Saliva and serum ghrelin and obestatin in iron deficiency anemia patients

Introduction

Iron deficiency anemia (IDA), also known as sideropenic anemia, is one of the most common diseases in infancy and adults [1]. It is associated with a reduced quality of life [1], [2]. This disease occurs when there is insufficient intake of iron-containing foods or insufficient absorption of iron or chronic bleeding, and hemoglobin (Hb), which bears iron, cannot be formed. The cost of caring for a patient with IDA continues to grow with the increasing incidence of the diseases [1]. In the US, between 2 and 5% of adolescent females have IDA, while in developing countries [2] and in Turkey [3], IDA is much higher, with estimates of up to 10% of the populations. One to two milligrams of dietary iron intake is enough to maintain iron homeostasis [1]. A number of other studies also exhibited hormonal alterations in subjects with IDA [4], [5]. Further, more recently, Isguven et al. [6] and Akarsu et al. [7] reported that total ghrelin decreased with IDA and concluded that appetite loss in iron deficiency is a result of decrement of appetite hormone ghrelin [6], [7].

Ghrelin is a growth hormone secretagogue lipo-peptide hormone secreted from numerous human and mouse organs such as stomach [8] (reviewed [9]), kidney [10] and teeth [11]. This hormone is composed of 28 amino-acids and is mainly found in two forms in biological fluids. One of its forms bears caprylic acid on the third amino-acid serine at the N-terminal side, known as acylated (active form) ghrelin. The other form is des-acylated, which does not bear caprylic acid, and also known to be the inactive form of this peptide hormone (reviewed [9]). Ghrelin levels have been extensively studied in various diseases such as preeclampsia [12], obesity (decreased [13], [14], [15]), cancer tissues (lost [10], [16], decreased [10], [16] or increased [17]), diabetes (decreased [18]), anorexia nervosa (increased [19]) and hepatic and renal diseases (increased [20]). Besides other physiological functions, acylated ghrelin’s well-known effects are stimulating food intake and growth hormone secretion [9], [21]. The other form of ghrelin (des-acylated ghrelin) is not totally active [21], as it causes cell proliferation.

Obestatin is another peptide hormone of the endocrine system that is produced, like ghrelin, by cells lining the stomach [22], the small intestine, kidney and salivary and mammary glands of humans (reviewed [9]). Both peptides are cleaved from preproghrelin. Obestatin was considered an antagonist of ghrelin [22]. However, several recent studies performed in rats and mice under various experimental conditions did not show it as an antagonist of ghrelin [23] (reviewed [24]). Further studies showed that obestatin was also involved in inhibiting thirst, improving memory and affecting cell proliferation (reviewed [24]). As indicated above, ghrelin and obestatin might play a crucial role or roles in a quite diverse range of physiologic functions in diseases, including IDA. As it has been shown that ghrelin decreased in children with IDA, it was concluded that loss of appetite was a result of decreased ghrelin [6], [7]. However, in both groups in those studies [6], [7], they measured only total ghrelin in children with IDA and did not follow up with iron supplementation, whereas appetite stimulation could be achieved with acylated ghrelin [21]. Thus, it was necessary to measure acylated ghrelin rather than total ghrelin if a relationship between ghrelin and appetite loss is to be found. Also, to the best of our knowledge, no study has investigated acylated ghrelin and obestatin secretetion, or their relationship to hematological parameters in adult females with IDA. In the present study, therefore, our goals were to assess acylated ghrelin and obestatin concentrations in adult females with IDA before and after iron supplementation. The association between both hormones and hematological parameters was also explored.

Materials and methods

Informed consent was obtained from all volunteer participants after the approval (meeting number: 13, issue no.: 03 and dated 02.02.2016) of the local Ethics Committee School of Medicine of Firat University. We have complied with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects. The study included 30 female patients with IDA (mean age 31.7±10.7 years) who applied to the outpatient clinic of the Internal Medicine Department in the Firat University Hospital, and 30 age- and sex-matched healthy individuals (mean age 30.2±8.0 years). Patients’ Hb, hematocrit (Hct), red blood cell (RBC) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), serum iron and ferritin levels and total iron-binding capacity (TIBC) were measured. Low Hb, Hct, RBC count, MCV, MCH, MCHC, serum iron, ferritin levels and high TIBC were taken as the criteria for an iron-deficient patient.

At the beginning of the study, none of the participants used ferroglycine sulfate. However, later, patients with IDA were orally treated with ferroglycine sulfate 568 mg (Ferro-Sanol^® duodenale capsule, Adeka, İstanbul, Turkey) daily for 1 month. For the control group, the following criteria were considered: age and body mass index (BMI) matched with the patients; no use of drugs, no diabetes and/or family history of diabetes, not taking alcohol and no use of tobacco product (past or present); no gastrointestinal disease in the individual or in the family; no operation performed in the gastrointestinal system; no liver or kidney dysfunction, not walking more than 1 km a day; no history of obesity, no health complaints during the previous month; and, for women, being in the luteal phase. The patient group included subjects who met the control group criteria, except for having an IDA. Venous blood (5 mL) and saliva (1 mL) samples were taken before treatment after an overnight fast, and also 1 week and 1 month after drug treatment. Samples were collected once only from the control subjects. Saliva was taken into a clean container containing 500 KIU/mL aprotinin, as previously described [25]. Each saliva sample was also treated with 1/10 vol 1 N HCl and stored at −20°C pending measurements of ghrelin. Each blood sample was divided into two portions: one half was put into tubes containing 500 KIU/mL aprotinin and the other half into untreated tubes. The tubes were centrifuged for 5 min at 1792 g to separate the serum, which was treated with 1/10 (vol/vol) 1 N HCl and stored at −20°C pending measurements of ghrelin and obestatin levels. Untreated tubes were used to analyze the other biochemical parameters (Hb, Hct, ferritin, TIBC). Hb, Hct, ferritin and TIBC levels were analyzed on the same day in an Olympus AU2700 auto analyzer using Olympus kits (Olympus Corp., Tokyo, Japan). Serum and saliva obestatin levels were determined by enzyme immunoassay (EIA) in an enzyme-linked immunosorbent assay (ELISA) reader (TRITURUS, Barcelona, Spain) using a kit (EIA, S-1285; Peninsula Laboratories LLC, CA, USA). Serum and saliva ghrelin levels were also measured by ELISA in an ELISA reader using a sensitive human ghrelin ELISA kit (SPI; Cat# A05119, Montigny le Bretonneux, France). Saliva “assay validity” test (linarites, recovery, inter and intra assay values) of the above-mentioned kits was previously tested as specified by Aydin’s detailed study [18]. Intra-assay coefficient of variation (CV) value and inter-assay CV value for ghrelin and obestatin were 10%, 12% and 8%, 10%, respectively.

Results

The hematological findings and demographic characteristics of healthy and IDA individuals are presented in Table 1. Patients’ Hb, Hct and ferritin levels remarkably increased while TIBC decreased when compared with the initial levels. Subjects’ glucose values were 84.1±3.20 mg/dL (controls); 91.44±12.20 mg/dL (before treatment); 87.07±7.90 mg/dL (after treatment). Subjects’ BMIs were 24.12±4.80 (controls); 23.51±5.82 (before treatment); 24.27±5.64 (after treatment). When weight after treatment was compared, an increase was seen in the BMI of patients compared with their weight at the start, but again this was not statistically significant. Saliva and serum ghrelin and obestatin levels were measured at the start, after 1 week and 1 month later. Serum and saliva ghrelin and obestatin levels were significantly lower than the control values (p<0.001) (Figures 1–4). Serum ghrelin and obestatin concentrations increased with iron treatment when compared with the initial values, but this increment was not statistically significant (Figures 2 and 4). Even though patients gained some weight, ghrelin tended to increase, but once again this increment was not statistically significant. While it is known that ghrelin levels reduce obesity [13], [15], an exception is the Prader-Willi syndrome where increased ghrelin levels seem to be involved in the development of an obese phenotype [26]. Saliva ghrelin and obestatin increased with treatment when compared with the control (basal) values, but this increase was not statistically significant (Figures 1 and 3).

Figure 1:

Saliva ghrelin levels before treatment and after treatment.

Values are presented as mean±SD. p<0.05 vs. corresponding control vs. before and after treatment.

Figure 2:

Serum ghrelin levels before treatment and after treatment.

Values are presented as mean±SD. p<0.05 vs. corresponding control vs. before and after treatment.

Figure 3:

Saliva obestatin levels before treatment and after treatment.

Values are presented as mean±SD. p<0.05 vs. corresponding control vs. before and after treatment.

Figure 4:

Serum obestatin levels before treatment and after treatment.

Values are presented as mean±SD. p<0.05 vs. corresponding control vs. before and after treatment.

Discussion

Iron is a necessary element for all cells and plays an important role in energy metabolism, gene regulation, neurotransmitter synthesis, cell growth and differentiation, cofactor of enzymic reactions (e.g. dehydratases), protein synthesis, oxygen binding and transport (Hb and myoglobin) [1], [27], [28]. Its absorption primarily occurs in the duodenum, and is then used for Hb synthesis, mitochondrial enzymes and cytochromes-heme containing proteins that promote oxidative phosphorylation within the mitochondria. Hb in RBCs carries oxygen to the tissues from the lungs. But if the iron levels are too low, the number of RBCs and Hb production fall below normal, resulting in IDA [1]. This anemia is also associated with some bioactive peptide hormonal changes [4], [7]. In this study, both saliva and serum basal hormone acylated ghrelin concentrations in subjects with IDA were significantly lower when compared with those in the control group without IDA. After iron supplementation, ghrelin concentration tended to increase compared with their basal ghrelin concentrations. These results are in agreement with the findings by Isguven et al. [6] and Akarsu et al. [7], who previously noted that total ghrelin was decreased in children with IDA. Like them, we also assumed that loss of appetite correlated with a decreased level of ghrelin in adult females with IDA. But we measured acylated ghrelin rather than total ghrelin amount they had measured. While appetite stimulation in organisms is due to acylated ghrelin, it is more important to distinguish acylated ghrelin from total ghrelin before making the above assumption [21] (reviewed [9]).

We also observed that serum glucose concentrations were slightly higher in patients with IDA than controls. Iron absorption is increased by the presence of glucose, so increased glucose in patients with IDA might be a compensatory mechanism that triggers increased iron absorption. Our glucose results are in agreement with those obtained by Coban et al., who reported that, before iron treatment, the mean HbA_1c level in patients with IDA was higher than in the healthy control group [29]. It is well known that when glucose increases, ghrelin decreases [18], making it possible that this ghrelin decrement with IDA might be a result of increased glucose increment, and therefore gives another explanation why ghrelin decreases with IDA.

Ghrelin was originally recognized via its role in growth hormone releasing (hence its name), but it has become evident that it has many roles, including appetite stimulation, gastric motility and acid secretion [8]. Gastric acid secretion has also been demonstrated to facilitate iron absorption (reviewed [30]). Also, ghrelin significantly increased H(+)-K(+)-ATPase activity of gastric mucosal cells [31]. If this is so, decreased ghrelin may not increase gastric acid secretion. Hence, the low iron level observed with IDA in our investigation might be a result of a low concentration of ghrelin. The low iron level might be a result of insufficient iron in the diet, along with impaired uptake by the gastrointestinal systems. We found that the ferritin level increased with iron supplementation, as expected, even though ghrelin tended to increase with this supplementation; however, the increase was not statistically significant. Superoxide radicals can be generated from free iron-liberated ferritin, which then catalyze the production of further hydroxyl radical by increasing the amount of free iron [27]. A similar effect can occur with Fe-S proteins in superoxide dismutase (SOD) deficiency, with the degradation of mitochondria by radical damage leading to further production of radicals and resulting in oxidative stress [32]. This observation is in agreement with that by Day et al., who observed that chronic iron administration elevates vascular oxidative stress [27]. Here, ghrelin seems to be highly effective in protecting against reactive oxygen species (ROS) because of its antioxidant properties that upregulate free-radical scavenger [33], and one that appears to involve anti-inflammatory activity [34]. Therefore, the low ghrelin (even after iron supplementation) might be consistent with the view that the body is attempting to deal with these problems by consuming ghrelin [33]. Hydroxyl radical, produced by the iron-catalyzed Haber-Weiss reaction, might also damage gastric mucosal cells [27], [35]. This hormone is produced mainly by P/D1 cells lining the fundus of the stomach (reviewed [33]). If, as assumed, gastric mucosal cells (the main producers of ghrelin) are damaged by ROS, this might reduce ghrelin secretion. However, further investigations will be necessary to determine whether there is a link between ROSs, Fe metabolism and ghrelin level.

We also observed that both saliva and serum obestatin were clearly lower in IDA patients compared with the control subjects; and after iron supplementation, obestatin concentration tended to increase, like ghrelin, even though serum obestatin showed fluctuations when compared with the basal obestatin level after 1 week (slightly decreased) and 1 month (slight increase). Serum obestatin and ghrelin are produced by many organs, including kidney and liver, although most is produced by P/D1 cells lining the fundus of the human stomach and X/A cells of rats [8]. Serum obestatin could be lowered as a result of changes in the synthesis in the stomach. This assumption is supported by decreased ghrelin, a peptide derived from the same prohormone. Both hormones are encoded by the same gene [24], therefore parallel increases or decreases in ghrelin and obestatin can be expected, as reported here. In the case of obestatin, we have not found a similar study in IDA to compare our results.

Our investigation has the following limitations. First, the limiting factor was the small sample size and the restriction of the measurements to 1 month. A longer follow-up period would be useful to determine more accurately how hormonal levels change with iron treatment. Furthermore, total anti-oxidant capacity should also be measured in patients with IDA because ghrelin shows antioxidant properties.

Conclusions

Briefly, we can conclude that ghrelin and obestatin can be analyzed in serum and saliva by using ELISA. Testing these hormones in saliva is simple and non-invasive. Serum monitoring in this disease could be replaced by measuring saliva level of these hormones. Ghrelin and obestatin might affect intestinal absorption of iron and mobilization from hepatic stores, where ghrelin and obestatin are also expressed. A significant decrease in ghrelin and obestatin is probably the consequence of fundus damage following iron treatment. For this reason, determination of ghrelin and obestatin concentrations may also reflect iron storage of the body, and might possibly be an indicator of damage to the stomach, as well as being an indicator of appetite loss in iron-deficient women. However, further research involving a larger prospective study is needed to elucidate the mechanisms leading to decreased serum ghrelin and obestatin in IDA.