Oxalate content of legumes, nuts, and grain-based flours

JOURNAL OF FOOD COMPOSITION AND ANALYSIS Journal of Food Composition and Analysis 18 (2005) 723 729 www.elsevier.com/locate/jfca Short Communication Oxalate content of legumes, nuts, and grain-based flours Weiwen Chai, Michael Liebman Department of Family and Consumer Sciences (Human Nutrition), University of Wyoming, University Station, P.O. Box 3354, Laramie, WY 82071, USA Received 14 January 2004; accepted 15 July 2004 Abstract About 75% of all kidney stones are composed primarily of calcium oxalate and hyperoxaluria is a primary risk factor for this disorder. Since absorbed dietary oxalate can make a significant contribution to urinary oxalate levels, oxalate from legumes, nuts, and different types of grain-based flours was analyzed using both enzymatic and capillary electrophoresis (CE) methods. Total oxalate varied greatly among the legumes tested, ranging from 4 to 80 mg/100 g of cooked weight. The range of total oxalate of the nuts tested was 42 469 mg/100 g. Total oxalate of analyzed flours ranged from 37 to 269 mg/100 g. The overall data suggested that most legumes, nuts, and flours are rich sources of oxalate. r 2004 Elsevier Inc. All rights reserved. Keywords: Dietary oxalate; Kidney stones; Legumes; Nuts; Flours 1. Introduction About 75% of all kidney stones are composed primarily of calcium oxalate (Williams and Wandzilak, 1989) and hyperoxaluria is a primary risk factor for this disorder (Goldfarb, 1988; Robertson and Hughes, 1993). Urinary oxalate originates from a combination of absorbed dietary oxalate and endogenous formation from oxalate precursors such as ascorbic acid and glyoxylate (Williams and Wandzilak, 1989). Restriction of dietary oxalate intake has been Corresponding author. Tel.: +1-307-766-5597; fax: +1-307-766-5686. E-mail address: liebman@uwyo.edu (M. Liebman). 0889-1575/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2004.07.001

724 W. Chai, M. Liebman / Journal of Food Composition and Analysis 18 (2005) 723 729 suggested as a treatment to prevent recurrent nephrolithiasis in some patients (Massey et al., 1993). Legumes, nuts, and different types of grain-based flours are commonly consumed throughout the world. Soybeans and other legumes such as lentils, red kidney beans, and white beans have been previously analyzed for oxalate (Massey et al., 2001; Ho now and Hesse, 2002). The oxalate content of nuts has been reported to be relatively high (Massey et al., 1993) and there are published values in the literature for almonds, cashews, hazelnuts, peanuts, pecans, pistachios, and walnuts (Hodgkinson, 1977; Brinkley et al., 1981, 1990; Noonan and Savage, 1999; Ho now and Hesse, 2002). However, comprehensive reports of oxalate concentrations in either legumes or nuts have not been published. In addition, there are few reported data on the oxalate contents of different types of flour products. Thus, the purpose of the present study was to determine the oxalate content of various types of legumes, nuts, and flours. 2. Materials and methods 2.1. Samples The following types of legumes were assessed for oxalate: azuki beans, anasazi beans, black beans, garbanzo beans, great northern beans, large lima beans, mung beans, navy beans, october beans, pink beans, pinto beans, red kidney beans, small red beans, small white beans, soybeans, lentils, green split peas, yellow split peas, and blackeye peas. The types of nuts assayed were almonds, cashews, hazelnuts, macadamia nuts, peanuts, pecans, pine nuts, pistachio nuts, and walnuts. The types of finely ground flours were barley flour, buckwheat flour, brown rice flour, dark rye flour, semolina flour, soy flour, unbleached white flour, whole wheat flour, and corn meal. All legumes, nuts, and flours were purchased from local establishments in Laramie, Wyoming. 2.2. Sample preparation Legumes were soaked overnight, the water discarded, and then boiled in distilled deionized water until they reached a soft consistency. The cooking times computed from when the water began to boil were: 2 h for all kinds of beans tested; 1 h and 30 min for blackeye peas; 1 h and 15 min for green split peas and yellow split peas; and 30 min for lentils. For moisture determinations, cooked legumes, nuts, and all flour samples were dried to constant weight at 80 1C in an Imperial II incubator for 48 h. Dried legumes were ground in a Pavoni coffee mill until the particles could not be further homogenized. Nuts, in their original pre-dried state, were also ground in a Pavoni coffee mill until the particles could no longer be further homogenized. Predried flour samples were used for oxalate extraction. 2.3. Total and soluble oxalate extraction Total oxalate from all test foods and soluble oxalate from a sampling of the test foods were extracted according to a previously described method (Ross et al., 1999). A 5 g sample

W. Chai, M. Liebman / Journal of Food Composition and Analysis 18 (2005) 723 729 725 of dried, finely ground food was accurately weighed into a 500 ml beaker. Approximately 50 ml of 2 M H 3 PO 4 (for total oxalate) or 50 ml of distilled deionized water (for soluble oxalate) were added. The contents were then mixed and the beaker was placed in a shaking water bath at 80 1C for 30 min. Aqueous samples containing the extracted oxalate were then quantitatively transferred to 100 ml volumetricflasks, cooled, made up to volume with distilled deionized water, and mixed. The diluted extractions were centrifuged at 3000 rpm for 20 min and the supernatant was filtered through Whatman number 1 filter paper. The use of H 3 PO 4 to extract total oxalate has been previously reported (Holmes et al., 1995). The use of 2 M H 3 PO 4 in the present study rather than 2 M HCL used by Ross et al. (1999) was necessary to prevent distortion of the oxalate peak by Cl molecules in the subsequent capillary electrophoresis (CE) analysis. One type of legume (navy beans), one type of nuts (almonds), and one type of flour (corn meal) were randomly selected for assessing the degree of methodological variation associated with oxalate extraction. Two extractions were carried out for each of these food samples followed by the determination of oxalate by the enzymaticmethod subsequently described. In addition to the method described in the previous section, total oxalate from cooked soybeans and lentils was also extracted using the method of Ohkawa (1985). A 10 g slurry (5 g sample with 5 g distilled deionized water) was homogenized with 20 ml 2 M HCL and the mixture was centrifuged at 10 000 rpm for 5 min. The supernatant was transferred to a 100 ml volumetric flask. The extraction was repeated two additional times. The supernatant was collected in the same flask, diluted to volume (100 ml) with distilled deionized water, and analyzed for oxalate by the enzymaticmethod subsequently described. 2.4. Sample analysis using capillary electrophoresis (CE) method The filtered samples were diluted 10-fold with distilled deionized water. Standard curves were constructed with known concentrations of oxalate added to a solution containing 65 mg/l NaCl, 11 mg/l Na 2 SO 4 and 5% (volume/volume) 2 M H 3 PO 4. A Biofocus (Bio-Rad Company, CA) 3000 CE system with a negative power supply was used to analyze oxalate content. Samples were detected at a wavelength of 254 nm. Separation was conducted on a 75 mm internal diameter 60 cm length polyimide-coated fused-silica capillary (Polymicro Technologies, Phoenix, AZ). The background electrolyte solution contained 10 mmol/l sodium chromate (Aldrich Chemical Company, Inc, Milwaukee, WI) and 0.5 mmol/l tetradecyl-trimethyl-ammonium bromide (Sigma Chemical Co, St. Louis, MO). The ph (8.1) was not adjusted. The electrolyte solution was degassed using vacuum before use. Samples were introduced at 10 KV for 10 s. Separations were conducted at a constant voltage of 16 KV. The capillary was washed for 1 min with 0.1 mol/l KOH, 1 min with 0.1 mol/l HCL, and 2 min with the electrolyte solution between samples (Holmes, 1995). The oxalate peak was identified and calculated by comparison of the retention time and peak area to the standard oxalate curve. An oxalate recovery determination was conducted by adding known levels of oxalic acid (3.0 or 6.0 mg) to 100 ml of a 10-fold diluted almond sample which had been extracted with 2 M H 3 PO 4 as previously described. Each of these samples was analyzed by CE in duplicate.

726 W. Chai, M. Liebman / Journal of Food Composition and Analysis 18 (2005) 723 729 2.5. Sample analysis using enzymatic method Oxalate from filtered food samples was also measured in duplicate by using a Sigma oxalate kit (Sigma Diagnostics, St. Louis, MO). The method is based on the oxidation of oxalate by oxalate oxidase followed by detection of the H 2 O 2 produced during the reaction (Li and Madappally, 1989). Lyophilized (control) urine samples (Sigma Diagnostics, St. Louis, MO) providing predetermined oxalate levels of 20 30 mg/l were analyzed with each assay to ensure good quality control. 2.6. Statistical analysis Pearson correlation coefficients (r) were computed to assess the strength of the association between oxalate levels measured by CE and enzymaticmethods and to determine whether there was an association between oxalate and moisture contents within each of the three types of foods tested. All P values o0.05 were considered to designate statistical significance. 3. Results and discussion The measurement of oxalate by the CE method agreed well with its measurement by the enzymatic method. The Pearson correlation coefficient (r) between oxalate levels as determined by the two methods for all food samples was 0.99. However, the CE method did not provide an accurate measurement when the oxalate concentration of a sample was relatively low (i.e., p1.5 mg/l). The CE oxalate recovery determination yielded recoveries of 98% and 94% for the addition of 3.0 and 6.0 mg oxalic acid to the almond acid extract, respectively. There was also a good agreement in total oxalate contents between the two extracts obtained from each of the three randomly selected food samples (i.e., 52 and 58 mg/100 g for navy beans; 491 and 503 mg/100 g for almonds; and 52 and 55 mg/100 g for corn meal). No significant correlations between oxalate and moisture contents were observed for any of the three types of foods analyzed (legumes, nuts, and flours). Data from this study indicated that nuts contain high levels of total oxalate, ranging from 42 to 469 mg/100 g (approximately equivalent to 12 131 mg/serving of 28 g) (Table 1). Kidney stone patients who form calcium oxalate-containing stones are advised to limit their intake of foods which contain 410 mg oxalate per serving, with total oxalate intake not to exceed 50 60 mg/day (Chicago Dietetic Association, 2000). Using these guidelines, none of the nuts assessed could be recommended for kidney stone patients. The values for total oxalate contents of almonds and pecans were about 3.5 and 5 times higher than the 131 mg/100 g and 12 mg/100 g reported in the previous literature (Brinkley et al., 1981, 1990). The difference may be due to the different methods used for oxalate extraction and analysis. Variations among almond or pecan cultivars may also partly contribute to the different oxalate levels reported. The oxalate values obtained for peanuts (140 mg/100 g) and cashews (262 mg/100 g) were close to the 116 and 231 mg/100 g previously reported (Brinkley et al., 1990; Noonan and Savage, 1999). The oxalate values reported by Ho now and Hesse (2002) for almonds (383 mg/100 g), hazelnuts (167 mg/100 g), and pistachios (57 mg/100 g) were similar to the values presently reported (Table 1).

W. Chai, M. Liebman / Journal of Food Composition and Analysis 18 (2005) 723 729 727 Table 1 Oxalate content of various types of nuts a Nuts Oxalate content (mg/100 g wet weight) Moisture content (g/100 g wet weight) Enzymatic method CE method Mean of the two methods Almonds (roasted) 491 447 469 1.7 Cashews (roasted) 263 260 262 1.3 Hazelnuts (raw) 221 223 222 3.9 Pine nuts (raw) 199 196 198 1.9 Peanuts (roasted) 131 148 140 1.1 Walnuts (raw) 77 70 74 2.6 Pecans (raw) 66 62 64 2.6 Pistachio nuts 51 46 49 1.9 (roasted) Macadamia nuts (raw) 43 40 42 1.5 a Sample n=1 for all types of nuts; wet weight refers to original store-bought weight prior to Imperial II incubator drying. There was a wide oxalate range of 4 80 mg/100 g in various types of cooked legumes (Table 2). The consumption of legumes such as anasazi beans, small white beans, great northern beans, pink beans, black beans, navy beans, soybeans, and small red beans should be considered carefully for kidney stone patients since total oxalate in 1 serving (1 cup, approximately 170 g) of these cooked legumes exceeds 50 mg. Legumes containing low levels of total oxalate such as green split peas, yellow split peas, and blackeye peas could be recommended for kidney stone patients. Total oxalate content of soybeans and lentils were much lower than values reported by Massey et al. (2001). However, the presently reported values for lentils, red kidney beans, and white beans were similar to those reported by Ho now and Hesse (2002). Oxalate from cooked soybeans and lentils was also extracted according to the method reported by Ohkawa (1985) to assess whether different extraction methods would significantly affect oxalate levels. The two extraction methods yielded almost identical oxalate results. Holmes et al. (1995) reported that altering extraction conditions by increasing acid concentration, temperature, and time of incubation, or re-extraction of the pellet did not increase oxalate yield. Almonds and black beans were further analyzed for soluble oxalate since they are commonly consumed and contain high total oxalate levels. The soluble oxalate content of almonds was 153 mg/100 g which was about 31% of the total oxalate content. The soluble oxalate content of black beans was 4 mg/100 g of cooked weight which was about 5% of the total. Thus, the proportion of soluble oxalate in almonds was about 6-fold greater than that in black beans. Since soluble oxalate in foods appears to be more bioavailable than insoluble oxalate (Albihn and Savage, 2001), almond consumption could be considered to be a much higher risk for individuals with hyperoxaluria as compared to the consumption of black beans. Future research should estimate the proportions of soluble oxalate in other high oxalate-containing nuts and legumes and determine whether the pattern is similar to that found in almonds and black beans. The analyzed flours contained relatively high levels of total oxalate, ranging from 37 to 269 mg/ 100 g (Table 3). These results may be of use to kidney stone patients since there are few reported

728 W. Chai, M. Liebman / Journal of Food Composition and Analysis 18 (2005) 723 729 Table 2 Oxalate content of various types of cooked legumes a Legumes Oxalate content (mg/100 g wet weight) Moisture content (g/100 g wet weight) Enzymatic method CE method Mean of the two methods Anasazi beans 85 75 80 71 Small white beans 77 78 78 66 Great northern beans 77 72 75 69 Pink beans 75 75 75 64 Black beans 73 71 72 66 Navy beans 58 56 57 65 Soybeans 57 55 56 70 Small red beans 36 33 35 64 Pinto beans 29 25 27 74 October beans 28 27 28 66 Azuki beans 26 23 25 63 Red kidney beans 19 13 16 68 Garbanzo beans 9 b 9 65 Mung beans 8 b 8 71 Lentils 8 b 8 72 Large lima beans 8 b 8 67 Green split peas 6 b 6 66 Yellow split peas 5 b 5 69 Blackeye peas 4 b 4 59 a Sample n=1 for all types of legumes; wet weight refers to cooked (boiled and drained) weight prior to Imperial II incubator drying. b CE was not able to measure oxalate because of the low oxalate concentration of the sample. Table 3 Oxalate content of various types of flours a Flours Oxalate content (mg/100 g wet weight) Moisture content (g/100 g wet weight) Enzymatic method CE method Mean of the two methods Buckwheat 271 267 269 6.8 Soy 187 179 183 4.5 Whole wheat 68 66 67 6.7 Barley 59 53 56 7.0 Corn meal 55 52 54 6.6 Dark rye 52 49 51 6.7 Semolina 48 48 48 7.2 Unbleached 41 38 40 6.4 white Brown rice 38 35 37 6.8 a Sample n=1 for all types of flours; wet weight refers to original store-bought weight prior to Imperial II incubator drying.

W. Chai, M. Liebman / Journal of Food Composition and Analysis 18 (2005) 723 729 729 data on the oxalate levels of various types of flours. Diets which are heavily based on flour products may increase pre-disposition to calcium oxalate-containing kidney stones in susceptible individuals. Oxalate bioavailability can be defined as the percentage of oxalate absorbed from an oxalatecontaining food. The ability of various oxalate-containing foods to increase urinary oxalate excretion and pre-disposition to stone formation depends on both oxalate content and bioavailability (Brinkley et al., 1981). Thus, it is also important for future studies to determine the oxalate bioavailability of high oxalate-containing legumes, nuts, and flours. Acknowledgements This study was supported by a grant from the VP Foundation, Graham, North Carolina. References Albihn, P.B.E., Savage, G.P., 2001. The bioavailability of oxalate from oca (Oxalis tuberosa). Journal of Urology 166, 420 422. Brinkley, L., MgGuire, J., Gregory, J., Pak, C.Y.C., 1981. Bioavailability of oxalate in foods. Urology 17 (6), 534 538. Brinkley, L.J., Gregory, J., Pak, C.Y.C., 1990. A further study of oxalate bioavailability in foods. Journal of Urology 144, 94 96. Chicago Dietetic Association, 2000. Manual of Clinical Dietetics. American Dietetic Association, Chicago, IL, p. 475. Goldfarb, S., 1988. Dietary factors in the pathogenesis and prophylaxis of calcium nephrolithiasis. Kidney International 34, 544 555. Hodgkinson, A., 1977. Oxalic Acid in Biology and Medicine. Academic Press, New York, pp. 193 212. Holmes, R.P., 1995. Measurement of urinary oxalate and citrate by capillary electrophoresis and indirect ultraviolet absorbance. Clinical Chemistry 41 (9), 1297 1301. Holmes, R.P., Goodman, H.O., Assimos, D.G., 1995. Dietary oxalate and its intestinal absorption. Scanning Microscopy 9 (4), 1109 1120. Ho now, R., Hesse, A., 2002. Comparison of extraction methods for the determination of soluble and total oxalate in foods by HPLC-enzyme-reactor. Food Chemistry 78, 511 521. Li, M.G., Madappally, M.M., 1989. Rapid enzymaticdetermination of urinary oxalate. Clinical Chemistry 35, 2330 2333. Massey, L.K., Roman-Smith, H., Sutton, R.A.L., 1993. Effect of dietary oxalate and calcium on urinary oxalate and risk of formation of calcium oxalate kidney stones. Journal of the American Dietetic Association 93, 901 906. Massey, L.K., Palmer, R.G., Horner, H.T., 2001. Oxalate content of soybean seeds (glycine max: leguminosae), soyfoods, and other edible legumes. Journal of Agricultural and Food Chemistry 49, 4262 4266. Noonan, S.C., Savage, G.P., 1999. Oxalate content of foods and its effect on humans. Asia Pacific Journal of Clinical Nutrition 8 (1), 64 74. Ohkawa, H., 1985. Gas chromatographic determination of oxalic acid in foods. Journal of the Association of Official Analytical Chemists 68 (1), 108 111. Robertson, W.G., Hughes, H., 1993. Importance of mild hyperoxaluria in the pathogenesis of urolithiasis new evidence from studies in the Arabian peninsula. Scanning Microscopy 7, 391 401. Ross, A.B., Savage, G.P., Martin, R.J., Vanhanen, L., 1999. Oxalate in oca (New Zealand yam) (Oxalis tuberosa Mol.). Journal of Agricultural and Food Chemistry 47, 5019 5022. Williams, H.E., Wandzilak, T.R., 1989. Oxalate synthesis, transport and the hyperoxaluricsyndromes. Journal of Urology 141, 742 747.