Non-Peer ReviewedThe objectives of this study are to determine the effect of genotype and environment on iron
bioavailability in a set of five pea varieties differing in phytate concentration using the in vitro
digestion/Caco-2 human cell assay (Glahn 2009), to determine whether iron bioavailability in
field pea is heritable by evaluating recombinant inbred lines (RILs) differing in phytate
concentration using in vitro digestion/Caco-2 human cell assay, and to determine the effect of
the pea low phytate trait on chicken performance and iron bioavailability in chicken. In a
previous study, two low phytate pea lines (1-2347-144 and 1-150-81) were developed from
CDC Bronco at the Crop Development Centre, University of Saskatchewan (Warkentin et al.
2012). As a powerful chelator of iron, phytate can reduce the iron bioavailability in diets. The
low phytate peas may have increased iron bioavailability compared to the normal phytate
peas. In the first objective of this project, the iron bioavailability of pea seeds of the two low
phytate lines, their parent CDC Bronco and two other popular pea varieties in western Canada
(CDC Meadow and CDC Golden), derived from 3 replicate field experiments conducted in
2009 and 2010 at SPG (Saskatchewan Pulse Growers land), Outlook and Rosthern, were
assessed using the in vitro digestion/Caco-2 cell culture. The result shows that the iron
bioavailability of the two low phytate lines is significantly higher than the other three normal
phytate varieties, although their iron concentrations have not significant difference. The low
phytate line 1-2347-144 and CDC Meadow were crossed to develop RILs

Bett, K.

Classen, H.

Glahn, R.

Liu, X.

Warkentin, T.D.

English

University of Saskatchewan Research Archive

Iron Bioavailability in Low Phytate Pea Xiaofei Liu1, Kirstin Bett1, Hank Classen2, Raymond Glahn3 and Thomas D. Warkentin1*    1 Department of Plant Sciences, Crop Development Centre, University of Saskatchewan   2 Department of Animal and Poultry Science, University of Saskatchewan    3 Robert W Holley Ctr Ag & Health, USDA-ARS, Cornell University, Ithaca, New York   * Corresponding author: tom.warkentin@usask.ca  Key words: breeding and genetics, iron bioavailability, field pea, low phytate  Abstract:  The objectives of this study are to determine the effect of genotype and environment on iron bioavailability in a set of five pea varieties differing in phytate concentration using the in vitro digestion/Caco-2 human cell assay (Glahn 2009), to determine whether iron bioavailability in field pea is heritable by evaluating recombinant inbred lines (RILs) differing in phytate concentration using in vitro digestion/Caco-2 human cell assay, and to determine the effect of the pea low phytate trait on chicken performance and iron bioavailability in chicken. In a previous study, two low phytate pea lines (1-2347-144 and 1-150-81) were developed from CDC Bronco at the Crop Development Centre, University of Saskatchewan (Warkentin et al. 2012). As a powerful chelator of iron, phytate can reduce the iron bioavailability in diets. The low phytate peas may have increased iron bioavailability compared to the normal phytate peas. In the first objective of this project, the iron bioavailability of pea seeds of the two low phytate lines, their parent CDC Bronco and two other popular pea varieties in western Canada (CDC Meadow and CDC Golden), derived from 3 replicate field experiments conducted in 2009 and 2010 at SPG (Saskatchewan Pulse Growers land), Outlook and Rosthern, were assessed using the in vitro digestion/Caco-2 cell culture. The result shows that the iron bioavailability of the two low phytate lines is significantly higher than the other three normal phytate varieties, although their iron concentrations have not significant difference. The low phytate line 1-2347-144 and CDC Meadow were crossed to develop RILs.   Preliminary results:  In the first objective, soluble iron concentration was tested using atomic absorption spectroscopy (AAS). Soluble iron concentration is not equal to iron bioavailability, because the highly soluble form of iron, like mono-ferric phytate, can have little availability. In each environment (2009 SPG, 2009 Outlook, 2009 Rosthern and 2010 Rosthern), there was no significant difference in iron concentration between these five varieties (Table 1).     Table 1. Iron concentration (ppm) of 5 varieties grown in field trials at SPG, Outlook, and Rosthern in 2009, and at Rosthern in 2010.  Variety 2009 SPG 2009 Outlook 2009 Rosthern 2010 Rosthern 1-150-81 42.7 a 39.3 a 40.9 a 38.1 a 1-2347-144 40.6 a 37.4 a 40.9 a 40.4 a CDC Bronco 43.1 a 34.9 a 40.0 a 39.4 a CDC Golden 44.7 a 35.2 a 44.2 a 42.1 a CDC Meadow 44.2 a 38.4 a 43.7 a 40.5 a Mean 43.1 37.0 41.9 40.1 LSD0.05 5.3 3.8 3.1 4.1  The inorganic phosphorus concentration was determined using modified Chen’s reagent method (Chen et al. 1956). Table 2 shows that the concentration of inorganic phosphorus in the two low phytate lines was approximately 3 times greater than the concentration in the other varieties in each environment.  Table 2. Inorganic-P concentration (µg/gm) of 5 varieties grown in field trials at SPG, Outlook, and Rosthern in 2009, and at Rosthern in 2010.  Variety 2009 SPG 2009 Outlook 2009 Rosthern 2010 Rosthern 1-150-81 67 a 89 a 67 a 89 a 1-2347-144 64 a 82 a 61 a 83 a CDC Bronco 19 b 20 b 19 b 19 b CDC Golden 22 b 22 b 21 b 20 b CDC Meadow 25 b 22 b 24 b 19 b Mean 39 47 38 46 LSD0.05 10.4 6.6 9.4 7.9  Phytate concentration was tested using Modified Wade’s reagent method (Gao et al. 2007). Table 3 indicates that the two low-phytate lines contained approximately 0.5 to 0.7 times the phytate concentration of the normal phytate varieties.     Table 3. Phytate concentration (µg/gm) of 5 varieties grown in field trials at SPG, Outlook, and Rosthern in 2009, and at Rosthern in 2010.  Variety 2009 SPG 2009 Outlook 2009 Rosthern 2010 Rosthern 1-150-81 72 b 73 b 87 b 101 c 1-2347-144 64 b 67 b 85 b 94 c CDC Bronco 129 a 146 a 142 a 154 ab CDC Golden 142 a 145 a 146 a 163 a CDC Meadow 133 a 137 a 150 a 141 b Mean 108 114   122 131 LSD0.05 17.9 8.2 11.8 9.4  Figure 1 below indicates that iron bioavailability of the two low-phytate lines (1-150-81 and 1-2347-144) was 1.4 to 1.9 times higher than the other three normal phytate varieties (LSD =	  4.3, 2.52, 2.96, 2.23 respectively).     Figure 1. Mean iron bioavailability (ng ferritin/mg protein) of 5 varieties grown in field trials at SPG, Outlook, and Rosthern in 2009, and at Rosthern in 2010.  In conclusion, filed pea lines 1-150-81 and 1-2347-144 had lower phytate concentration, higher inorganic phosphorus concentration, and greater iron bioavailability in human cells 0.00	  5.00	  10.00	  15.00	  20.00	  25.00	  30.00	  35.00	  ng	  ferri(n	  /	  mg	  protein	  pea	  varie(es	  2009	  SPG	  a a bc c b 0.00	  5.00	  10.00	  15.00	  20.00	  25.00	  30.00	  35.00	  ng	  ferri(n	  /	  mg	  protein	  pea	  varie(es	  2009	  Outlook	  a a c c b 0.00	  5.00	  10.00	  15.00	  20.00	  25.00	  30.00	  35.00	  ng	  ferritn	  /	  mg	  protein	  pea	  varie(es	  2009	  Rosthern	  a cd bc d ab 0.00	  5.00	  10.00	  15.00	  20.00	  25.00	  30.00	  35.00	  ng	  ferritn	  /	  mg	  protein	  pea	  varei(es	  2010	  Rosthern a a b b	   b	  than normal phytate varieties. In this case, low phytate is a technique for biofortification of the pea crop. In the future study, the iron bioavailability of RILs developed from a cross between 1-2347-144 and CDC Meadow will be tested, and the iron bioavailability of low phytate and normal phytate pea varieties will be conducted in an in vivo chicken feeding study.   References:  Warkentin, T.D., Delgerjav, T., Arganosa, G., Rehman, A.U., Bett, K.E., Anbessa, Y., Rossnagel, B., and Raboy, V. (2012) Development and characterization of low-phytate pea.  Crop Sci. 52:74-78.    Chen P.S.,Toribara, T.Y. and Warner, H. 1956. Micro-determination of phosphorus. Analytical Chemistry. 28:1756-1758.   GaoY., Shang C., SaghaiMarrof M.A.,Biyashev R.M., Grabau E.A., Kwanyuen P., Burton J.W. and Buss G.R. 2007. A modified colorimetric method for phytic acid analysis in soybean. Crop Science.47(5): 1797-1803.  Glahn R.P. 2009. The use of caco-2 cells in defining nutrient bioavailability: application to iron bioavailability of foods. In: McClements D. and Decker E. Designing functional foods: measuring and controlling food structure breakdown and nutrient absorption. Cambridge, UK: Woodhead Publishing Limited. p. 340-361.   

Iron bioavailability in low phytate pea

eCommons@USASK

Non-Peer ReviewedPhytate is the main storage form of phosphorus in the seeds of most crops. Phytate is not well digested by monogastrics and it chelates iron, zinc and some other micronutrients. To increase the nutritional value of pea seeds, two low phytate lines (1-150-81 and 1-2347-144) were developed from CDC Bronco in previous research. In this study, an in vitro digestion/Caco-2 cell culture bioassay was used to simulate the iron absorption of peas in humans, as the cell line originated from human colon adenocarcinoma cells. The iron bioavailability of the two low-phytate lines was 1.4 to 1.9 times higher than that of three normal phytate varieties, while having the same total iron concentration. In vivo studies were used to evaluate iron absorption of chickens fed low phytate and normal phytate pea diets. The diets containing the two low-phytate pea lines had no significant effect on chicken body weight and hemoglobin level, compared with the diets containing normal phytate pea cultivars, however, iron deficiency was suspected in all diets used

Iron bioavailability in low-phytate pea

Iron Bioavailability in Low-phytate Pea Xiaofei Liu(1), Hank Classen(2), Kirstin Bett(1), Raymond Glahn(3) and Thomas D. Warkentin(1)* (1) Crop Development Centre/Department of Plant Sciences, University of Saskatchewan (2) USDA-ARS, Cornell University, Ithaca, NY, USA. (3) Department of Animal and Poultry Science, University of Saskatchewan, * Corresponding author: tom.warkentin@usask.ca Key Words: iron bioavailability, field pea, low phytate, Caco-2 cell culture, in vivo chicken study Abstract: Phytate is the main storage form of phosphorus in the seeds of most crops. Phytate is not well digested by monogastrics and it chelates iron, zinc and some other micronutrients. To increase the nutritional value of pea seeds, two low phytate lines (1-150-81 and 1-2347-144) were developed from CDC Bronco in previous research. In this study, an in vitro digestion/Caco-2 cell culture bioassay was used to simulate the iron absorption of peas in humans, as the cell line originated from human colon adenocarcinoma cells. The iron bioavailability of the two low-phytate lines was 1.4 to 1.9 times higher than that of three normal phytate varieties, while having the same total iron concentration.  In vivo studies were used to evaluate iron absorption of chickens fed low phytate and normal phytate pea diets. The diets containing the two low-phytate pea lines had no significant effect on chicken body weight and hemoglobin level, compared with the diets containing normal phytate pea cultivars, however, iron deficiency was suspected in all diets used. Introduction: Field pea is an annual cool-season herbaceous legume crop. Canada is the leading field pea producing country. Saskatchewan is the top producing province of field pea within Canada1. Field pea contains high level of protein, lysine, slowly digestible starch and fibres. However, it also contains phytate, a main storage form of phosphorus in seeds, which is also a strong chelator which can bind with K, Zn, Fe and some other micronutrients. Phytate is poorly digested by human and monogastric animals, due to the limited enzyme, phytase. Iron (Fe) is an essential element for human, iron-deficiency and iron-deficiency anemia can cause poor work performance and decreased immunity.  In previous study, two low-phytate lines (1-150-81 and 1-2347-144) were developed from CDC Bronco2, which are confirmed to have increased inorganic phosphorus level. In this paper, two studies are included to evaluate if the two low-phytate lines also have additional benefit to increase iron bioavailability. Materials and Methods: The first study is to determine the effect of genotype and environment on iron bioavailability in varieties differing in phytate concentration. Two low-phytate lines (1-150-81 and 1-2347-144) and other three normal phytate cultivars (CDC Bronco, CDC Golden and CDC Meadow) were tested to compare the concentration of inorganic-phosphorus, phytate-phosphorus, and total iron using modified Chen’s reagent method3, modified Wade’s reagent method4 and atomic absorption spectroscopy (AAS), respectively. The iron bioavailability was tested using the in vitro digestion/Caco-2 cell culture model5. The second study is to determine the effect of the pea low phytate trait on chicken performance and iron bioavailability in chicken. The same two low-phytate lines (1-150-81 and 1-2347-144) and the other two normal phytate cultivars (CDC Bronco and CDC Meadow) were mixed in the diets respectively with total 4 treatments. Birds were distributed using RCBD with six replications per treatment and 4 birds per replication. Before day 7, the birds were fed with commercial diet and the treatments (ingredients: Peas: 87.01%, Canola oil: 7.2%, Salt: 0.44%, Calcium Phosphate: 0.67%, Limestone: 2.1%, Vit/Min premix (FE free): 0.5%, Choline Chloride: 0.1%, Celite: 1.5%, DL Methionine: 0.48%) started from day 7 and end on day 35. In the end of each week the bird body weight and feed intake were recorded and blood samples were collected for hemoglobin test. Results and Discussion: In study one, the concentration of inorganic-phosphorus in the two low phytate lines was approximately three times greater than in the other varieties in each environment, meanwhile, the two low-phytate lines contained approximately 0.5 to 0.7 times the phytate-phosphorus concentration of the normal phytate varieties. The iron bioavailability of the two low-phytate lines was 1.4 to 1.9 times higher than that of three normal phytate varieties, while having the same total iron concentration. Also environmental effect on iron bioavailability is significant (p-value=0.0153).  The chicken study showed that there was no significant difference of bird’s body weight and the hemoglobin level among four treatments. Meanwhile, the mortality rate and the sick bird rate were higher compared with other trials which were done previously. Meanwhile the hemoglobin level is around 9.1g/dl, which is also lower than 10.5 ~11.5g/dl in the study of Hoekenga et al. (2011)6. Based on the symptom of the sick birds, an iron-deficiency was believed that happen to the birds due to no additional iron added in diet.  So the next steps of this study will be to determine the ideal iron level in the diets for iron bioavailability comparison, and to test another objective that iron bioavailability is heritable in RILs (PR-15) (1-2347-144 X CDC Meadow).   References:  1. Agriculture and Agri-Food Canada.2008. Crop profile for field peas in Canada – March 2008 (42 pp) – synopsis [Online] Available: http://www4.agr.gc.ca/AAFC-AAC/display-afficher.do?id=1181751017184&lang=eng[Accessed on April 14, 2011]. 2. Warkentin, T.D., Delgerjav, T., Arganosa, G., Rehman, A.U., Bett, K.E., Anbessa, Y., Rossnagel, B., and Raboy, V. (2012) Development and characterization of low-phytate pea.  Crop Sci. (In press). 3. Chen P.S.,Toribara, T.Y. and Warner, H. 1956. Micro-determination of phosphorus. Analytical Chemistry. 28:1756-1758.  4. GaoY., Shang C., SaghaiMarrof M.A.,Biyashev R.M., Grabau E.A., Kwanyuen P., Burton J.W. and Buss G.R. 2007. A modified colorimetric method for phytic acid analysis in soybean. Crop Science.47(5): 1797-1803. 5. Glahn R.P. 2009. The use of caco-2 cells in defining nutrient bioavailability: application to iron bioavailability of foods. In: McClements D. and Decker E. Designing functional foods: measuring and controlling food structure breakdown and nutrient absorption. Cambridge, UK: Woodhead Publishing Limited. p. 340-361. 6. Hoekenga O.A., Lung’aho M.G., Tako E., Kochian L.V. and Glahn R.P. 2011. Iron biofortification of maize grain” Plant Genetic Resources: Characterization and utilization. 1-3.  

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Iron bioavailability in low phytate pea

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