Effect of iron fortification and prebiotics on iron biomarkers in anemic rats

Efeito da fortificação com ferro e dos prebióticos nos biomarcadores de ferro em ratos anêmicos

Abstract

One of the biggest public health problems globally is that of iron deficiency anemia. The present research aimed to determine the effect of prebiotics along with iron fortification on iron biomarkers in female anemic rats as some evidence suggests that prebiotics convert increase the solubility of iron, thereby enhancing its absorption. A total of 126 Sprague Dawley rats were fed with sixteen different types of fortified feed containing prebiotics (Inulin + Galacto Oligosaccharides) and Iron Fortificants (Sodium Ferric Ethylenediaminetetraacetate + Ferrous Sulphate). The duration of the trials was 3 months aimed at determining the effect of iron fortification and prebiotics on different iron biomarkers including Hemoglobin (Hb), Hematocrit, Red Blood Cell (RBC) count, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration (MCHC). The trials resulted in statistically significant improved iron biomarkers among female anemic rats (P-value < 0.05). It was concluded that iron fortification and prebiotics in combination were able to increase the levels of iron biomarkers in female anemic rats.

Keywords:  hemoglobin; hematocrit; iron deficiency anemia; iron biomarkers; prebiotics; iron fortificants

Resumo

Um dos maiores problemas de saúde pública no mundo é a anemia por deficiência de ferro. A presente pesquisa teve como objetivo determinar o efeito dos prebióticos associados à fortificação com ferro nos biomarcadores de ferro em ratas anêmicas, uma vez que algumas evidências sugerem que os prebióticos podem aumentar a solubilidade do ferro, melhorando assim sua absorção. Foram utilizados 126 ratos Sprague Dawley alimentados com dezesseis tipos diferentes de rações fortificadas contendo prebióticos (Inulina + Galacto-Oligossacarídeos) e Fortificantes de Ferro (Etilenodiaminotetracetato Férrico de Sódio + Sulfato Ferroso). A duração dos ensaios foi de 3 meses, com o objetivo de determinar o efeito da fortificação de ferro e prebióticos em diferentes biomarcadores de ferro, incluindo Hemoglobina (Hb), Hematócrito, Contagem de Glóbulos Vermelhos (RBC), Volume Corpuscular Médio (VCM), Hemoglobina Corpuscular Média (MCH) e Concentração Média de Hemoglobina Corpuscular (CHCM). Os ensaios resultaram em biomarcadores de ferro significativamente melhorados entre ratas anêmicas (valor P <0,05). Concluiu-se que a fortificação com ferro e a combinação de prebióticos foram capazes de aumentar os níveis de biomarcadores de ferro em ratas anêmicas.

Palavras-chave:  hemoglobina; hematócrito; anemia por deficiência de ferro; biomarcadores de ferro; fortificantes de ferro; prebióticos

1. Introduction

Micronutrients are required in minute amounts, yet their role is essential in maintaining overall health and wellbeing. Micronutrient deficiencies including iron deficiency is one amongst the significant health issues in both high- and low-income countries. This is primarily because in general people do not include enough iron as per their requirements and also, rich iron sources in diet are generally expensive (Keats et al., 2021). Estimates showed that approximately 30% of the world's population is affected by iron deficiency, considering the most vulnerable group of reproductive age women. Moreover, other physiological states such as pregnancy, lactation, infancy or adolescence impose the higher risk for developing Iron Deficiency Anemia (IDA) owing to higher iron requirements during these stages of life. Individuals who suffer from a hemorrhage may also suffer from IDA because of significant iron loss (Stevens et al., 2022).

Iron is primarily involved in oxygen transport, energy metabolism and cellular proliferation (Mahan and Raymond, 2017). Also, the role of iron is indispensable for DNA synthesis, electron transport, mitochondrial function, and to regulate different enzymatic processes (Muckenthaler et al., 2017). The iron acquisition is tightly regulated by hepcidin homoeostasis emphasizing on the release of iron from enterocytes and macrophages to maintain plasma iron concentration within the normal levels (Yiannikourides and Latunde-Dada, 2019). However, the bio-availability iron is mainly dependent on different forms of iron (heme or non-heme) and its absorption. For example, a daily diet providing 13-18 mg of iron, only a small amount (1-2 mg/day of iron) is absorbed in the body, while approximately 30 mg/day iron is required for production of red blood cells (Miret et al., 2003) . Considering this higher requirements, adequate dietary iron is essential to maintain optimal health and to prevent IDA The recommended dietary allowance for men and postmenopausal women is 8 mg/day while for premenopausal women, it is 18 mg/day (Mann et al., 2023).

Various approaches including iron supplementation, fortification of condiments and staple foods with iron and dietary diversification are widely used to combat the IDA. Supplementation results in rapid replenishment of iron stores in the deficient individuals. Food fortification on the other hand enhances the iron content in various foods, thereby addressing iron deficiency. (Bathla and Arora, 2022). In concomitant, few other studies have suggested that prebiotics such as Inulin and Galacto Oligosaccharides play an important role to enhance iron absorption through improving total iron binding capacity, hematocrit, hemoglobin and transferrin saturation factor (Sundberg, 2011; Kobayashi et al., 2011). It has been reported that probiotic bacteria such as propionibacterium freudenreichii appears to enhance iron absorption in proximal colon and duodenum due to production of short chain fatty acids (Bougle et al., 2002). Similarly, another study reported significant positive effect of lactulose and Inulin on mean corpuscular volume, hemoglobin and hematocrit in anemic animal models (Masanetz et al., 2011). However, none of the studies conducted so far have comprehensive recommendations regarding which types of prebiotics and which iron fortificants in which particular combinations may have the best results in terms of improving various iron biomarkers or decreasing anemia. As such, our current research was an attempt to optimize various treatment combinations of prebiotics and iron fortificants in different dosages.

Moreover, in low/middle income countries, multifactorial causes such as deficiency of iron, folate, vitamin B12 and parasitic diseases are the leading cause of anemia (Addis Alene and Mohamed Dohe, 2014). Studies have shown that pregnant women from developing countries like Pakistan are highly vulnerable for pregnancy complications due to low Hb levels and iron status. Yet, there are no robust human trials which have looked into the combined effect of iron fortification and prebiotics in this region. (Akhtar et al., 2013). National Nutrition Survey (NNS) of 2018 reported that more than half of the Pakistani children (53.7%) and around 42% women of reproductive age are suffering from anemia (UNICEF, 2018). Considering these alarming facts, further innovative research is needed to address the challenge of nutritional deficiencies including iron deficiency anemia in low/middle income countries.

2. Materials and Methods

2.1. Experimental animals and diet

The current study incorporated 126 female Sprague Dawley rats who were aged 6 to 8 weeks. The rats were obtained from the National Institute of Health (NIH), Islamabad. For the purchase of relevant raw materials and chemicals, well-reputed international companies were selected. Overall, rats were divided into n = 18 groups whereby n = 16 groups were interventional while n = 2 groups were controls. In terms of feed to be fed to 16 different groups, 16 different feeds each with varying amounts of iron fortificants and prebiotics were formulated (Table 1). Iron fortificants were added to the standard diet of rats while prebiotics were dissolved in water; the rats were given this feed orally.

Table 1
Treatment Plan (Iron Fortificants & Prebiotics Based Feed Groups).

Groups	Diet Plan	Human Equivalent Dose (HED) for Pre-biotic
G+	Positive Control (only basal diet fed to healthy rats)	-
G-	Negative Control (only basal diet fed to anemic rats)	-
G1	Basal Diet + 722 mg/kg Inulin + 10 ppm NaFeEDTA	100 mg/kg = 6 grams
G2	Basal Diet + 722 mg/kg Inulin + 20 ppm NaFeEDTA	100 mg/kg = 6 grams
G3	Basal Diet + 963 mg/kg Inulin + 10 ppm NaFeEDTA	133 mg/kg = 8 grams
G4	Basal Diet + 963 mg/kg Inulin + 20 ppm NaFeEDTA	133 mg/kg = 8 grams
G5	Basal Diet + 722 mg/kg Inulin + 15 ppm FeSO4	100 mg/kg = 6 grams
G6	Basal Diet + 722 mg/kg Inulin + 30 ppm FeSO4	100 mg/kg = 6 grams
G7	Basal Diet + 963 mg/kg Inulin + 15 ppm FeSO4	133 mg/kg = 8 grams
G8	Basal Diet + 963 mg/kg Inulin + 30 ppm FeSO4	133 mg/kg = 8 grams
G9	Basal Diet + 722 mg/kg GOS + 10 ppm NaFeEDTA	100 mg/kg = 6 grams
G10	Basal Diet + 722 mg/kg GOS + 20 ppm NaFeEDTA	100 mg/kg = 6 grams
G11	Basal Diet + 963 mg/kg GOS + 10 ppm NaFeEDTA	133 mg/kg = 8 grams
G12	Basal Diet + 963 mg/kg GOS + 20 ppm NaFeEDTA	133 mg/kg = 8 grams
G13	Basal Diet + 722 mg/kg GOS + 15 ppm FeSO4	100 mg/kg = 6 grams
G14	Basal Diet + 722 mg/kg GOS + 30 ppm FeSO4	100 mg/kg = 6 grams
G15	Basal Diet + 963 mg/kg GOS + 15 ppm FeSO4	133 mg/kg = 8 grams
G16	Basal Diet + 963 mg/kg GOS + 30 ppm FeSO4	133 mg/kg = 8 grams

Human equivalent dose (HED) Equation 1 was used to calculate the doses for prebiotics.

$$HED\left(mg/kg\right)=AnimalDoseinmg/kg\times {\frac{\left(AnimalWeightinkgs\right)}{\left(HumanWeightinkgs\right)}}^{0.33}$$ (1)

Exact doses were, however, calculated after weighing the actual rats.

2.2. Experimental protocol

Bio-evaluation trials were conducted on the anemic female rats so as to evaluate the combined efficacy of iron fortification and prebiotics. Each group has n = 7 rats, including the positive and negative controls. All the guidelines for handling animals were followed using National Research Council Guidelines (ILAR, 1986). Stainless steel cages were employed for adequate housing of rats. A temperature of 23 ±2 °C was maintained through the trials period while the relative humidity was set at 55 ±5%. During the course of trials, 1 12-hour light-dark cycle was maintained.

Before the start of actual trials, a standard feed was fed to the rats for a period of 1 week whose purpose was to acclimatize the rats. Following the one-week time period, anemia was induced in the interventional groups and negative control group using iron binder, triapine. Once it was ensured that anemia had been induced (by determining the hemoglobin levels), baseline values for all the biomarkers of interest were determined. Rats were considered to become anemic once their hemoglobin levels fell below 11 g/dL as the normal range of hemoglobin in rats is defined as 11-19 g/dL (Jacob Filho et al., 2018). As soon as the rats became anemic, iron binders were not added further in their diet and subsequently, the rats were treated with iron and prebiotics based fortified feed on a daily basis for next 3 months. The fortified feed was added to their regular diet. During the trials, it was ensured to adjust the doses of prebiotics by taking the mean weight of rats at the beginning and end of every week. Any rats that deviated significantly from the mean weight were excluded and the average of remaining rats was taken. Overall, values for all the iron biomarkers of interest were determined at 0 day, 30^th day, 60^th day, and 90^th day. For this purpose, blood samples were collected from saphenous vein from overnight fasted rats.

2.3. Analytical procedures

Hematological analysis was performed for all the collected blood samples from various groups. Six major iron biomarkers were studied which included Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Red Blood Cell (RBC) count, Mean Corpuscular Volume (MCV), Hemoglobin (Hb), and Hematocrit. Standard protocols were used to determine the respective iron biomarkers in blood samples using BC-700 hematology analyzer (Al Haj et al., 2011).

2.4. Statistical analysis

Statistical analysis was performed using SPSS version 23.0 to determine the level of significance. As per the nature of the study, factorial design was the most appropriate study design and the results were considered to be significant at P - value < 0.05 (Daniel and Cross, 2018). Moreover, we calculated the Means ±Standard deviations of all the parameters across different groups and study intervals. For the purpose of comparison of Means ±Standard deviations of various parameters, we used one-way ANOVA.

2.5. Ethical considerations

The ethical review committee (ERC) of the University of Veterinary & Animal Sciences, Lahore was approached to get the approval of the study which was given vide letter No. DR/996.

3. Results

3.1. Effect of iron and prebiotic fortified feed on Hb levels (g/dL)

Mean square values for iron biomarkers for rats fed with varying fortified diets were recorded (Table 2), while changes in the mean values for Hb levels along with their respective standard deviations have been shown in Table 3.

Table 2
Mean squares regarding various iron biomarkers for anemic female rats fed with iron and prebiotic fortified feed.

SOV	df	Hb	Hematocrit	RBC Count	MCV	MCH	MCHC
Groups	17	54.90*	41.00*	8.76*	106.60*	36.01*	23.50*
Study Intervals	3	250.05*	94.00*	13.70*	46.68*	102.32*	18.06*
Groups × Study Intervals	51	3.97*	6.44*	0.62*	9.18*	8.28*	1.96*
Error	432	0.16	0.12	0.05	0.62	0.14	0.87
Total	503

• ^*
  Significant (P-value < 0.05).

Table 3
Effect of fortified diets on Hemoglobin levels (g/dL).

Treatments/Groups	Days				Means
	0	30	60	90
G+	16.94 ± 0.75a	16.80 ± 0.87a	16.87 ±1.04a	16.40 ±0.76a	16.75 ±0.24a
G-	9.82 ±0.14b	9.47 ± 0.21g	8.94 ± 0.20h	8.04 ± 0.13h	9.06 ± 0.77g
G1	9.92 ± 0.15b	11.72 ± 0.62cdef	12.77 ± 0.63defg	13.44 ± 0.65ef	11.96 ± 1.53def
G2	9.85 ± 0.15b	11.78 ± 0.22cdef	12.86 ± 0.33defg	13.37 ± 0.28efg	11.96 ± 1.56def
G3	9.94 ± 0.14b	12.00 ± 0.19bcd	13.40 ± 0.24cd	14.30 ± 0.26bcd	12.41 ± 1.90cd
G4	9.87 ± 0.17b	12.58 ± 0.34b	14.42 ± 0.42b	15.70 ± 0.24a	13.14 ± 2.53b
G5	9.95 ± 0.16b	11.67 ± 0.71def	12.62 ± 0.72defg	13.32 ± 0.68efg	11.89 ± 1.46def
G6	9.91 ± 0.16b	11.20 ± 0.45ef	12.22 ± 0.44fg	12.84 ± 0.42fg	11.54 ± 1.28ef
G7	10.00 ± 0.23b	11.12 ± 0.27f	12.00 ± 0.28g	12.64 ± 0.30g	11.44 ± 1.14f
G8	10.07 ± 0.34b	12.50 ± 0.31bc	13.84 ± 0.37bc	14.60 ± 0.35b	12.75 ± 1.99bc
G9	9.88 ± 0.25b	11.50 ± 0.28def	12.54 ± 0.40efg	13.20 ± 0.22efg	11.78 ± 1.45def
G10	10.11 ± 0.35b	11.82 ± 0.38bcdef	12.88 ± 0.43def	13.52 ± 0.36def	12.08 ± 1.49cdef
G11	10.08 ± 0.32b	11.90 ± 0.24bcdef	13.02 ± 0.26de	13.65 ± 0.25cde	12.16 ± 1.57cde
G12	9.95 ± 0.24b	12.15 ± 0.26bcd	13.38 ± 0.27cd	14.38 ± 0.34bc	12.46 ± 1.91bcd
G13	9.97 ± 0.33b	11.42 ± 0.36def	12.72 ± 0.32defg	13.53 ± 0.25ef	11.91 ± 1.56def
G14	9.94 ± 0.21b	11.71 ± 0.40cdef	13.00 ± 0.37cde	13.75 ± 0.45cde	12.10 ± 1.67cdef
G15	10.07 ± 0.27b	12.02 ± 0.23bcd	13.05 ± 0.22cde	13.70 ± 0.44cde	12.21 ± 1.59cde
G16	10.14 ± 0.36b	11.94 ± 0.40bcde	13.08 ± 0.35cde	13.93 ± 0.47bcde	12.27 ± 1.64cd

• Means carrying the same letters in a column are not significantly different. G+ = Unfortified feed (healthy control); G- = Unfortified feed (anemic control); G1 = Basal Diet+722mg/kg Inulin+10ppm NaFeEDTA; G2 = Basal Diet+722mg/kg Inulin+20ppm NaFeEDTA; G3 = Basal Diet+963 mg/kg Inulin+10ppm NaFeEDTA, G4 = Basal Diet+963mg/kg Inulin+20ppm NaFeEDTA; G5 = Basal Diet+722 mg/kg Inulin+15ppm FeSO₄; G6 = Basal Diet+722 mg/kg Inulin+30ppm FeSO₄; G7 = Basal Diet+963 mg/kg Inulin+15 ppm FeSO₄; G8 = Basal Diet+963 mg/kg Inulin+30ppm FeSO₄; G9 = Basal Diet+722 mg/kg GOS+10ppm NaFeEDTA; G10 = Basal Diet+722mg/kg GOS+20ppm NaFeEDTA; G11 = Basal Diet+963 mg/kgGOS+10ppm NaFeEDTA; G12 = Basal Diet+963mg/kgGOS+20ppm NaFeEDTA; G13 = Basal Diet+722 mg/kgGOS+15ppm FeSO₄; G14 = Basal Diet+722 mg/kg GOS+30 ppm FeSO₄; G15 = Basal Diet+963mg/kg GOS+15ppm FeSO₄; G16 = Basal Diet+963 mg/kg GOS+30 ppm FeSO₄.

The results showed the maximum Hb value of 13.14 ± 2.53 g/dL in Group G_4, fed with basal diet + 963 mg/kg Inulin + 20 ppm NaFeEDTA. Following by, G₈ and G₁₂ group reported the Hb values of 12.75 ± 1.99 g/dL and 12.46 ± 1.91 g/dL respectively. Furthermore, the subsequent maximum values for Hb were found in G₃, D₁₆ and G₁₅ groups.

Minimum values of Hb were found in group G₉ (Basal Diet + 722 mg/kg GOS + 10 ppm NaFeEDTA), group G₆ (Basal Diet + 722 mg/kg Inulin + 30 ppm FeSO₄) and group G₇ (Basal Diet + 963 mg/kg Inulin + 15 ppm FeSO₄) showing the Hb levels at 11.78 ± 1.45 g/dL, 11.54 ± 1.28 g/dL and 11.44 ± 1.14 g/dL. On the other hand, the anemic control group G_- showed Hb levels of 9.06 ± 0.77 g/dL while healthy control group G₊ showed a Hb value of 16.75 ± 0.24 g/dL.

As the study intervals progressed, a considerable improvement in Hb levels was also observed. Maximum improvement was observed in group G₄ (9.87 ± 0.17 to 15.70 ± 0.24) g/dL, followed by group G₈, G₁₂ and G₃ over a period of 90 days. For groups G₈, Hb levels ranged from 10.07 ± 0.34 to 14.60 ± 0.35g/dL, G₁₂ 9.95 ± 0.24 to 14.38 ± 0.34g/dL and G₃ improved from 9.94 ± 0.14 to 14.30 ± 0.26g/dL at 0 to 90^th day.

3.2. Effect of iron and prebiotic fortified feed on hematocrit levels (%)

Mean Hematocrit values and their respective standard deviations have been presented in Table 4 and results found a significant increase in hematocrit levels in consideration to both groups and study intervals (P-value < 0.05).

Table 4
Effect of fortified diets on Hematocrit levels (%).

Treatments/Groups	Days				Means
	0	30	60	90
G+	37.87 ± 0.78a	38.10 ± 0.73a	37.91 ± 0.58ab	37.84 ± 0.46c	37.93 ± 0.12a
G-	34.28 ± 0.17b	32.87 ± 0.20h	31.80 ± 0.26i	30.42 ± 0.55i	32.34 ± 1.64h
G1	34.32 ± 0.11b	35.20 ± 0.12efg	35.58 ± 0.15g	34.20 ± 0.27h	34.82 ± 0.67g
G2	34.28 ± 0.35b	35.17 ± 0.32fg	35.48 ± 0.26g	33.77 ± 0.75h	34.67 ± 0.79g
G3	34.37 ± 0.23b	36.60 ± 0.30bc	37.60 ± 0.22b	39.13 ± 0.19b	36.92 ± 2.00bc
G4	34.14 ± 0.13b	36.85 ± 0.15b	38.21 ± 0.17a	40.16 ± 0.47a	37.34 ± 2.53ab
G5	34.15 ± 0.23b	35.22 ± 0.27efg	35.74 ± 0.18fg	36.35 ± 0.19def	35.36 ± 0.93fg
G6	34.12 ± 0.21b	35.31 ± 0.32edfg	35.38 ± 0.41g	36.11 ± 0.36efg	35.23 ± 0.82fg
G7	34.21 ± 0.12b	35.07 ± 0.24g	34.77 ± 0.35h	35.58 ± 0.43g	34.90 ± 0.57g
G8	34.17 ± 0.28b	36.45 ± 0.26bc	37.40 ± 0.37bc	38.72 ± 0.60b	36.68 ± 1.92bcd
G9	34.25 ± 0.19b	35.70 ± 0.22def	36.41 ± 0.23de	37.11 ± 0.49d	35.86 ± 1.22def
G10	34.38 ± 0.20b	35.71 ± 0.21de	36.37 ± 0.18e	37.16 ± 0.72d	35.90 ± 1.18def
G11	34.22 ± 0.18b	35.64 ± 0.29defg	36.48 ± 0.61ef	37.08 ± 0.87de	35.85 ± 1.24def
G12	34.22 ± 0.17b	36.08 ± 0.13cd	36.91 ± 0.16cd	37.81 ± 0.43c	36.25 ± 1.53cde
G13	34.35 ± 0.15b	35.24 ± 0.24efg	35.65 ± 0.31g	36.41 ± 0.72efg	35.41 ± 0.86fg
G14	34.37 ± 0.19b	35.40 ± 0.21efg	35.82 ± 0.22fg	36.31 ± 0.26def	35.47 ± 0.83efg
G15	34.37 ± 0.17b	35.38 ± 0.22efg	35.75 ± 0.26fg	36.27 ± 0.21def	35.44 ± 0.80efg
G16	34.30 ± 0.14b	35.25 ± 0.16efg	35.58 ± 0.20g	36.08 ± 0.25fg	35.30 ± 0.75fg

• Means carrying the same letters in a column are not significantly different.

The maximum value of hematocrit (%) was seen in group G₄ (37.34 ± 2.53%) treated with Basal Diet + 963 mg/kg Inulin + 20 ppm NaFeEDTA, followed by group G₃ (Basal Diet + 963 mg/kg Inulin + 10 ppm NaFeEDTA) and group G₈ (Basal Diet + 963 mg/kg Inulin + 30 ppm FeSO₄) with respective values of 36.92 ± 2.00% and 36.68 ± 1.92%.

Minimum values of hematocrit were found to be in group G₇ (34.90 ± 0.57%, Basal Diet + 963 mg/kg Inulin + 15 ppm FeSO₄), group G₁ (34.82 ± 0.67%, Basal Diet + 722 mg/kg Inulin + 10 ppm NaFeEDTA) and group G₂ (34.67 ± 0.79%, Basal Diet + 722 mg/kg Inulin + 20 ppm NaFeEDTA) respectively. Anemic control group G_- showed a negative trend in terms of hematocrit values with a mean of 32.34 ± 1.64% than healthy control group G₊ (37.93 ± 0.12%).

With the progression of study intervals, a significant improvement in hematocrit levels could also be observed. Maximum improvement was observed in group G₄ ranged from 34.14 ± 0.13% at baseline to 40.16 ± 0.47% at the termination of the study followed by G₃, G₈ and G₁₂ groups. For G₃, G₈ and G₁₂ groups, hematocrit levels ranged from 34.37 ± 0.23% to 39.13 ± 0.19%, 34.17 ± 0.28% to 38.72 ± 0.60% and 34.22 ± 0.17% to 37.81 ± 0.43% at 0 and 90^th day, respectively.

3.3. Effect of iron and prebiotic fortified feed on red blood cell count levels (mil/mm³)

Mean values regarding Red Blood Cell Count in Table 5 observed the maximum value in group G₄ (6.20 ± 1.13mil/mm³), followed by groups G₈, G₃, G₁₂ and G₁₁ with consecutive values of 5.52 ± 0.74mil/mm³, 5.43 ± 0.73mil/mm³, 5.25 ± 0.58mil/mm³ and 5.18 ± 0.24mil/mm³.

Table 5
Effect of fortified diets on RBC count levels (mil/mm³).

Treatments/Groups	Days				Means
	0	30	60	90
G+	7.05 ± 0.51a	7.00 ± 0.45a	6.98 ± 0.37a	7.02 ± 0.27a	7.01 ± 0.03a
G-	4.79 ± 0.15b	4.63 ± 0.07d	4.60 ± 0.10e	4.55 ± 0.05f	4.64 ± 0.10h
G1	4.66 ± 0.23b	4.77 ± 0.29cd	4.98 ± 0.21de	5.06 ± 0.22de	4.87 ± 0.18fgh
G2	4.63 ± 0.30b	4.79 ± 0.29cd	5.09 ± 0.14cd	4.90 ± 0.37ef	4.85 ± 0.19gh
G3	4.61 ± 0.26b	5.04 ± 0.28cd	5.93 ± 0.30b	6.15 ± 0.29b	5.43 ± 0.73cd
G4	4.73 ± 0.19b	5.93 ± 0.15b	6.84 ± 0.44a	7.28 ± 0.20a	6.20 ± 1.13b
G5	4.54 ± 0.23b	4.88 ± 0.28cd	5.19 ± 0.18cd	5.06 ± 0.21de	4.92 ± 0.28fgh
G6	4.66 ± 0.22b	4.90 ± 0.25cd	5.27 ± 0.23cd	5.16 ± 0.23de	5.00 ± 0.27efg
G7	4.69 ± 0.26b	4.82 ± 0.28cd	5.10 ± 0.29cd	5.20 ± 0.25de	4.95 ± 0.24efgh
G8	4.67 ± 0.26b	5.19 ± 0.16c	5.87 ± 0.25b	6.35 ± 0.27b	5.52 ± 0.74c
G9	4.73 ± 0.18b	5.05 ± 0.09cd	5.09 ± 0.17cd	5.27 ± 0.15de	5.04 ± 0.22efg
G10	4.67 ± 0.17b	5.00 ± 0.13cd	5.06 ± 0.19cd	5.22 ± 0.16de	4.99 ± 0.23efg
G11	4.86 ± 0.13b	5.14 ± 0.15c	5.29 ± 0.20cd	5.43 ± 0.14d	5.18 ± 0.24def
G12	4.60 ± 0.19b	4.97 ± 0.20cd	5.52 ± 0.21bc	5.90 ± 0.22bc	5.25 ± 0.58cde
G13	4.70 ± 0.17b	5.00 ± 0.11cd	5.10 ± 0.18cd	5.34 ± 0.15d	5.04 ± 0.27efg
G14	4.61 ± 0.18b	4.84 ± 0.23cd	5.03 ± 0.16de	5.27 ± 0.22de	4.94 ± 0.28efgh
G15	4.78 ± 0.18b	5.00 ± 0.19cd	5.23 ± 0.20cd	5.48 ± 0.28cd	5.12 ± 0.30defg
G16	4.79 ± 0.11b	5.03 ± 0.12cd	5.23 ± 0.13cd	5.49 ± 0.19cd	5.14 ± 0.30defg

• Means carrying the same letters in a column are not significantly different.

During the efficacy trials conducted, a considerable improvement in red blood cell count levels was also seen amongst the groups with time progression. As per the results, maximum improvement in RBC count levels in group G₄ recorded at 4.73 ± 0.19mil/mm³ at start and improved to 7.28 ± 0.20mil/mm³ at the end of the study.

3.4. Effect of iron and prebiotic fortified feed on mean corpuscular volume levels (fL)

Mean Corpuscular Volume values given in Table 6 showed the maximum value in group G₄ (48.47 ± 2.48fL), followed by groups G₈ (47.46 ± 1.70fL), G₁₂ (47.03 ± 1.53fL), G₁₃ (46.46 ± 0.88fL), G₃ (46.30 ± 1.56fL), G₁₅ (46.28 ± 0.74fL) and G₆ (46.19 ± 0.61). Lowest mean values for MCV were recorded in groups G₉ (46.04 ± 0.59fL), G₁₁ (45.99 ± 0.41fL) and G₁₀ (45.88 ± 0.48fL). During the progression of efficacy trials which involved female Sprague Dawley anemic rats, a considerable increase in mean MCV levels from the start of trials to the termination of study was also recorded during the 90 days of study interval (Table 6).

Table 6
Effect of fortified diets on Mean Corpuscular Volume Levels (fL).

Treatments/Groups	Days				Means
	0	30	60	90
G+	53.58 ± 0.35a	53.14 ± 0.40a	53.01 ± 0.38a	52.67 ± 0.44a	53.10 ± 0.38a
G-	45.28 ± 0.27b	44.27 ± 0.61f	43.01 ± 0.43f	38.22 ± 3.45e	42.69 ± 3.12f
G1	45.28 ± 0.24b	46.07 ± 0.23de	46.44 ± 0.26e	46.82 ± 0.27cd	46.15 ± 0.66de
G2	45.30 ± 0.23b	45.97 ± 0.28e	46.38 ± 0.30e	46.82 ± 0.31cd	46.11 ± 0.65de
G3	45.21 ± 0.21b	47.02 ± 0.24c	48.15 ± 0.23c	44.84 ± 5.25d	46.30 ± 1.56de
G4	45.41 ± 0.19b	47.77 ± 0.22b	49.50 ± 0.34b	51.21 ± 0.67ab	48.47 ± 2.48b
G5	45.28 ± 0.25b	46.05 ± 0.29de	46.37 ± 0.30e	46.92 ± 0.59cd	46.15 ± 0.69de
G6	45.37 ± 0.22b	46.14 ± 0.24de	46.44 ± 0.26e	46.82 ± 0.24cd	46.19 ± 0.61de
G7	45.30 ± 0.22b	46.10 ± 0.21de	46.50 ± 0.17de	46.84 ± 0.15cd	46.18 ± 0.66de
G8	45.30 ± 0.31b	47.00 ± 0.34c	48.34 ± 0.33c	49.21 ± 0.35bc	47.46 ± 1.70bc
G9	45.31 ± 0.20b	45.85 ± 0.17e	46.35 ± 0.22e	46.65 ± 0.21cd	46.04 ± 0.59de
G10	45.24 ± 0.16b	45.82 ± 0.17e	46.10 ± 0.18e	46.37 ± 0.08cd	45.88 ± 0.48e
G11	45.47 ± 0.29b	45.91 ± 0.27e	46.20 ± 0.33e	46.41 ± 0.21cd	45.99 ± 0.41de
G12	45.12 ± 0.14b	46.55 ± 0.18cd	47.88 ± 0.22c	48.60 ± 0.16bc	47.03 ± 1.53cd
G13	45.45 ± 0.17b	46.04 ± 0.16e	46.97 ± 0.14d	47.40 ± 0.24cd	46.46 ± 0.88cde
G14	45.31 ± 0.23b	45.88 ± 0.21e	46.44 ± 0.30e	46.91 ± 0.33cd	46.13 ± 0.69de
G15	45.40 ± 0.22b	46.00 ± 0.23e	46.65 ± 0.24de	47.10 ± 0.21cd	46.28 ± 0.74de
G16	45.22 ± 0.16b	45.84 ± 0.21e	46.48 ± 0.24de	46.95 ± 0.25cd	46.12 ± 0.75de

• Means carrying the same letters in a column are not significantly different.

3.5. Effect of iron and prebiotic fortified feed on MCH and MCHC levels (g/dL)

Figure 1a and 1b has shown the significant positive variations for MCH as well as MCHC among groups, study intervals and their interaction. The maximum value for MCH was recorded in group G₄ (21.44 ± 3.27pg) and group G₈ (20.28 ±2.24pg). Furthermore, groups G₊, G₁₄ and G₁₃ in turn showed the mean values of 19.07 ± 0.45pg, 18.59 ± 0.86pg and 18.56 ± 0.94pg. On the other hand, least values were recorded in groups G₇, G₁ and G_- as 18.22 ± 0.34pg, 18.17 ± 0.46pg and 16.41 ± 1.10pg, respectively.

Figure 1
Effect of Fortified Diets on MCH (a and b) and MCHC (c and d) among Anemic Female Rats. Data are presented as Means ± SD. P-value < 0.05 is considered to be significant. Means carrying the same letters are not significantly different.

Regarding MCHC, maximum mean value was seen in group G₊ at 33.15 ± 0.19g/dL, followed by group G₄ at 31.65 ± 1.15g/dL (Figure 1c and 1d). Lowest values for this trait were recorded in groups G₂, G₁ and G_- with a mean values of 30.39 ± 0.28g/dL, 30.24 ± 0.17g/dL and 28.22 ± 1.34g/dL consecutively.

During the rat trials, it was observed that serum MCH and MCHC levels increased considerably from 0 day to up to the 90^th day. Group G₄ with a maximum value showed a mean levels ranged from 30.27 ± 0.27g/dL at 0 day to 32.94 ± 0.29g/dL at 90^th day. For groups G₁₂ and G₈, mean values varied from 30.12 ± 0.21g/dL and 30.40 ± 0.47g/dL at the time of initiation of trials to 32.35 ± 0.20g/dL and 32.32 ± 0.38g/dL at the termination of research.

4. Discussion

Our results suggested that the levels of Hb, Hematocrit, RBCs, MCV, MCH, MCHC were significantly improved in anemic rates when treated with a combined iron and prebiotic fortified diet for a period of 90 days. Also, a significant positive increase was observed for studied iron biomarkers with the progression of study duration.

In our study, two prebiotics, Inulin and Galacto Oligosaccharides were used at two different levels of 722 mg/kg body weight and 963 mg/kg respectively along with two iron salts (fortificants). The NaFeEDTA was used as iron fortificants at 10 and 20 ppm levels while FeSO₄ was used at 15 and 30 ppm levels. All treated study groups showed a positive improvement in iron absorption, however, the groups treated with Inulin reported the better outcomes as compared to Galacto oligosaccharides in terms of enhancing iron absorption in anemic rats. In agreement to our results, Coudray et al. (1997) performed a studied the effects of an Inulin diet and a sugar beet diet on absorption of minerals such as Iron, Calcium, Zinc and Magnesium and concluded fiber from Inulin increase the absorption of various minerals in the body (Scheuchzer, 2022). Similarly, another study by Tako et al (2008), determined the effect of dietary Inulin on gene expression of various intestinal iron transporters and iron binding proteins. The results observed an improvement of Duodenal Cytochrome B (Dcytb), Divalent metal transporter 1 (DMT 1), serum ferritin, ferroportin, and transferrin receptor levels in group consuming Inulin supplemented diet (Tako et al., 2008). In concomitant, few studies have shown that Galacto oligosaccharides (GOS) and Fructo oligosaccharides that prebiotics if added along with iron supplementation enhance absorption of iron significantly among anemic subjects (Zhang et al., 2017).

Dietary iron absorption is primarily performed through enterocyte cells of small intestine primarily involved in the dietary iron absorption and maintain iron homeostasis in the body (Piskin et al., 2022) though different dietary factors including effect of prebiotics on iron absorption depend on type, dose, duration consumption of prebiotics, iron fortificants, along with age, inflammatory and iron status of the population (Husmann et al., 2022).

The role of prebiotics to increase iron absorption can be explained by fermentation process in colon due to short chain fatty acids followed by i) lowering the pH of luminal content, ii) increase iron solubility through reduction of Fe+++ to Fe ++ and iii) increase the surface area for iron absorption through stimulating the proliferation of epithelial cells (Weinborn et al., 2017). Similarly, inulin appears to increase the amount of butyrate which has been recognized as mediators of iron absorption (Zakrzewska et al., 2022). However, the few other studies that Inulin do not directly enhance the bioavailability of Iron and shows a positive impact when used by probiotic cells (Laparra et al., 2014; Dabour et al., 2019). Further research is needed to elucidate the interaction between pre and probiotics in relation to iron absorption (Birmani et al., 2019; Bhatnagar et al., 2020).

5. Conclusion

The results of the study suggested that prebiotics Inulin and Galacto Oligosaccharides along with the iron fortificants improve the levels of hemoglobin, hematocrit and several other iron biomarkers in anemic rats. Specifically in our study, group G4 with 963 mg/kg of Inulin along with 20 ppm NaFeEDTA and group G8 with 963 mg/kg of Inulin along with 30 ppm FeSO₄ showed the best results in terms of improving various iron biomarkers in anemic rats. In our recommendation, these dosages should be further tested in large scale research studies to further warrant this claim. Also, such research should further be conducted with human models to find out if the same effect is pronounced or not. Further research is also required to determine the influence dose of prebiotics, duration of the study and influence of other dietary factors to treat IDA.

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Publication Dates

• Publication in this collection
  27 Jan 2025
• Date of issue
  2024

History

• Received
  23 Mar 2024
• Accepted
  06 Oct 2024