Effectiveness of treatment of iron deficiency anemia in rats with squid ink melanin–Fe

Abstract

Iron deficiency anemia (IDA) is the one of the most common nutritional problems and is encountered all over the world. This study analysed the effects of squid ink melanin–Fe (SM–Fe) on IDA in rats. Forty weanling SD male rats were used and thirty-two rats were fed an iron-deficient diet for 4 weeks. Then SM–Fe (dosages of iron is 6 mg kg⁻¹ BW) was given to the IDA rats once a day for 3 weeks by intragastric administration, with FeCl₃ and FeSO₄ (dosages of iron is 6 mg kg⁻¹ BW) as positive controls. While the IDA model group and the control group were administrated distilled deionized water each day for 3 weeks. The content of haemoglobin (Hb), serum iron (SI), total iron binding capacity (TIBC), serum ferritin (SF), transferrin receptor (sTfR), erythropoietin (EPO), and iron content in the liver and spleen were measured. The results showed that the content of Hb, SI, SF, EPO, iron content in the liver and spleen were significantly increased in the iron supplement groups (SM–Fe, FeCl₃ and FeSO₄) compared with the model group (P < 0.05), while TIBC and sTfR were significantly decreased in the iron supplement groups compared with the model group (P < 0.05). In comparison with the FeCl₃ and FeSO₄ groups, a higher bioavailability of iron and fewer side effects were observed in the SM–Fe group. The present study indicated that SM–Fe is an effective source of iron supplement for IDA rats and might be exploited as a new iron fortifier.

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Introduction

Iron deficiency anemia (IDA) is one of the highest incidence nutritional-deficiency diseases, which is encountered all over the world, especially in infants and children. The World Health Organization (WHO) estimates that 39% of children younger than 5 years, 48% of children between 5 and 14 years, 42% of all women, and 52% of pregnant women in developing countries are anemic,¹ with half having IDA.² According to the WHO, the frequency of iron deficiency in developing countries is about 2.5 times that of anemia, which is not iron deficiency related.¹

IDA can result in a wide variety of adverse outcomes including reduced psychomotor development in infants,³ poor cognitive and motor development in children,^4,5 poor pregnancy outcomes,⁶ decreased immune function,⁷ tiredness and poor work performance.⁸

IDA is presently one of the most important nutritional problems that remains to be solved. Targeted iron supplementation, iron fortification of foods, or both, can control iron deficiency in populations.⁹ Several iron compounds have been used with this preventive objective. One of them, ferrous sulfate contains a high iron content with better absorption, and to a certain extent, may help relieve the symptoms of iron deficiency and anemia, it causes many adverse reactions, such as diarrhoea, epigastric discomfort and constipation.¹⁰ Iron bioavailability and side effects from its therapeutic use have stimulated studies on other iron compounds.

A squid contains a 1.3% ink sac of its total weight. About 300 to 400 thousand tons of squid are processed each year in China and a considerable portion are processed in Zhoushan of the Zhejiang province. The ink sac is discarded, which pollutes the environment and depletes a potentially highly useful resource. It is documented that squid ink has many biological properties, such as immunity regulation, anti-tumor, anti-oxidant, anti-radiation and anti-bacterial activities.^11–13 The main components of squid ink are melanin and a non-sulfated glycosaminoglycan.¹⁴

Melanin accounts for about 15.5% of fresh squid ink, and is a highly irregular polymer consisting of an indole structure,¹⁵ which can resist free radical chain reactions as an acceptor of free radicals,^16,17 and chelate many kinds of metal ions.^18,19 Since squid melanin is a natural product and can be combined with a high content of Fe, it may be developed as a safe and efficient iron supplement. Therefore, in this study, high-purity squid ink melanin (SM) was extracted from squid ink sac, and squid ink melanin–Fe (SM–Fe) was prepared, and the objective of this investigation was to compare the effects of SM–Fe, FeCl₃ and FeSO₄ on IDA in rats and to determine whether SM–Fe can effectively normalize the body iron status in IDA rats. The possible mechanism of SM–Fe on the effects was studied as well.

Materials and methods

Preparation of the SM–Fe

The ink was obtained from squid (Ommastrephes bartrami) from Zhou-Shan Fishery Company (Zhejiang, China) and stored at −40 °C before use. The melanin was isolated and purified by high-speed centrifugation.²⁰ Then melanin was added into 10 mmol L⁻¹ FeCl₃ solution at the ratio of 1 : 250 (w/v) followed by slow stirring at 4 °C for 24 h. The mixture was centrifuged at 8000g for 30 min, and crude SM–Fe was collected. It was then purified by successive washings with distilled water and centrifugation, the pellet was lyophilized using a freeze dryer (see Fig. 1 for the IR spectra of the melanin and melanin–Fe). The iron content of SM–Fe was 85 mg g⁻¹, as determined by atomic absorption spectrophotometry (Perkin-Elmer AA700, USA) with an air-acetylene flame. SM–Fe was stored at −20 °C and prepared as a suspension with distilled deionized water for use.

Fig. 1 The IR spectra of the melanin and melanin–Fe. Compared to the native melanin, the intensity of the protonated NH (3200 cm⁻¹), phenolic OH (3400 cm⁻¹) and COOH groups (1710 cm⁻¹) bands decreased notably in melanin–Fe, which indicated the chemical binding of Fe ions with the melanin.

Experimental animals

All experimental procedures involving animals received the approval from the Animal Care and Use Committee of Zhejiang University. Guidelines and the Policy on the care and use of laboratory animals were followed at all times. Forty male Sprague-Dawley (SD) rats with an initial weight of 80 ± 5 g were purchased from Shanghai-Silaike Co., and were housed in stainless steel cages, in a temperature-controlled room (24 ± 1 °C, 55 ± 5% humidity) with a 12 h light/dark cycle, and distilled deionized water was provided continuously. After adaptation for 3 days, the rats were randomly divided into the control group and the IDA model groups. Eight rats were selected randomly and given normal diets as the control group. Others were given low iron diets for 4 weeks to generate an IDA animal model.^21,22 The whole experimental process was strictly controlled to avoid iron stain. Hb levels were tested weekly, and IDA was defined as Hb values less than 90 g L⁻¹.

Thirty-two IDA rats were randomly assigned into 4 groups of eight animals, each with equal mean Hb values: the IDA model group, the positive groups (FeSO₄, dosage of iron is 6 mg kg⁻¹ BW; FeCl₃, dosage of iron is 6 mg kg⁻¹ BW), SM–Fe group (dosage of iron is 6 mg kg⁻¹ BW). Rats in the positive groups were administered FeSO₄ solution or FeCl₃ solution, rats in the SM–Fe group was administered the SM–Fe suspension, while rats in the control and model groups were administered distilled deionized water (placebo). All supplements were freshly prepared every day and intragastric administration was performed once at 10:00 AM each day for 3 weeks.

Table 1 presents a summary of the iron deficient diets supplied during the experimental period. The diets and mineral and vitamin supplements provided were prepared according to recommendations of the AIN (American Institute of Nutrition), containing 5.2 mg iron kg⁻¹ diet.²²

Table 1 Ingredient composition of the IDA model diets

Ingredient	g kg^−1 diet
a AIN 76 mineral mix had no iron addition.
Cornstarch	630.0
Casein, low trace element	200.0
Fiber	50.0
Corn oil	50.0
Mineral mix^a	35.0
Sucrose	20.0
Vitamin mix	10.0
DL-Methionine	3.0
Choline bitartrate	2.0

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Tissue and blood collection

After overnight food deprivation, rats were first anesthetized with barbital sodium and blood samples were collected from the abdominal aorta into anticoagulant blood vessels for immediate Hb analysis and into centrifuge tubes for separation of the serum for future analyses. The rats were killed by decapitation and the organs were removed and weighed. The serum was separated by centrifugation at 3000g for 15 min and stored at −20 °C for future analyses. The wet weights of the heart, liver, spleen and kidneys were recorded and tissues were rapidly frozen in liquid nitrogen, and then stored at −80 °C for future analyses.

Chemical analyses

The hemoglobin (Hb) concentrations of the collected blood samples were determined by the cyanmethemoglobin method.²³ Serum iron (SI) concentrations and the total iron binding capacity (TIBC) were determined spectrophotometrically using test kits (Nanjing Jiancheng Bioengineering Inst., Nanjing, PR China). The concentrations of serum ferritin (SF), transferrin receptor (sTfR) and erythropoietin (EPO) were measured by the enzyme-linked immunoassays (ELISAs) kits (R&D Systems Inc, USA). The iron content in the rat liver and spleen were determined by atomic absorption spectrophotometry (Perkin-Elmer AA700, USA) with an air-acetylene flame. After the samples had been dried in a vacuum dryer for 48 h and wet digested with a mixture of nitric acid and perchloric acid (3 : 1, v/v). Appropriate dilutions were made by using deionized water. Iron in the diets was measured similarly without prior drying of the samples.

Statistical analysis

All the data were analysed by SPSS 18.0 software for Windows (SPSS Inc., Chicago, IL, USA). The differences between the groups among treatments in each group were determined by one-way analysis of variances ANOVA. Duncan's multiple-range tests were performed. Comparisons of the means with P values equal or less than 0.05 were considered significantly different. Data were expressed as mean ± SD.

Results

The Hb changes of rats

The Hb content in the experiment groups and the control groups were not significantly different at the beginning of the experiment (P > 0.05) (Table 2). The Hb content in the rats fed the iron-deficient diet groups were significantly lower than the control group after 4 weeks depletion (P < 0.05). Therefore, the rats fed the iron-deficient diet induced anemia. After the IDA rats were fed iron supplements (FeCl₃, FeSO₄ and SM–Fe) for 3 weeks, the Hb content approached normal values, and did not differ from the control group (P > 0.05).

Table 2 The Hb content of rats before depletion, after depletion, and after repletion^a

Groups	Dosage Fe (mg kg^−1 bw)	HB (g L^−1) (before depletion)	HB (g L^−1) (after depletion)	HB (g L^−1) (after repletion)
a a and b, means in the same row with no common superscripts differ significantly (P < 0.05).
Control	0	115.31 ± 6.01^a	125.13 ± 9.38^b	132.81 ± 13.07^b
Model	0	115.36 ± 7.42^a	86.07 ± 3.40^a	83.88 ± 6.3^a
FeSO4	6	115.09 ± 8.43^a	85.80 ± 5.27^a	127.32 ± 4.77^b
FeCl3	6	115.22 ± 7.14^a	85.45 ± 4.76^a	126.79 ± 6.17^b
SM–Fe	6	114.91 ± 16.67^a	85.09 ± 4.17^a	127.95 ± 6.68^b

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The body weight changes of rats

The body weight changes of the rats are shown in Table 3. Initially, the mean body weight of the rats in the experiment groups and the control group did not differ (P > 0.05). After 4 weeks of low iron dietary treatment, the mean body weight of the rats in the IDA groups were significantly lower than the control group (P < 0.05). After 3 weeks repletion, the mean body weight of the SM–Fe group, the FeCl₃ group and the FeSO₄ group were higher than the IDA model group (P < 0.05), but still lower than the control group (P < 0.05).

Table 3 The body weight changes of rats before depletion, after depletion, and after repletion^a

Groups	Dosage Fe (mg kg^−1 bw)	Body weight (before depletion)	Body weight (after depletion)	Body weight (after repletion)
a a–c, means in the same row with no common superscripts differ significantly (P < 0.05).
Control	0	86.96 ± 7.29^a	261.25 ± 35.79^b	340.75 ± 13.45^c
Model	0	90.35 ± 1.86^a	195.13 ± 24.18^a	239.13 ± 9.40^a
FeSO4	6	90.75 ± 5.35^a	196.25 ± 11.57^a	258.75 ± 15.42^b
FeCl3	6	90.23 ± 3.92^a	195.39 ± 12.73^a	258.25 ± 14.32^b
SM–Fe	6	90.03 ± 4.74^a	194.50 ± 18.39^a	258.78 ± 16.25^b

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Effects of SM–Fe on organ indices in IDA rats

Table 4 shows the relative tissue (heart, liver, spleen and kidney) weights in each group. The relative weights of the heart, liver and kidney were greater in the IDA rats compared with the control group (P < 0.05). The relative weights of the heart and kidney were greater in the SM–Fe group, the FeCl₃ group and the FeSO₄ group compared with the control group (P < 0.05). The relative weights of the spleen in the control group, the IDA model group and the iron supplement groups did not differ (P > 0.05).

Table 4 Effects of SM–Fe on organ indices in IDA rats^a

Groups	Dosage Fe (mg kg^−1 BW)	Heart (g per 100 g)	Liver (g per 100 g)	Spleen (g per 100 g)	Kidney (g per 100 g)
a a and b, means in the same row with no common superscripts differ significantly (P < 0.05).
Control	0	0.32 ± 0.01^a	3.57 ± 0.49^a	0.27 ± 0.04^a	0.90 ± 0.04^a
Model	0	0.38 ± 0.04^b	4.06 ± 0.52^b	0.30 ± 0.11^a	1.31 ± 0.24^b
FeSO4	6	0.36 ± 0.03^b	3.68 ± 0.23^ab	0.27 ± 0.03^a	1.17 ± 0.07^b
FeCl3	6	0.36 ± 0.05^b	3.67 ± 0.32^ab	0.27 ± 0.04^a	1.16 ± 0.09^b
SM–Fe	6	0.36 ± 0.04^b	3.69 ± 0.51^ab	0.27 ± 0.07^a	1.17 ± 0.14^b

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Effects of SM–Fe on SI, TIBC, liver and spleen iron of the IDA model rats

The content of SI, TIBC, liver and spleen iron are shown in Table 5. The iron content of the serum, liver and spleen in the IDA rats were significantly lower than the control group (P < 0.05). The iron content of the serum, liver and spleen in the SM–Fe group, the FeCl₃ group and the FeSO₄ group rats were significantly higher than the IDA model group (P < 0.05), and did not differ from the control group (P > 0.05). Serum TIBC in the IDA rats was significantly higher than the control group and iron supplement groups (the SM–Fe group, the FeCl₃ group and the FeSO₄ group) (P < 0.05).

Table 5 Effects of SM–Fe on SI, TIBC, liver and spleen iron of the IDA model rats^a

Groups	Dosage Fe (mg kg^−1 bw)	SI (mg L^−1)	TIBC (mg L^−1)	Liver iron (mg kg^−1)	Spleen iron (mg kg^−1)
a a and b, means in the same row with no common superscripts differ significantly ( P < 0.05).
Control	0	16.34 ± 1.19^b	28.84 ± 1.10^a	597.57 ± 23.02^b	1046.77 ± 161.06^b
Model	0	9.73 ± 1.43^a	35.43 ± 1.43^b	285.64 ± 38.92^a	607.94 ± 22.28^a
FeSO4	6	16.28 ± 1.55^b	28.87 ± 1.36^a	575.57 ± 50.38^b	1032.48 ± 120.84^b
FeCl3	6	16.13 ± 1.37^b	28.95 ± 1.29^a	573.22 ± 37.57^b	1029.74 ± 97.45^b
SM–Fe	6	16.35 ± 1.52^b	28.77 ± 1.23^a	583.57 ± 35.66^b	1047.97 ± 94.59^b

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Effects of SM–Fe on SF, sTfR and EPO in the IDA model rats

The concentrations of the SF, sTfR and EPO are shown in Table 6. The SF and EPO content in the IDA model group were significantly lower than the control group and iron supplement groups (SM–Fe group, the FeCl₃ group and the FeSO₄ group) (P < 0.05), SF and EPO content in the iron supplement groups were lower than the control group (P < 0.05). The sTfR content in the iron supplement groups (SM–Fe group, the FeCl₃ group and the FeSO₄ group) were significantly lower than the IDA model group (P < 0.05), but still higher than the control group (P < 0.05).

Table 6 Effects of SM–Fe on SF, sTfR and EPO of the IDA model rats^a

Groups	Dosage Fe (mg kg^−1 bw)	SF (ng ml^−1)	sTfR (μg ml^−1)	EPO (IU L^−1)
a a–d, means in the same row with no common superscripts differ significantly ( P < 0.05).
Control	0	104.91 ± 5.71^c	4.04 ± 0.17^a	14.63 ± 0.85^c
Model	0	25.52 ± 2.71^a	7.17 ± 0.17^d	8.03 ± 0.58^a
FeSO4	6	73.16 ± 2.68^b	5.64 ± 0.21^b	9.92 ± 0.42^b
FeCl3	6	72.94 ± 2.08^b	5.84 ± 0.21^c	9.77 ± 0.52^b
SM–Fe	6	77.35 ± 2.64^b	5.16 ± 0.11^b	10.33 ± 0.65^b

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Discussion

The objective of the present study was to evaluate the effects of SM–Fe supplements on IDA rats. After iron deprivation (5.2 mg kg⁻¹ of diet), hematologic parameters in the IDA group were dramatically different from those of the control group, with a low mean blood Hb concentration. Similarly, SI, SF and EPO concentrations were lower in the IDA group, while TIBC and sTfR concentrations increased markedly due to progressive Fe depletion from the body stores. All these findings were expected and consistent with induced severe IDA in rats. The degree of iron deficiency produced by our iron-restricted diet was severe enough to impair weight gain and to reduce final body weight, as found previously.^22,24

The IDA rats had greater absolute and relative wet heart weights, demonstrating the presence of cardiac hypertrophy.²⁵ Also, increases in the wet heart weights and size of the cardiac muscle cells were observed in anemic rats compared with the controls.²⁶ In this study, the relative heart weights in rats fed with SM–Fe, FeCl₃ and FeSO₄ were significantly greater than the control group.

Iron is necessary for hemoglobin synthesis, it is logical to assume that any iron inadequacy would be reflected in a slower hemoglobin synthetic rate. A marked decrease in hemoglobin and serum iron observed in the rats fed the iron-deficient diet showed depletion of hemoglobin iron.

The liver and spleen are the main storage sites for iron.²⁷ The IDA model rats had lower iron contents in the liver and spleen than the control rats and iron supplement rats. It is then suggested that a marked decrease of iron concentration in the liver and the spleen, showing the depletion of storage iron, and the addition of iron can ameliorate iron storage.

Ferritin is a protein that functions in vivo as an iron storage compound. Although ferritin is primarily intracellular, it can be detected in serum. Serum ferritin closely reflects the level of body stores, where the level of serum ferritin is low for an iron deficiency. Serum ferritin responds quickly to iron treatment or iron deficiency.²⁸ The concentration of serum ferritin reflects the size of the storage iron compartment, if the subject is not in an inflammatory state.²⁹ In this study, the IDA rats had lower serum ferritin than the SM–Fe, FeCl₃ and FeSO₄ supplement rats. While the IDA rats supplemented with SM–Fe, FeCl₃ and FeSO₄ showed increased serum ferritin.

The measurement of sTfR concentration in the serum has a diagnostic value for the assessment of IDA.^30,31 The amount of sTfR in circulation has been shown to vary with individual iron status. Serum sTfR concentration increased even in mild iron deficiency of recent onset.³² The present study found that the IDA model rats had a higher sTfR concentration than the control rats. When supplemented with iron, the sTfR concentration decreased significantly.

EPO is an important factor for red blood cell production, it can stimulate the proliferation of burst forming unit-erythroid (BFU-E) and differentiation of colony forming unit-erythroid (CFU-E), essential for the amplification and terminal differentiation of erythroid progenitors and precursors.³³ Regulation is believed to rely on a feed-back mechanism measuring blood oxygenation.³⁴ The EPO level in the blood is quite low in the absence of anemia. The present study found that the EPO concentration in the IDA rats was lower than for the control rats. While supplement with SM–Fe, FeCl₃ and FeSO₄ could enhance EPO production and promote erythropoiesis via increasing Hb levels in the IDA rats.

Conclusion

In summary, the present study suggests that SM–Fe is an effective iron supplement and may, through upgrading SF and EPO, enhance iron bioavailability. Further research is required to apply SM–Fe to practical use as a safe and new natural iron fortifier.

Acknowledgements

This research was financially supported by a public service project of Zhejiang Province (2011C22026), a science and technology project of the Science and Technology Bureau of Zhoushan Putuo District of Zhejiang Province, and the postdoctoral scientific research project for preferential financial aid in Zhejiang Province. We thank all of the people who offered help in this study.

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