepatic cells but only mild alteration in Hb [13, 23, 26]. Consistently, knocking out Cp did not abolish sFeOx in transgenic mice [11]. The most convincing experimental evidence supporting the existence of additional plasma ferroxidases mostly come from a seminal study by Prohaska [27] and other two more recent works [11, 25]. Overall, these separate studies reached the conclusion that other proteins (such as holotransferrin or a not still identified 700-kDa macromolecules [11]) or biological polyanions, such as bicarbonate, phosphate, and citrate, which are prevalent in biological fluids [25], can play a major role in iron metabolism. The finding of an inverse association between the sFeOx and Hb in low Hb subset is more puzzling, because it appears to refute the previously proposed influence of ferroxidase function on iron metabolism. However, we interpreted this unexpected behavior as a clue that in a condition of relatively low concentration of Hb, a different type of sFeOx/Hb interaction might become Bstatistically^ predominant. It is indeed tempting to speculate that a decrease in the oxygen carrier concentration might stimulate sFeOx as a mechanism involving Cp that responds to the increased iron demand of hematopoietic tissues. In line with this postulation, we found that sFeOx variance is now explained by Cp, which in turn significantly increased along with sFeOx in low Hb as compared to the other subsample. Our thesis is also supported by both in vitro and animal studies where a hypoxia state is able to increase the synthesis of Cp through a hypoxia-inducible factor 1-alpha (HIF1-alpha)-dependent mechanism [28, 29]. Thus, as also proposed elsewhere, in conditions where high rates of cellular iron efflux are required or in the presence of low oxygen concentration, the enzymatic machinery of Cp might be essential to stimulate the iron efflux and increase iron loading into transferrin [24]. If confirmed by further epidemiological and clinical data, this role of Cp in elderly individuals with low Hb levels (when it is not associated with iron or vitamin deficiency) might suggest that the supplementation of copper could help to restore the concentration of the erythrocyte protein within the normal range. Indeed, dietary copper is a wellknown activator of Cp stability, turnover, and mostly iron oxidizing activity [8, 15, 30]. Undoubtedly, care has to be taken to avoid excessive supplementation of this trace element in order to avoid toxicity events. We acknowledge that our study has some limitations. First, the design of the study was cross-sectional, thereby precluding our ability to establish any cause/effect relationship between sFeOx and Hb. A longitudinal approach could be more valuable to ascertain the real nature of this link. Second, the small sample size may have affected the reliability and the clinical significance of the observed associations. Third, we are aware

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that besides vitamin B12, folic acid, ferritin, and copper, there are several other determinants of Hb and sFeOx that should be assessed. For instance, low levels of the iron carrier, transferrin, could have negatively influenced the postulated effect of sFeOx on Hb production. Moreover, although serum ferritin is widely regarded as an excellent marker of iron deficiency/ overload [31], a more accurate data of iron status could have been achieved with the evaluation of total iron and, mostly, transferrin saturation. Finally, the assessment of a marker of inflammation such as high sensitivity C-reactive protein (hsCRP) might be important to rule out any possible confounding effects of systemic inflammation on our statistical results. Relevant to this regard, it is well-acknowledged that an inflammatory state, even at low grade, is able to stimulate the hepatic production of Cp [32]. At the same time, systemic inflammation, even in the absence of underlying autoimmune disorders or infections, has been frequently found in association with anemia, in particular in the elderly [33]. It remains to be clarified if inflammation does influence merely the synthesis of the protein or also interferes with its enzymatic activity, as suggested by two studies that showed reactive oxygen species-induced inactivation of Cp [34, 35].

Conclusion In our opinion, this study suggests that, at least in the elderly, circulating ferroxidase enzymes or polyanions might mediate the flux of iron toward erythropoietic tissues. Moreover, the observed diphasic relationship between sFeOx and Hb might be a clue that Cp and non-Cp ferroxidases have different Bspecific weight^ in sFeOx, with Cp more prone to act in stress-like conditions (low oxygenation). However, we are truly aware that further experiment al and clini calepidemiological studies are needed to support the present findings and to corroborate our speculations. In particular, future efforts should be addressed to elucidate the real quantitative impact of sFeOx on Hb levels and a role of Cp as possible player in the prevention of anemia and its clinical sequelae. Acknowledgments The authors want to thank Monica Squezanti, Manuela Spagnolo, and Juana M Sanz for their meaningful contribution in data collection and elaboration. Compliance with ethical standards This study conforms to the Code of Ethics of the World Medical Association (Declaration of Helsinki) and was conducted according to the guidelines for Good Clinical Practice (European Medicines Agency, http://www.ema.europa.eu). Signed informed consent, which was written in compliance with local and national ethical guidelines, was obtained from each patient prior to the inclusion into the study. Personal data and medical history were collected by a structured interview from participants. Funding The study was supported by BLocal Research Project^ grant from University of Ferrara.

Ann Hematol (2016) 95:1333–1339 Conflicts of interest The authors declare that they have no conflict of interest.

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