Bioavailability of Zinc in Bread Wheat Grown in the Calcareous Soils of the Kurdistan Region of IRAQ


 A survey of wheat grain from 120 fields in Sulaimanyah province, Kurdistan region, Iraq was undertaken in 2017. We evaluated the concentrations of phosphorus (P), Zn and phytic acid (PA) and the estimated Zn bioavailability in wheat grain grown in the calcareous soils of the region. Concentrations of P in wheat grain were higher than the recommended value of 3570 mg kg− 1; PA was within the range of other countries but Zn concentration in 88% of wheat grain samples fell below the value recommended for human nutrition of 41.6 mg kg− 1. Bioavailable Zn intake (mg d− 1) was estimated assuming bread consumption of 300 g day− 1 in the region. Considering PA and Zn intake suggested only 21.5 ± 2.9% of whole grain flour Zn was bioavailable. Thus the effective Zn intakes from whole wheat was only 1.25–2.05 mg d− 1 for the local population. Typical dietary Zn was below recommended levels (11 mg d− 1), due to low soil Zn uptake by wheat and the large concentrations of PA in wheat grains, probably from over-use of phosphate fertilizer.


Introduction
In calcareous soils, the bioavailability of zinc (Zn) limits crop production due to the presence of CaCO 3 as an adsorbent for Zn 2+ ions, high pH values which encourage Zn 2+ adsorption on Fe oxides and humus, and the application of P fertilizer which may co-precipitate with Zn 2+ (Duffner et (Fageria, 2001; Mousavi and Galavi, 2012). To explain the interactions between P and Zn, (Fageria, 2001; Mousavi and Galavi, 2012) reported three mechanistic hypotheses including: (i) interference by P at the level of plant metabolism including effects on uptake, utilization and translocation of Zn, (ii) variation in the P/Zn ratio due to changes in dry matter production with P application and (iii) soil P and Zn interactions. Phosphate can also cause a decrease in the bioavailability of Zn within wheat grains, by decreasing the total concentration of Zn and increasing the molar ratio of  (Bouis et al., 2011;Chen et al., 2017). However, the total Zn concentration of wheat grain is not the only factor controlling dietary assimilation of Zn. The presence of 'anti-nutrient' compounds, such as PA, is one of the major considerations when evaluating the nutritional quality of crops (Poblaciones and Rengel, 2016) and the proportion of Zn that is readily absorbed (Magallanes-Lopez et al., 2017). To determine Zn bioavailability in foods, it is necessary to measure the molar ratio of PA to Zn. A decrease in this molar ratio increases Zn bioavailability.
Wheat variety, growing conditions, and location are all factors that may cause differences in the bioavailability of Zn (Gabaza et al., 2018). The wheat growing area in the Kurdistan Region amounts to about 570,000 ha; approximately 500,000 t is harvested every year and wheat production is mostly rainfed (Mazid, 2015). Therefore, the aims of this survey were: (i) to determine the bioavailability of P and Zn in wheat grain; (ii) to evaluate PA concentration, the PA/Zn molar ratio and the estimated Zn bioavailability (to humans) in wheat cultivars consumed in Sulaimanyah province; (iii) to evaluate the nutrient status of wheat grain grown in the calcareous soils of Kurdistan.

Wheat survey in Sulaimanyah province
A total of 120 wheat grain samples were collected from elds in ten locations in Sulaimanyah province, Kurdistan region, Iraq during May-June 2017. The number of samples taken from each location was dependent on the size of a location area. Locations in selected for wheat grain sampling (Table 1) were chosen on the basis of agricultural activity (predominantly wheat production), wheat variety and geographical location. Adana and Aras are the two main wheat varieties grown in the Kurdistan region along with other varieties such as Rashidi, Barcelona, and Wa a ( Table 1). In each eld selected, wheat grain samples were collected from ve sampling points within an area of 100 m 2 . The ve samples were combined to produce a composite sample and sealed in plastic bags for transport to a laboratory at the Halabja Technical College of Applied Science. Wheat grain samples (n = 120) were collected in a Wtransect with each sample consisting of 15-20 cereal heads. Wheat heads were removed from the straw and threshed by hand to remove the outer grain husk, mixed, and a representative sample retained.
Approximately 10 g of each sample was then stored in a plastic bag labelled and transported to the University of Nottingham, UK. A portion of each sample was nely ground using a centrifugal mill (Retsch, Model ZM200) prior to analysis.

Determination of Phytic acid (PA)
Phytic acid (PA) was determined using a Megazyme kit (K-PHYT, Megazyme, and Bray, Ireland) (Electronic appendix 1). The molar ratios of PA and Zn were determined by converting from gravimetric concentrations (phytate = 660.8 g mol − 1 and Zn = 65.38 g mol − 1 ) and dividing PA concentration by Zn concentration. Equation 3 has two predictor variables: total daily intake of Zn (TDZ; millimoles d − 1 ) and PA (TDP) and the response variable is total absorbed Zn (TAZ). The model has three parameters, including maximal absorption (A MAX =0.091), a Zn-transporter binding equilibrium dissociation constant (K R =0.680) and a Zn-phytate binding equilibrium dissociation constant (K P =0.033).

Statistical analysis
The data were analyzed using SPSS 24.0 for Windows. A one-way analysis of variance (ANOVA) followed by a least signi cant difference (LSD) test (p < 0.05) was performed to test the signi cance of treatments.
The relationships between variables were determined via Pearson's correlation analysis.

Phosphorus and Zn concentrations in wheat grain
At harvest, the average concentrations of P (mg kg − 1 ) in wheat grain ranged from 2890 ± 694 to 3630 ± 490 for PIR and BAR locations respectively; there was a signi cant variation (p < 0.05) between locations ( Fig. 1). About 40% of wheat grain samples contained higher concentrations than the recommended value of 3570 mg kg − 1 of P (USDA Food Composition Databases, 2018), re ecting the high solubility of P in some of the soils probably due to the application of P fertilizer in each growing season by farmers.
Alam and Azam Shah (2002) conducted a greenhouse experiment to study the in uence of phosphate fertilization on P concentration in wheat gown in calcareous soils; they found that application of 40 kg kg − 1 of P as SSP increased P concentration in wheat grain from 1978 to 2942 mg kg − 1 .
The average Zn concentration in wheat grain (mg kg − 1 ) ranged from 26.3 ± 7.37 to 35.0 ± 6.11 for CHA and HAL locations respectively (Fig. 2). 3.2 Phytic acid and PA/Zn molar ratio in wheat grain Figure 3 presents PA concentrations (g 100g − 1 ) in wheat grain samples; signi cant differences (p < 0.05) were found between the ten sampling locations. In the present survey, the average PA content in the whole wheat grains ranged from 0.54 ± 0. 16   The regional average PA/Zn molar ratio for whole wheat grain ranged between 15.7 ± 5.06 and 30.6 ± 6.18 for HAL and BAR locations respectively (a 2-fold variation) as shown in (Fig. 4) Anatolia, Turkey. They reported that the application of 23 kg Zn ha − 1 to soils reduced the PA contents of wheat grain from 1.07 to 0.91 g 100g − 1 and PA/Zn molar ratio from 126 to 55. These results were due to a reduction in P concentration in wheat grain from 3900 to 3500 mg kg − 1 and increasing Zn concentration from 14 to 23 mg kg − 1 . Probably in the calcareous soils of Kurdistan, P de ciency occurs due to high calcium content. Therefore, when samples collected and farmers interviewed. They apply phosphate fertilizer to improve yield which causes an increase in PA content in wheat grain and in the resulting PA/Zn ratio.
The PA/Zn molar ratio has been used as an indicator of Zn bioaccessibility by the International Zinc Nutrition Consultative Group (IZiNCG). Considering the PA/Zn molar ratios observed in the current survey, and in previous studies on calcareous soils, it is apparent that ratios are not ideal when durum wheat is the staple food and principal source of dietary Zn. The relatively high PA/Zn molar ratio in the current survey (average of 24.3 ± 8.59) suggests that wheat bioforti cation with Zn and decreasing the application of P fertilizer are urgent priorities in the Kurdistan region. Developing cereal cultivars with low PA contents and a high a nity for Zn would also be useful strategies.

Estimated Zn bioavailability in wheat grain
Estimated Zn bioavailability (Eq. 3), in mg d − 1 assuming consumption of 300 g wheat per day, ranged from 1.84 ± 0.08 for BAR to 2.65 ± 0.05 for HAL locations respectively, as shown in ( On average, about 23.3 ± 3.48% of grain Zn was actually bioavailable. Low bioavailability of Zn in the wheat was probably due to high PA/Zn molar ratio caused by a high concentration of P in wheat grains and low Zn concentrations. Li et al. (2015) conducted a two-year eld experiment to investigate the effects of P and Zn fertilization on Zn bioavailability in wheat grain. They found that estimated Zn bioavailability in whole grain was greater in the Zn-alone treatments than in combined P + Zn treatments. Table 3 Estimated Zn bioavailability in mg d -1 for consumption of 300 g wheat grain (Eq. 3). Samples were collected from ten different locations (

Conclusions
In approximately 88% of samples the concentration of Zn in wheat grain was lower than the recommended value, as expected from the low availability of Zn in the Kurdistan regional soils. By contrast the P concentrations were higher than the recommended level in about half of the samples. The composition of the wheat grain, could reasonably be explained as a consequence of large annual applications of P fertilizer. The PA and PA/Zn molar ratio in wheat grain samples were in the range reported globally. However, the bioavailability to human populations of Zn in wheat grain was low due to high PA concentrations and PA/Zn molar ratios. Results of this survey suggest that foliar application of Zn in wheat farms could have a signi cant positive effect on cereal-based food quality grown in the calcareous soils of the Kurdistan region. The problem of low Zn in wheat in the region is not one of yield restriction but rather of human nutrition. Concentration of phosphorus in wheat grain samples (n=120) collected at ten locations (Table 1) in Sulaimanyah province, Iraq.

Figure 2
The concentrations of Zn in wheat grain samples (n=120) collected at ten different locations in Sulaimanyah province, Iraq.

Figure 3
Page 14/15 Range of Phytic acid (PA) concentrations in wheat grain samples (n=120), collected from ten different locations in Sulaimanyah province, Kurdistan region, Iraq.

Figure 4
Phytic acid to Zn molar ratio in wheat grain samples (n=120), collected at ten different locations in Sulaimanyah province, Kurdistan region, Iraq.