Practical 4: Determination of Phytate in Food and Effect of Phytic Acid on Calcium Absorption

Name: Hung Wing Tung (2009053464)Group No.: 3Date: 1/4/2011

BIOL2537 Laboratory in Nutritional Science

10

AbstractThe phytate content in different food samples and the effect of phytic acid on calcium absorption in a total of 54 University students were determined. To determine phytate content, 5 g of 8 food samples were diluted and 1 mL of diluted samples were extracted and allowed to react with NH4Fe(SO4)2. After boiling and cooling, 0.1 mL KCNS and 2 mL of tertiary amyl alcohol were added separately. Absorbance of sample mixtures at 465 nm was read after 60 minutes for comparison with standard phytate. Bran flake and oat bran meal were found to have the highest phytate content, while corn flake and white bread were having the lowest. The influence of phytic acid on calcium absorption were investigated by feeding 2 groups of subjects with corn flake (low phytate) and bran flake (high phytate) respectively after milk consumption. Urine excreted within 4 hours were collected and weighed, then diluted and reacted with 0.5% Lanthanum Chloride. Absorbance of urine sample mixtures was read using the Atomic Absorption Spectrophotometer for comparison of standard calcium. Calcium excreted in 4 hours for low phytate group was 44.75 24.68 mg/mL (mean SD), while that for high phytate group was 41.50 19.86 mg/mL. Intake of high phytate food did not significantly lower the calcium excretion (p = 0.6055). Therefore, it is concluded that the difference in phytic acid content in foods does not have a significant effect on calcium absorption.

IntroductionMicronutritent deficiency is a global problem, with developing countries being the most susceptible to1. Although a rich source of these trace elements can be found in unrefined cereals2, the cereals also contain phytic acid 3. Phytic acid is an organic phosphorous compound (myo-inositol hexaphosphate, InsP6) commonly occurred in cereals (up to 5% by weight)4. It reduces the bioavailability of many essential trace elements, including iron, zinc, calcium, manganese and magnesium by formation of insoluble salts2. This causes reduced mineral absorption.

There have been several studies on the effect of phytate intake on calcium absorption, showing that phytate did influence calcium absorption5-8. For example, McCance and Widdowson found that the apparent calcium absorption in subjects fed brown bread was lower than subjects fed white bread8. Studies conducted by Heaney, Weaver & Fitzsimmons and Weaver CM et al. using radioisotope technique also found that calcium absorption in subjects fed with low phytate diets was better than those fed with high phytate diets8,9.

Inadequate absorption of calcium can result in deficiency in long term. Deficiency of calcium can lead to poor bone and teeth formation, osteoporosis, difficulties in blood clotting, etc10, which greatly affects ones health. In developing countries, since consumption of cereals has already contributed 90% of daily energy intake of the people11, calcium deficiency is dominant. Therefore, to prevent deficiency of calcium and other essential minerals, it is important to examine the content of phytic acid in foods, especially cereal products. In this study, the relationship between phytic acid and calcium absorption will also be investigated.

Materials and Methods

Part A: Determination of phytate in food

Collection of Materials8 types of food samples (white bread, whole-meal bread, honey oat bran, corn flake, bran flake, oat meal, oat bran meal and bean curd) were purchased from the School of Biological Sciences, University of Hong Kong for experimental uses. A list of reagents (0.5M HNO3, tertiary amyl alcohol, NH4Fe(SO4).12H2O, 10% KCNS and phytate standard 100 g/mL) and spectrophotometers for reading absorbance were also provided by the School.

Phytate Extraction5 g of each food samples were accurately weighed. They were then extracted with 50 mL of 0.5M HNO3 and shaken for 3 hours. Each food sample mixture was centrifuged by a bench centrifuge and the supernatant of food each sample was filtered through No. 54 Whatman paper. The filtrates were frozen in 4 mL aliquots.

Determination of phytateA series of standard solutions which contained 0, 10, 25, 50, 70 and 100 g/mL of phytate was run. Meanwhile, the aliquots of filtrates for different food samples were extracted and diluted with various amount of distilled water (summarized in Table a), so thateach sample was calculated to contain 50 100 g phytate.

Table a. Dilution required for food samples to get a range of 50-100 g phytateFood SampleVolume of Sample added (L)Volume of Water added (L)

White bread10000

Whole-meal bread100900

Honey oat bran bread100900

Corn flake10000

Barn flake20980

Oat meal100900

Oat bran meal40960

Bean curb100900

1 mL of phytate standards and diluted food samples were allowed to mixed with 1 mL of NH4Fe(SO4)2 in 0.5M HNO3 containing 50 g Fe/mL respectively. The mixtures were then placed in a hot water bath and boiled for 20 minutes. After cooling, 2 mL of tertiary amyl alcohol was added into each mixture, followed by 0.1 mL of 10% KCNS solution in the fume cupboard. Resulting mixtures were covered the by parafilm and mixed gently. Contents in the mixtures were then allowed to separate for 60 minutes.Red amyl alcohol layers formed on top of each mixture were pipetted for reading the absorbance at 465 nm. 2 mL of tertiary amyl alcohol was used as the blank. The phytate content in each food sample was then determined from the standard curve prepared using phytate standards.

Part B: Effect of Phytic Acid on Calcium Absorption

Collection of MaterialsPacks of corn flake and bran flake were purchased by the School of Biological Sciences, University of Hong Kong. Besides, urine bottles for collection of 4-hour urine, 0.5% Lanthanum Chloride and the Atomic Absorption Spectrophotometer for reading absorbance were also provided by the School.

Subjects54 students from the University of Hong Kong voluntarily participated in this study. On the day of experiment, this group of students was required to empty their bladders beforeconsumption of the test diet. They were randomly assigned to take in either 50 g of corn flake (low phytate) or 50 g of bran flake (high phytate) after drinking 1 box High Calcium Diary Farm Milk. They had to collect all urine excreted within 4

hours of consumption. During the experimental period, consumption of other food was not allowed.

Determination of Calcium in urineThe total volume of urine excreted from each subject was measured. 0.4 mL of urine sample was pipetted. The sample was then mixed and diluted with 3.6 mL of 0.5% Lanthanum Chloride to add up a total volume of 4 mL. Absorbance of the sample was read using the Atomic Absorption Spectrophotometer. Hence, the concentration of calcium excreted in urine for each subject was determined from the calcium standard curve.

Results Analysis

Part A: Determination of phytate in foodAbsorbance of phytate standards at 465 nm were read, the readings were summarized in the following:

Table b. Absorbance of phytate standards at 465 nmConcentration of phytate (ug/mL)Absorbance at 465nm

02.086

101.816

251.650

501.455

751.150

1000.953

Hence, the standard curve of phytate was outlined for the determination of phytate content in different food samples.

Referring to the equation y = -0.0107 x + 1.9815 of the curve in Fig a, the phytate concentration in food extract (g/mL) was estimated. The original phytate content in the food samples (mg/g) was then calculated as follows:

*Original phytate in food sample (mg/g) = (Phytate concentration estimated from standard curve x 50 mL x dilution factor / 5 g) / 1000

The values were summarized in Table c. on the next page.

Table c. Original phytate content of the food samplesFood SampleDilution FactorAbsorbance at 465 nmPhytate Concentration Estimated from Standard Curve (g/mL)Original Phytate Concentration (mg/g)

White bread1X1.18774.250.74

Whole-meal bread10X1.35358.745.87

Honey oat bran bread10X1.75321.362.14

Corn flake1X1.46748.080.48

Barn flake50X0.98293.4146.71

Oat meal10X1.27765.846.58

Oat bran meal25X0.97394.2523.56

Bean curb10X1.59236.43.64

The original phytate content in the food samples obtained were illustrated in the bar chart below for comparison:

Part B: Effect of Phytic Acid on Calcium Absorption

Personal results

Table d. Effects on personal calcium absorption after consumption of high phytate dietType of food consumedVolume of urine in 4 hour (mL)AbsorbanceTotal Calcium excreted in 4 hours (mg)

Bran flake99.240.01812.11

Referring to the nutrition facts of the High Calcium Diary Farm Milk, the milk contains 170 mg/100 mL of calcium, and the total volume of milk is 236 mL. Hence, percentage of ingested calcium excreted was calculated as follows:

% of ingested calcium excreted= total calcium excreted in 4 hours (mg) / total calcium ingested from milk (mg) x 100%= (12.11 / 170 x 2.36) x 100%= 3.02%

Class results (Statistical Analysis)

Table e. Effects on class calcium absorption after consumption of low and high phytate dietsType of food consumedCalcium excreted in 4 hour (mg/mL)

Corn flake (low phytate)44.75 24.68

Bran flake (high phytate)45.45 23.80

Values are means SD of the mean (n=27 subjects in each group).

Since 2 data in the group fed with bran flake (high phytate group) were greater than mean 2 SD, they were conceived as statistically irrelevant and ruled out. The new means and SD were obtained after discarding the 2 data.

Table f. Effects on class calcium absorption after consumption of low and high phytate dietsType of food consumedCalcium excreted in 4 hour (mg/mL)

Corn flake (low phytate)44.75 24.68

Bran flake (high phytate)41.50 19.86

Values are means SD of the mean (n=27 subjects in low phytate group; n=25 subjects in high phytate group).

The new set of data was then analyzed statistically by conducting t-test (paired data, 2 tails). The p-value obtained was 0.6055, which was > 0.05 (did not differ statistically).

From the formula % of ingested calcium excreted = total calcium excreted in 4 hours (mg) / total calcium ingested from milk (mg) x 100%, the percentage of ingested calcium excreted from different groups were calculated and summarized in the Table g.

Table g. Percentages of ingested calcium excreted after consumption of low and high phytate diets

Type of food consumed% of ingested calcium excreted

Corn flake (low phytate)11.15% 6.15%

Bran flake (high phytate)10.34% 4.95%

Values are means SD of the mean (n=27 subjects in low phytate group; n=25 subjects in high phytate group).

Discussion

Part A: Determination of phytate in foodPhytate or phytic acid in food can bind with ferric ions in dilute mineral acid to precipitate a complex ferric phytate11. The residual iron in the mineral acid will react with thiocyanate (CNS) to give a deep red solution, while the complex will not have any reaction11. Therefore, the color intensity of the solution mixture is positively related to the amount of phytate in food (i.e. the absorbance of the mixture is negatively related to the phytate content in food).

Comparison of Phytate Content in Different Food SamplesWith reference to Fig b., the phytate content of the food were ranked as follows in descending order: bran flake (46.71 mg/g), oat bran meal (23.56 mg/g), oat meal (6.58 mg/g), whole-meal bread (5.87 mg/g), bean curb (3.64 mg/g), honey oat bran bread (2.14 mg/g), white bread (0.74 mg/g) and lastly corn flake (0.48 mg/g). Among all the food samples, bran flake had the highest phytate content, while corn flake had the lowest phytate.

Part B: Effect of Phytic Acid on Calcium AbsorptionCalcium in blood has the ability to bind with phytic acid to form Ca-phytate4, which is a large molecule that cannot pass through the glomerulus in kidney. Therefore, upon uptake of high phytate food, more calcium in blood will bind to the phytic acid and less will be excreted in urine. This binding also reduces the bioavailability of calcium for absorption theoretically.

Calcium excreted in urine can be determined by direct dilution with Lanthanum Chloride using an Atomic Absorption Spectrophotometer. Lanthanum serves to remove the anions bound to calcium so that the calcium will be more available for analysis13. The operating mechanism of an Atomic Absorption Spectrophotometer is illustrated in the following. According to Willis14, the light emitted from the cathode of spectrophotometer is first focused at the center of the flame, so as to aspirate the sample. Then the light is refocused onto the entrance slit to pass the appropriate resonance line (Ca: 4227 A). The signal from a photomultiplier behind the exit slit is amplified by an alternating current amplifier and read on a microammeter. By monitoring the amplifier gain, the absorbance of the atomized sample is known. Since the absorbance is positively related to the concentration of calcium in the urine sample, total calcium excreted in urine can be determined against the calcium standard curve.

Comparison between Individual and Class results in Total Calcium ExcretionMy results in total calcium excreted after consumption of bran flake (high phytate) (12.11 mg) was lower when compared to the class results for high phytate group, which was 41.50 19.86 mg (mean SD). This could be attributed to biological variation in calcium retention and difference in volume of urine excreted. The variations among subjects which affected the overall class results will be discussed in the next section.

Limitations of the Experiment (Sources of Error)The negative relationship between phytate intake and calcium absorption has been shown in many previous studies5-8. In this study, however, consumption of food with great difference in phytate content (bran flake and corn flake) did not show a significant difference in total calcium excreted in urine (p > 0.05). Thus, there must be some errors leading to this contradictory result. Biological variations were believed to be the most determining factor. First, different subjects had different calcium concentration in blood, and hence different amount of calcium excretion. Different individual absorptive efficiencies could account for a great part of the class results variations15. The concentration of parathyroid hormone (PTH) secreted to blood also altered the amount of calcium excreted16. Apart from biological variations, there was also a possibility of non-adherence to the diets among subjects. For example, subjects did not take in all test meals assigned or they consumed other food during the experimental period, which could influence the amount of dietary phytate or calcium. Likewise, some subjects might not adhere to the instructions of urine collection that they did not collect all the urine within 4 hours of consumption. Besides, poor handling of urine samples by some experimenters, such as wrong dilution of urine samples, might also occur and affect the final absorbance reading.

Common High Phytate Foods in Hong KongPhytate can be found in a wide variety and by-products of cereals and nuts4. Some common high phytate foods in Hong Kong are barley (0.97 1.08% of dry weight4), oat (0.84 1.01% of dry weight4), peanuts (5% of phytate was found in an aleurone grain-containing protein-rich fraction4), rice (primarily in the outer layers of rice grain, and to be more specific, 2% of outside kernel was found to contain 23 times more phytate than the intact kernel4), soya beans (1.00 1.47% of dry weight4) and wheat (0.62 1.35% of dry weight in whole kernel, the bran portion was found to contain 4.59 5.52% of phytate4).

Dietary Habits Which Have a Higher Risk of Mineral DeficiencyApart from a high phytate diet (e.g. diet containing many unrefined cereals), there are also many diet routines that may cause mineral deficiency. Here are some examples of them:

A vegetarian diet may cause deficiency in calcium16 since a wide range of vegetables (e.g. spinach, eggplants), fruits (e.g. strawberries, blackberries) and nuts (e.g. peas, peanuts) contain oxalates that can chelate calcium. Since the resulting complex is insoluble and cannot be absorbed in the intestine, fecal calcium excretion will increase. Oxalates also reduce iron, zinc and copper absorption by chelation16. Besides, consumption of fruits and vegetables which contain a high concentration polyphenols (e.g. apples, broccoli) can also reduce iron and zinc absorption16.

A high sodium diet (e.g. eating a lot of salty or processed food) can impair calcium absorption by increasing urinary excretion16. It was found that 20 60 mg of calcium will be lost in urinary excretion daily given a sodium load of 2.3 g per day17.

Other factors such as alcoholism and high intake of caffeine may contribute to mineral deficiency. Although there is no clear mechanism on the effect of alcohol on calcium absorption, study has shown that alcohol consumption is significantly related to bone loss in men18. Caffeine, on the other hand, can reduce the renal reabsorption of calcium and increase its excretion in urine16.

References1. Lukaski HC (2000) Magnesium, zinc, and chromium nutriture and physical activity. Am J Clin Nutr 72: 585S 93S.2. Fredlund K et al. (2006) Absorption of zinc and retention of calcium: Dose-dependent inhibition by phytate. Journal of Trace Elements in Medicine and Biology 20: 49 57.3. Reddy NR, Sathe SK & Salunkhe DK (1982) Phytates in legumes and cereals. Adv Food Res 28: 1 92.4. Maga AJ (1982) Phytate: Its Chemistry, Occurrence, Food Interactions, Nutritional Significance, and Methods of Analysis. J. Agric. Food Chem 30 (1): 1 9.5. Weaver CM et al. (1991) Human calcium absorption from whole-wheat products. J Nutr 121: 1769 75.6. McCance RA & Widdowson EM (1942) Mineral metabolism of healthy adults on white and brown bread dietaries. J Physiol 101: 44 85.7. Heaney RP, Weaver CM & Fitzsimmons ML (1991) Soybean phytate content: effect on calcium absorption. Am J Clin Nutr 53: 745 7. 8. Weaver CM et al. (1993) Absorbability of calcium from common beans. J Food Sci 58: 1401 3.9. Meschino J (2002) Calcium: requirements, bioavailable forms, physiology and clinical aspects. Dyn Chiropr 20: 18.10. Ahmed A et al. (2008) Bioavailability of Calcium, Iron and Zinc Fortified Whole Wheat Flour Chapatti. Plant Foods Hum Nutr 63: 7 13.11. Morris ER & Ellis R (1976) Isolation of Monoferric Phytate from White Bran and Its Biological Value as an Iron Source to the Rat. J Nutr 106: 753 60.12. Graf E (1983) Calcium binding to phytic acid. J. Agric. Food Chem 31 (4): 851 55.13. Trudeau DL & Freier EF (1967) Determination of Calcium in Urine and Serum by Atomic Absorption Spectrophotometry (AAS). Clinical Chemistry 13: 101 14.14. Willis JB (1961) Determination of Calcium and Magnesium in Urine by Atomic Absorption Spectroscopy. Analytical Chemistry 33 (4): 556 59.15. Weaver CM (1992) Calcium bioavailability and its relation to osteoporosis. Soc Exp Biol Med 200: 157 60.16. Gropper SS, Smith JL & Groff JL (2009) Advanced Nutrition And Human Metabolism (5th Edition). Belmont, CA: Wadsworth.17. Massey L (1993) Dietary factors influencing calcium and bone metabolism: Introduction. J Nutr 123: 1609 10.18. Slemenda C et al. (1992) Long-term bone loss in men: Effects of genetic and environmental factors. Ann Intern Med 117: 286 91.