Effects of extruded whole-grain sorghum (Sorghum bicolor (L.) Moench) based diets on calcium absorption and bone health of growing Wistar rats

Abstract

Apparent calcium absorption, total bone mineral content and density, and mineral contents of the right femur were studied using a growing rat model. Twenty-four male Wistar rats were fed with diets based on extruded whole grain red (RSD) or white sorghum (WSD), and control diet (CD) up to 60 days. The animals fed with sorghum diets consumed less and gained less weight compared to those fed with CD, but the efficiency of all diets was similar. Calcium intake was lower in animals fed with sorghum diets, related to the lower total intake of these animals. Apparent calcium absorption in animals fed with RSD was lower than in those fed with CD (CD: 72.7%, RSD: 51.0%, WSD: 64.8%). No significant differences in bone mineral density of total body, spin, femur, distal femur, tibia and proximal tibia were observed among the groups. However, Ca and P contents in the right femur of the rats consuming RSD were lower, indicating a certain imbalance in the metabolism of these minerals.

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Introduction

Osteoporosis is a common disorder in both men and women of advanced age, which leads to an increased risk of bone fractures, significantly affecting the physical health of those who suffer from it.¹ To reduce the risk of fractures, it is essential to increase bone mass during growth. It is known that low Ca intake is associated with low bone mass, while high Ca intake has an unclear effect.²

Among cereals, sorghum (Sorghum bicolor (L.) Moench) is recognized for its adaptation to harsh environments and antioxidant properties.³ Also epidemiological studies show that the consumption of whole grains (WG) can reduce the risk of suffering from chronic diseases such as type 2 diabetes, cardiovascular diseases, etc.⁴ Besides the content of Ca in foods, its absorption is also an important factor influencing its bioavailability.⁵ In this regard, WG contain phytic and oxalic acids and phenolic compounds that are chelating agents that may decrease mineral bioavailability.⁶ Several technological processes have been applied to reduce the level of phytates: vegetable crops with reduced levels, germination,⁷ soaking,⁸ thermal processing (autoclaving, microwave cooking, boiling)⁹ and decorticating.¹⁰ Also, extrusion processes can reduce the content of anti-nutrient factors (e.g. phytates, tannins, and enzyme inhibitors).¹¹ Llopart et al.¹² studied the antioxidant effects of extruded WG-sorghum diets using a growing Wistar rat model. They reported that the low bioavailability of sorghum phenolics caused them to mainly exert acute antioxidant effects at the colon level. Despite these beneficial local effects, WG sorghum diets are rich in Ca absorption inhibitors (particularly in phenolic compounds and phytic acid), which can affect Ca bioavailability and bone health. However, there is no information about the effect of WG-sorghum diet on Ca absorption and bone health.

The aim of this work was to evaluate the effects of extruded WG-sorghum based diets on dietary Ca absorption and bone minerals of growing Wistar rats.

Materials and methods

Raw materials and extruded WG samples

Commercial red (RS) and white (WS) sorghum (Sorghum bicolor (L.) Moench), free of condensed tannins, were used for this study. Sorghum grains were ground in a Buhler MIAG roll mill (BUA AG, Uzwil, Switzerland) to produce WG grits with a particle size suitable for extrusion (1920–420 μm). The extrusion process was carried out according to Llopart et al.¹² Thus, red (RS) and white (WS) extruded WG-flours were obtained.

Composition of extruded WG-sorghum flours

An aliquot of extruded samples was milled using a Cyclone Samplemill (UD Corporation, Boulder, USA) with 1 mm mesh and analyzed. Proximate composition (moisture, ether extract, proteins, ash, and dietary fiber) was determined according to the AACC (American Association of Cereal Chemists)¹³ and the phytic acid (PA) content was determined according to the AOAC (Association of Official Analytical Chemists).¹⁴ Free (FPC) and bound (BPC) phenolic compounds were extracted according to Qiu et al.¹⁵ The content of phenolics in each extract was determined by the Folin–Ciocalteu method,¹⁶ using gallic acid as the standard. Total phenolic compounds (TPC) were calculated as the sum of FPC and BPC.

Animals and diets

Male Wistar rats (n = 24, 42.80 ± 4.87 g average initial body weight) were obtained from the Faculty of Biochemistry and Pharmacy of the University of Buenos Aires, Argentina (Animal Service Laboratory). Throughout the experiment, the rats were placed in a controlled room (12 h light/dark cycle) at 21 ± 1 °C and 60 ± 10% humidity, in individual stainless steel cages with free access to water and food. Each group (n = 8) was fed for 60 days with one of the following diets: (1) Control Diet (CD): rats fed with a semi-synthetic diet prepared according to the American Institute of Nutrition Diet (AIN 93)¹⁷ containing 5 g per 100 g cellulose; (2) Red Sorghum Diet (RSD): rats fed with AIN 93 diet containing 5 g per 100 g fibre from extruded WG-RS flour; and (3) White Sorghum Diet (WSD): rats fed with AIN 93 diet containing 5 g per 100 g fibre from extruded WG-WS flour (Table 1). The diets were isocaloric and supplied a similar amount of macronutrients, Ca (0.5 g per 100 g) and P (0.3 g per 100 g). The study was carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and was approved by the Committee of Health Guide for the Care and Use of Laboratory Animals of the Faculty of Biochemistry and Pharmacy of the University of Buenos Aires, Argentina.

Table 1 Composition of control diet (CD), red sorghum diet (RSD), and white sorghum diet (WSD)

Food components	Diets
	CD	RSD	WSD
Casein (g kg^−1)	200.00	128.20	134.60
AIN-93 mineral mix (g kg^−1)	35.00	34.00	34.00
AIN-93 vitamin mix (g kg^−1)	10.00	10.00	10.00
L-Cysteine (g kg^−1)	3.00	3.00	3.00
Vitamin A (mL kg^−1)	1.00	1.00	1.00
Soybean oil (g kg^−1)	69.00	64.60	63.50
Choline bitartrate (mL kg^−1)	7.10	7.10	7.10
Cellulose (g kg^−1)	50.00	—	—
RS (g kg^−1)	—	574.70	—
WS (g kg^−1)	—	—	553.10
Dextrin (g kg^−1)	624.90	177.40	193.70
Total energy (kcal kg^−1)	3745	3741	3736

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Body weight (BW) was recorded once a week while food intakes were recorded every three days during the experiment. Total intakes and efficiency of diets (weight gain/food intake) were calculated. Also, feces were collected and weighed every 15 days throughout the experiment.

At 60 days, the rats were killed after being anesthetized with an intraperitoneal injection of 50 mg per kg BW of ketamine hydrochloride + 10 mg per kg BW of xylazine. Then, right femurs were processed according to Albarracín et al.¹⁸

Bone mineral content and bone mineral density

Total skeleton bone mineral content (BMC), bone mineral density (BMD) and mineral densities of femur (F-MD), distal femur (dF-MD), tibia (T-MD), proximal tibia (pT-MD), and spine (S-MD) were determined in vivo, five days before the end of the experiment, under anesthesia (50 mg per kg BW of ketamine hydrochloride + 10 mg per kg BW of xylazine) as described by Albarracín et al.¹⁸

Apparent calcium absorption

Feces were collected, weighed during the last three days of the experiment, and then dried under infrared light and pounded. Also, Ca intake (Ca I) and Ca excreted (fecal Ca) were determined. Apparent calcium absorption (%Ca Abs_app) was calculated as follows:

%Ca Abs_app = [(Ca I − fecal Ca)/Ca I] × 100

Mineral contents of femur

The amounts of Ca and P were calculated as the percentage content of dried fat-free bone according to Weisstaub et al.¹⁹

Mineral analysis

Diets and feces were digested by wet ashing (nitric acid) using Parr bombs.²⁰ Calcium levels in diets, feces, and bones were determined by atomic absorption spectrophotometry (Analyst 300, PerkinElmer Corp, USA) with the addition of lanthanum chloride (65 g L⁻¹). Phosphorous concentration was measured according to the Gomori method.²¹

Statistical analysis

Data were presented as the arithmetic means ± standard error of the mean (SEM) for each treatment group (n = 8). Differences were tested by one-way analysis of variance (ANOVA) and statistical differences among the samples were determined using the least significant difference test (LSD) (p < 0.05).

Results and discussion

Characterization of extruded whole grain sorghum flours

Chemical composition of extruded WG-sorghum flours is shown in Table 2.

Table 2 Chemical composition, phytic acid (PA) content, free (FPC), bound (BFC) and total phenolic compounds (TPC) of extruded whole grain sorghum flours

Composition	Extruded sorghum flours
	Red sorghum	White sorghum
Mean ± SD. Values with different letters are significantly different (p < 0.05). GA: gallic acid.
Ash (g per 100 g)	1.59 ± 0.05	1.45 ± 0.07
Protein (g per 100 g)	11.22 ± 0.10	11.02 ± 0.02
Ether extract (g per 100 g)	2.72 ± 0.02	2.67 ± 0.10
Dietary fiber (g per 100 g)	8.07 ± 0.02	9.04 ± 0.03
PA (mg per 100 g)	603.36 ± 55.59^b	547.04 ± 26.96^a
TPC (mg GA per 100 g)	560.97 ± 5.85^b	505.33 ± 6.12^a
FPC (mg GA per 100 g)	116.31 ± 4.69^b	75.89 ± 3.03^a
BPC (mg GA per 100 g)	444.66 ± 19.31^a	429.48 ± 20.36^a

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The values were within the ranges reported in the literature for WG-sorghum flours.²² Regarding phenolics, TPC and FPC were higher in RS flour than in WS flour and more than 80% of them were found in the bound form, according to Awadelkareem et al.²³ Phytic acid was higher in RS flour than in WS flour, as was reported by Llopart and Drago.²² It has been reported that phytates have health promoting properties contributing to the prevention of heart disease, diabetes, and cancer.²⁴ However, phytates also have anti-nutrient properties since they can form insoluble complexes with minerals, reducing the absorption of cations such as Fe, Zn, Mg, and Ca.⁸

Food intake, efficiency, and feces excretion

Total food intake, body weight gain, efficiency, and feces excretion are shown in Table 3. Total food intake was higher in the rats consuming CD than WG-sorghum diets. Consequently, animals that consumed less food had a lower body weight gain at the end of the study. However, efficiency did not differ significantly among the diets. Similar results were observed in a previous study¹⁸ which used the same rat model and extruded WG maize based diet. Satiating effects of WG-sorghum were also reported by Stefoska-Needham et al.²⁵ These effects would be related to its high content of complex carbohydrates (fiber and starch), which are usually slowly digested increasing satiety and delaying hunger.²⁶ Nowadays, sorghum is considered to be a suitable ingredient in targeting satiety foods in order to assist with longer-term weight management.²⁷ Several studies in humans reported low body weight with the consumption of WG foods because of the promotion of satiety and a spontaneous decrease in food intake.^28–30

Table 3 Total food intake, body weight gain (BWG), efficiency and feces excretion of the rats fed with control diet (CD), red sorghum diet (RSD), and white sorghum diet (WSD)

Groups	Total intake (g per 60 days)	BWG (g per 60 days)	Efficiency (g BW per g diet)	Feces excretion (g solids per day)
Mean ± SEM (n = 8 per group). Values with different letters in a column are significantly different (p < 0.05).
CD	1115.62 ± 16.62^b	310.69 ± 4.78^b	0.26 ± 0.01	3.85 ± 0.13^b
RSD	985.71 ± 23.32^a	279.70 ± 6.17^a	0.29 ± 0.02	3.63 ± 0.10^b
WSD	931.35 ± 31.42^a	279.49 ± 7.30^a	0.28 ± 0.01	2.93 ± 0.10^a
p	0.0030	0.0055	0.2247	0.0000

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In relation to feces excretion, the rats fed with WSD excreted less feces. This group ate less compared to the CD group; therefore lower excretion was expected. However, the RSD group also ate less than the CD group but feces excretion did not differ significantly between them. RS has a higher content of phenolics than WS (Table 2) that can ferment in the colon, increasing the bacterial mass and consequently solid feces. It was reported that the consumption of RSD decreased the caecal pH to a higher extent compared to the consumption of WSD and CD (6.56, 6.84, and 7.28, respectively) confirming a higher fermentation.¹² Moreover, it is possible that phenolics and phytates of WG-RS react with other components of the diet resulting in less digestible compounds compared to those generated by WSD.³¹ Albarracín et al.³² also observed that feces excretion of the rats fed with a control diet with cellulose was significantly greater compared to extruded WG diets based on brown, soaked, and germinated rice. In addition, Macagnan et al.³³ observed a greater feces excretion with a control diet (cellulose) compared to diets containing fruit fibers.

Apparent calcium absorption

Table 4 shows Ca intake, fecal Ca, and %Ca Abs_app. Calcium intake was significantly lower in animals fed with WG-sorghum based diets than those fed with CD, which is related to the lower total intake in these animals. Also, animals consuming RSD showed the highest fecal Ca excretion and the lowest %Ca Abs_app compared to other diets. This could be due to the presence of inhibitors, such as PA and phenolic compounds, with higher contents in RS than in WS. There is strong evidence of the adverse effects of PA on Ca bioavailability.³⁴

Table 4 Ca daily intake, Ca daily fecal excretion and apparent Ca absorption of rats fed with control diet (CD), red sorghum diet (RSD), and white sorghum diet (WSD)

Groups	Ca daily intake (mg day^−1)	Ca daily fecal excretion (mg day^−1)	Apparent Ca absorption (%)
Mean ± SEM (n = 8 per group). Values with different letters in a column are significantly different (p < 0.05).
CD	144.22 ± 6.47^b	30.47 ± 0.71^a	72.71 ± 2.34^b
RSD	85.34 ± 6.31^a	44.36 ± 1.56^b	50.98 ± 1.85^a
WSD	67.98 ± 5.71^a	23.75 ± 2.31^a	64.75 ± 2.53^b
p	0.0001	0.0001	0.0000

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It has been reported that a PA/Ca molar ratio in the diet higher than 0.24 could compromise Ca bioavailability.³⁵ In this study, although the PA/Ca molar ratio was 3.22 and 3.81 for extruded WG red and white sorghum flours, respectively, when these flours were included in the diets, this ratio decreased to 0.072 and 0.066 for RSD and WSD, respectively. Thus, although PA can decrease Ca absorption,³⁶ free phenolic compounds could also inhibit it, as was observed for rats fed with RSD.

Mineral content and density of bones

No significant differences among the groups in relation to BMC and BMC related to body weight were observed (Table 5). When the BMC is expressed as a function of areas, BMD is obtained, which is a strong biomarker indicating the tendency to fracture.³⁷ No significant differences in the BMD of total body, spin, femur, distal femur, tibia, and proximal tibia among the groups were observed. These results suggest that the consumption during 60 days of these extruded WG-sorghum based diets did not affect the mineral content and density in comparison with CD, despite containing anti-nutrients such as PA and phenolic compounds and the lower Ca intake of the rats. Perhaps in a longer period of feeding, detrimental effects on the bone health would be observed. Albarracín et al.¹⁸ observed that the intake of extruded WG corn improved the BMC but not the BMD of animals. Body mineral density is usually determined in the column, femur, distal femur, tibia and proximal tibia. These areas are representative of bone, in addition to being the sites of frequent fractures associated with osteoporosis.³⁸

Table 5 Total skeleton bone mineral content (BMC) and bone mineral density of total body (BMD), spine (S-MD), femur (F-MD), distal femur (dF-MD), tibia (T-MD) and proximal tibia (pT-MD) at 60 days of rats fed with control diet (CD), red sorghum diet (RSD), and white sorghum diet (WSD)

Groups	BMC (mg)	BMC (mg per g BW)	BMD (mg cm^−2)	S-MD (mg cm^−2)	F-MD (mg cm^−2)	dF-MD (mg cm^−2)	T-MD (mg cm^−2)	pT-MD (mg cm^−2)
Mean ± SEM (n = 8 per group).
CD	4051.75 ± 204.35	11.79 ± 0.46	259.75 ± 2.09	242.50 ± 4.60	259.25 ± 7.85	252.50 ± 5.44	223.50 ± 2.95	219.50 ± 5.70
RSD	3962.38 ± 174.45	12.14 ± 0.44	262.38 ± 1.80	241.38 ± 5.81	265.75 ± 7.65	256.86 ± 8.53	221.86 ± 1.64	230.50 ± 4.68
WSD	4099.25 ± 313.21	12.78 ± 0.30	265.50 ± 1.27	232.00 ± 2.38	272.63 ± 4.66	252.38 ± 3.28	216.13 ± 2.33	218.38 ± 3.95
p	0.9243	0.3153	0.1473	0.2648	0.5016	0.8659	0.1223	0.1707

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Table 6 shows the composition of the right femur from animals consuming sorghum diets. The femur ash content and ash/OC ratio of animals consuming CD were lower than those fed with WG-sorghum diets, indicating that there was a lower mineral deposition in the collagen matrix of bone of the CD group. However, the femur of the rats consuming RSD presented a lower Ca content than the others, probably due to the lower apparent Ca absorption of this group. The mineral content of the femur is associated with bone strength and indirectly with the risk of fractures.³⁹ Calcium and phosphorus should be bioaccessible and in appropriate amounts in the diet for adequate bone mineralization. The Ca/P ratio was around 2.00 in the femur of the rats fed with CD and RSD but was higher in the rats fed with WSD. This Ca/P ratio (2.00) is the ratio found in bone of healthy human adults.⁴⁰

Table 6 Mineral composition of the right femur of the rats fed for 60 days with control diet (CD), red sorghum diet (RSD), and white sorghum diet (WSD)

Group	Ashes (mg 100 per g)	Ashes/OC	Femur Ca (mg per 100 g)	Femur P (mg per 100 g)	Femur Ca/P
Mean ± SEM (n = 8 per group). Values with different letters in a column are significantly different (p < 0.05). OC: Organic content.
CD	55.02 ± 0.33^a	1.22 ± 0.02^a	23.03 ± 0.56^b	11.93 ± 0.39^c	1.94 ± 0.06^a
RSD	59.48 ± 0.18^b	1.47 ± 0.01^b	14.42 ± 0.73^a	7.98 ± 0.27^a	1.81 ± 0.08^a
WSD	59.23 ± 0.19^b	1.45 ± 0.01^b	21.73 ± 0.76^b	9.06 ± 0.17^b	2.40 ± 0.04^b
p	0.0000	0.0000	0.0000	0.0000	0.0002

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Conclusions

Sorghum precooked flours have a great potential as food ingredients in numerous foods; hence it is interesting to study the impact of their consumption on health. Also, it could be used to develop special foods considering the satiating effects of WG-sorghum flours, since it was demonstrated that animals fed with extruded WG-sorghum (red or white) consumed less diet and gained less body weight, although the efficiency of all the studied diets were similar. Red sorghum consumption impacts apparent Ca absorption differently compared to white sorghum. However, no negative impact of extruded WG-sorghum was observed on bone health after 60 days of feeding, measured through BMC, BMD, and mineral density in different bones. Differences in the content of ash, organic matter, Ca, and P of the right femur among the diets would imply a certain imbalance in the metabolism of these minerals, particularly in those consuming RSD, since Ca and P contents were lower in this group. This could be related to the lower apparent absorption of this mineral. A study longer than 60 days could show a different impact on bone health. Moreover, it could be interesting to study the effects of refined sorghum flours on bone health.

Conflicts of interest

There are no conflicts of interest.

Acknowledgements

This work was supported by the Fund for Scientific and Technological Research (PICT 1282 project), the National Agency for Scientific and Technological Promotion, Argentina and the Universidad Nacional del Litoral (CAI+D 2016 PIC 50420150100092 project), Argentina. The authors are thankful to Ricardo Orzuza and Cecilia Mambrín for technical assistance.

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