Pii: s0271-5317(01)00313-x

Iron supplementation prevents the development of iron deficiency in rats with omeprazole-induced Edemilson Cardoso da Conceic¸a˜o, M.S.a,1, Tadao Shuhama, Ph.D.b, Clarice Izumi, Ph.D.c, Osvaldo de Freitas, Ph.D.a,c,* Departamentos ade Cieˆncias Farmaceˆuticas bFisica e Quı´mica, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo, cCentro de Quı´mica de Proteı´nas, Faculdade de Medicina de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Received 10 November 2000; received in revised form 14 April 2001; accepted 17 April 2001 Abstract
Gastric acidity is an important luminal factor for non-heme iron absorption. The effect of iron supplementation (1 mg Fe/kg body weight) was studied in rats submitted to hypochlorhydria by dailyoral administration of omeprazole (40 ␮mol/kg). Forty (40) rats received omeprazole (experimentalgroup) and 20 rats received vehicle (control group) for 4 weeks. At the end of this period, 10 animalsfrom each group were sacrificed. The remaining rats in the control group continued receiving vehiclealone for 2 additional weeks. The experimental group was divided into three subgroups of 10 rats each.
One subgroup received omeprazole alone, and the other subgroups received omeprazole plus ironsupplementation with iron sulphate (Feϩ2) or iron-peptide complex (Feϩ3) for 2 additional weeks.
After 4 weeks of treatment, the group that received omeprazole presented an increase of serumtransferrin and a decrease of hepatic iron levels. However, only after 6 weeks did a decrease ofhaemoglobin occur in this subgroup. Supplementation started during the 5th week prevented thedecrease of haemoglobin, improved the transferrin levels but did not cause hepatic iron to return tocontrol levels. These results suggest that iron deficiency due to hypochlorhydria could be preventedby iron supplementation and that the two iron sources were equally efficient in this respect. 2001Elsevier Science Inc. All rights reserved.
Keywords: Omeprazole; Hypochlorhydria; Iron supplementation; Iron deficiency * Corresponding author. Tel./fax: ϩ55-016-602-4289.
E-mail address: [email protected] (O. de Freitas).
1Present address of ECC is Curso de Farma´cia, Universidade Federal de Goia´s, Goiaˆnia, GO, Brazil.
0271-5317/01/$ – see front matter 2001 Elsevier Science Inc. All rights reserved.
PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 3 1 3 - X E. C. da Conceic¸a˜o et al. / Nutrition Research 21 (2001) 1201–1208 1. Introduction
Human anaemia may be related to iron deficiency caused by ingestion of an unbalanced diet or by the low bioavailability of ingested iron from diets with apparently adequateamounts of iron. Iron deficiency may also be associated with physiological processes such aspregnancy, menstruation, or growth, or with pathological processes such as haemorrhage,malabsorption syndrome and hypochlorhydria [1,2].
Gastric acidity is considered to be an important and necessary luminal factor for non-heme iron absorption [2– 4]. Substances from the diet that form soluble low molecular weightcomplexes with iron (ascorbic acid, amino acids and sugars) tend to facilitate iron absorptionin the neutral to alkaline pH milieu of the duodenum. In the case of ascorbic acid, a solublecomplex (ascorbate) is formed and ferric iron is partially reduced to ferrous iron, which ismore soluble [2,5,6]. This situation prevents the precipitation of ingested iron at the duodenalpH. On the other hand, a low gastric secretion output increases gastric pH, thus decreasingthe rate of iron dissociation from food complexes and favouring the formation of insolublecomplexes [3,7].
The reduced secretion of gastric acid may be the result of atrophy of the gastric glands in the mucosa of the stomach fundus. The decrease in secretion is proportional to the decreasein parietal cell mass. In atrophic gastritis, hypochlorhydria is caused by a loss of gastricglands. Approximately 30% of elderly people (Ͼ60 years old) are affected by hypochlorhy-dria, and the prevalence of atrophic gastritis increases with age [8,9].
Additionally, hypochlorhydria may be the consequence of treatment with drugs that affect gastric acid secretion, such as a histamine H receptor antagonist, prostaglandin analogues and proton pump inhibitors. The latter compounds are widely used in the therapeutictreatment of gastric ulcer, dyspepsia, esophageal reflux and hypersecretory situations [10,11].
When no response to histamine H receptors is obtained, omeprazole is utilised [12,13].
The chronic hypochlorhydria occurring after atrophic gastritis or therapeutic treatment may promote iron deficiency and iron deficiency anaemia. In this situation, iron supplemen-tation with a Feϩ3-peptide complex may prevent the development of iron deficiency. TheFeϩ3-peptide complex shows a solubility behaviour opposite to that of iron sulphate, i.e., itis soluble at acid pH and totally soluble in neutral to alkaline pH, keeping the ironcomplexed. Thus, it is expected that the iron absorption from the Feϩ3-peptide complex maybe independent of the gastric acid environment. In the present study we investigated the ironstatus of rats submitted to long-lasting treatment with omeprazole and the effect of ironsupplementation with Feϩ3-peptide complex or iron sulphate on these animals.
2. Material and methods
Omeprazole (20 mg/capsule, Merrel Lepetit) was purchased on the local market. Hy- droxyethylcelullose was from Union Carbide (Sa˜o Paulo, SP, Brazil). NaHCO O (19.34% iron) were from Merck (Darmstadt, Germany). Feϩ3-peptide complex E. C. da Conceic¸a˜o et al. / Nutrition Research 21 (2001) 1201–1208 containing 4.0% iron was prepared in our laboratory. The biochemical analyses wereperformed using commercial kits for serum iron from Companhia Equipadora de Laborato´-rios Modernos (Sa˜o Paulo, SP, Brazil), for haemoglobin from Labtest (Sa˜o Paulo, SP,Brazil), and for serum transferrin from APES Associates (Brussels, Belgium). Water wasMilli-Q purity and other reagents were of analytical grade. Standard animal diet was fromCentro Interunidades de Zootecnia Industrial (CIZIP), University of Sa˜o Paulo (Pirassu-nunga, SP, Brazil), and contained 23.10% protein, 47.90% carbohydrates and 4.45% lipids.
They were from the animal house of the Ribeira˜o Preto Campus, University of Sa˜o Paulo, and were maintained in accordance with the Guide for the Care and Use of LaboratoryAnimals [14]. Male Wistar rats (170 –225 g) with haemoglobin levels Ն12 ␮g/dL [15] werehoused in individual stainless-steel metabolic cages in a room with a 12 h light/dark cycle,at 25°C Ϯ 1°C. The animals were fed a normal diet containing 158 mg iron/kg diet andreceived tap water ad libitum. The animals were used in the experiments after a 3 dayadaptation period. In all experiments, animals were dosed by gastric gavage, in a volume ofless than 5% of their body weight. Some animals died during the experiment due to wrongadministration of gavage.
2.3. Preparation of omeprazole suspension The suspension was prepared according to Larsson et al. [16]. Briefly, the omeprazole granules were ground into a powder which was dispersed in vehicle (0.25%, w/v, hydroxy-ethylcellulose 4400 in 0.1 M sodium bicarbonate, pH 7.4).
2.4. Determination of basal pH and after dosing with omeprazole, vehicle and thecomponents of vehicle Animals were divided into 4 groups with homogeneous body weight and fasted for 12 h, while receiving 0.9% (w/v) NaCl containing 5% (w/v) glucose ad libitum. The animalsreceived 5.0 ml of MilliQ water by gastric gavage, and the stomach content was aspiratedimmediately after the procedure and its pH was determined. This value was considered as abasal pH. Then, each group received 2.5 ml of: a) Milli-Q water; b) vehicle consisting of0.25% (w/v) hydroxyethylcellulose 4400 in 0.1 M sodium bicarbonate, pH 7.4; c) 0.1 Msodium bicarbonate, pH 7.4; and d) vehicle ϩ omeprazole (corresponding to 40 ␮mol/kgbody weight). After 2 h, they received 5.0 ml of Milli-Q water and the stomach content wasaspirated and its pH determined.
Other rats were separated into two groups (control—n ϭ 20, and experimental—n ϭ 40), and orally dosed daily for 4 weeks with either omeprazole (dose corresponding to 40␮mol/kg body weight) dispersed in vehicle (experimental group) or with vehicle alone E. C. da Conceic¸a˜o et al. / Nutrition Research 21 (2001) 1201–1208 (control group). At the end of this 4 week period, after a 12 h fasting period, a blood samplewas obtained from the tail of each animal for haemoglobin and hematocrit determination.
Then, 10 rats from each group were sacrificed at random after superficial ether anaesthesiaand blood was collected by cardiac puncture. The liver was collected, immediately washedwith cold 0.9% (w/v) NaCl, and dried with paper towels. A liver fragment (Ϯ2.0 g) wasexcised and kept in borosilicate tubes at Ϫ20°C until the time for processing for tissue irondetermination. The serum was obtained after centrifugation and maintained at Ϫ80°C untilthe time for serum iron and transferrin level determination. The remaining rats continued tobe used in the experiment for an additional 2 weeks, receiving vehicle alone (control group,n ϭ 10), omeprazole alone (n ϭ 10), or omeprazole plus iron supplementation with 1 mgFe/kg body weight of Feϩ2 from FeSO (n ϭ 10), or Feϩ3 from an iron peptide complex (n ϭ 10) 2 hours after omeprazole administration. At the end of the experiment, all animals weresacrificed after a 12 h fasting period, and blood and liver were collected as described above.
Food ingestion was determined daily and weight gain weekly, throughout the experimentalperiod.
Haemoglobin was determined by the cyanomethaemoglobin method [17]. Serum iron was determined by a colorimetric method using ferrozine, pyridyl-phenyl sulfonic acid triazine[18]. Serum transferrin was determined by turbidimetry using human anti-transferrin anti-body. Hepatic iron was determined by atomic absorption spectroscopy after digestion underflame of a liver tissue sample with H SO and H O .
The data were analysed by Anova followed by the Duncan test (significance level of p Ͻ 0.05), using the Statistica for Windows software, version 4.5.
3. Results
Basal gastric pH (mean Ϯ SEM) ranged from 3.56 Ϯ 0.11 to 2.81 Ϯ 0.25. Two hours after the administration of Milli-Q water, 0.25% (w/v) hydroxyethylcellulose 4400 in 0.1 Msodium bicarbonate, pH 7.4, or 0.1 M sodium bicarbonate, pH 7.4, the gastric pH rangedfrom 2.64 Ϯ 0.13 to 3.24 Ϯ 0.27. The group treated with omeprazole at 40 ␮mol/kg bodyweight presented a gastric pH of 5.19 Ϯ 0.47 compared to 2.64 Ϯ 0.13 for the control group(Table 1).
Under the present experimental conditions daily oral administration of omeprazole (40 ␮mol/kg body weight) for 4 or 6 weeks did not alter food ingestion or body weight gain (datanot shown). No differences in serum iron (Table 2) or hematocrit were observed between theomeprazole-treated group and the controls throughout the experimental period (4 or 6 weeks)(data not shown). Also, no differences were observed in food ingestion or body weight gainbetween groups throughout the experimental period (data not shown).
E. C. da Conceic¸a˜o et al. / Nutrition Research 21 (2001) 1201–1208 Table 1Values of rat gastric basal pH and pH two hours after administration of MilliQ water (A), 0.25% (w/v)hydroxyethylcellulose 4400 in 0.1 M sodium bicarbonate, pH 7.4 (B), 0.1 M sodium bicarbonate, pH 7.4 (C),or 0.25% (w/v) hydroxyethylcellulose 4400 in 0.1 M sodium bicarbonate, pH 7.4 ϩ omeprazole(corresponding to 40 ␮mol/kg body weight) (D) Data are expressed as mean Ϯ SEM. The number of rats in each group is given in parenthesis.
After 4 weeks of treatment there was a significant (p Ͻ 0.05) increase of serum transferrin and a decrease of hepatic iron levels in the group treated with omeprazole (OMP) comparedto the control group receiving vehicle alone, although there were no differences in haemo-globin, serum iron (Table 2) or hematocrit (data not shown).
After 6 weeks, in addition to the increase in serum transferrin and the decrease in hepatic iron levels, already detected after 4 weeks, there was a decrease of haemoglobin levels in thegroup which received omeprazole compared to the control group (control 6 weeks). Hae-moglobin and serum transferrin levels, but not hepatic iron levels, in the groups supple-mented with iron (OMP ϩ Feϩ2 or OMP ϩ Feϩ3) returned to control group values. Therewas no difference between iron sources.
4. Discussion
No technique or animal model is perfect for the study of bioavailability of compounds for therapeutic or nutritional use. The rat model is limited as a model for human beings bydifferences in eating behaviour, energy expenditure per body area and heme iron absorption Table 2Effect of iron supplementation on iron metabolism parameters in rats on long-term treatment with omeprazole Data are expressed as mean Ϯ SEM. The number of rats in each group is given in parenthesis. The groups were supplemented with 1 mg Fe/kg from iron sulphate (Feϩ2) or iron peptide complex (Feϩ3). Different letters insuperscript in the same column represent a statistically significant difference during the same experimental period(Anova followed by the Duncan test, p Ͻ 0.05). OMP ϭ omeprazole; Feϩ2 ϭ FeSO ; Feϩ3 ϭ Feϩ3-peptide E. C. da Conceic¸a˜o et al. / Nutrition Research 21 (2001) 1201–1208 [15]. However, data obtained in parallel mineral studies on humans and rats were generallyconsistent [19].
In the present study, there were no significant differences in body weight gain between omeprazole-treated (with or without iron supplementation) and control groups, suggestingthat the treatment did not promote significant biochemical or physiological alterationsbeyond hypochlorhydria, allowing us to use this model for the evaluation of the effect ofhypochlorhydria on iron absorption.
Previous studies have shown that omeprazole administration (40 ␮mol/kg) is effective in inhibiting acid gastric production in the stomach without affecting other gastric functions[16,20,21]. In our study, the mean gastric pH of the rats 2 hours after omeprazole admin-istration was 5.2 Ϯ 0.47, similar to that reported for human beings after administration of 30mg/day omeprazole (86.85 ␮mol/day) [22].
Long-term treatment with omeprazole (40 ␮mol/kg body weight/day) promoted an in- crease in serum transferrin and a decrease in hepatic iron levels during the initial phase (4weeks). After 6 weeks, there was also a decrease of haemoglobin, which, however, did notcorrespond to anaemia (haemoglobin Ͼ12 mg/dL) but rather iron deficiency, that could beattributed to the hypochlorhydria induced by long term treatment with omeprazole. The otherparameters did not change.
Iron deficiency develops over 3 phases [23]. The first is characterised by iron stock depletion (decrease in serum ferritin); in the second, there is a decrease in iron transport(increased total iron binding capacity and decreased serum iron); in the third, there is areduction in haemoglobin biosynthesis (iron deficiency anaemia) [5,15]. Thus, we expecteda decrease of serum iron in the omeprazole-treated group, but this did not occur, possibly dueto the 12 h fasting period before sacrifice.
The iron supplementation (Feϩ2 or Feϩ3) started during the 5th week prevented the decrease of haemoglobin and normalised the serum transferrin levels compared to the controlgroup (vehicle). The lack of recovery of hepatic iron levels in the iron supplemented groupscould be attributed to the short-term supplementation. Both iron sources presented similarefficacy, except for hepatic iron, contrary to our initial hypothesis based on the differentsolubility characteristics of iron compounds. In vitro, iron sulphate is soluble at acid pH andthe iron-peptide complex is insoluble; at neutral to alkaline pH, the iron-peptide complex issoluble and iron sulphate is not, but in the 2–9 pH range, iron was present in the complexedform in the iron-peptide complex (Chaud et al., unpublished results). On the basis of thissolubility behaviour, we expected the iron from the iron-peptide complex to be more efficientsince its solubility is independent of acid pH. Probably the inhibition of gastric acid secretionby omeprazole was partial or the increase of intestinal iron solubility promoted by gastricacidity could be more important when the iron absorption is increased by iron deficiency.
These factors could explain the similar efficacy of both iron sources.
In humans with chronic corporal atrophic gastritis, which presented total absence of gastric acid secretion, the oral administration of ferrous acetyl transferrin (40 mg, twice aday) increased serum iron and reduced serum transferrin after a short-term treatment [24].
Hypo/achlorhydria is a relatively common occurrence in patients with chronic gastritis or gastrectomy and in atrophic gastritis which develops in the elderly since the prevalence ofhypochlorhydria increases with age [9]. On the other hand, the use of acid gastric secretion E. C. da Conceic¸a˜o et al. / Nutrition Research 21 (2001) 1201–1208 inhibitors (cimetidine, omeprazole) is very frequent in the treatment of peptic ulcer diseaseand reflux esophagitis, although these conditions are not considered as possible causativefactors in iron deficiency anaemia [25].
In conclusion, our results suggest that the hypochlorhydria due to long-term treatment with omeprazole at doses similar to those administered to humans with gastrointestinaldiseases reduces iron absorption from food in rats. This reduction induced iron deficiencythat could be prevented by Feϩ2 (iron sulphate) or Feϩ3 (iron-peptide complex) supplemen-tation.
Acknowledgments
This work was partially supported by fellowships from CAPES (ECC) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (OF).
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