Effect of Cooking on Soluble and Insoluble Oxalate Contents in Selected Pakistani Vegetables and Beans

Abstract

The present study evaluated the effects of cooking on the total, soluble and insoluble oxalate contents in six different types of locally consumed vegetables and beans (spinach, carrots, beet root, white bean, red bean and soybean). The foods were cooked in water until they reached the soft consistency (12–15 min for vegetable and 2 h for beans). The raw and cooked samples were analyzed for their soluble and insoluble oxalate contents using the HPLC techniques. The total oxalate content of raw spinach, carrot, beet root, white bean, red bean and soybean, were found to be 978 ± 5, 49 ± 7, 67 ± 12, 158 ± 16, 113 ± 15, and 497 ± 22 mg/100 g of fresh weight respectively. The total oxalate contents of these foods after boiling were 477 ± 8, 16 ± 9, 52 ± 14, 47 ± 17, 72 ± 17, and 224 ± 25 mg/100 g of fresh weight respectively. The results showed that boiling significantly (P < 0.05) reduced both soluble and insoluble oxalate contents. More losses were observed in the soluble than the insoluble oxalates. The reduction in soluble oxalate in different vegetables ranged from 16 to 66% whereas in beans ranged from 40 to 77%. The data suggests that the use of boiled vegetables can be an effective strategy for reducing the dietary intake of oxalates in individuals predisposed to the development of kidney stones.

Keywords:

• Vegetables
• Beans
• Raw
• Boiling
• Cooking
• Oxalate

INTRODUCTION

Vegetables and legumes constitute an important component of everyday diet of millions of people in Pakistan. Vegetables are regarded a good source of certain minerals, vitamins, dietary fibre, and some antioxidant phytochemicals,[1,2] whereas the legumes in addition to all these benefits also contribute towards meeting the daily protein requirements.[3–5] Increased intake of plant derived phenolics is thought to lower the risk of age-related chronic diseases such as cardiovascular disease and cancer.[6–8] However, these foods may contain certain anti-nutritional constituents, which can reduce the bioavailability of these nutrients.[9–11] Oxalates are widely found in many plant species and occur as end products of metabolism both in soluble and insoluble forms.[12 –13] Some plant species can accumulate significant amounts of oxalates in their tissues.[14] The oxalate content of foods can vary depending on the variety, growth, season, soil conditions, time of harvest and many other factors.[15–18] Plant foods are considered as the major dietary source of both the soluble and insoluble oxalates.[19] Some species and some cultivars may contain 400–600 mg/100 g fresh weight (FW), while others range from 700–900 mg/100 g FW.[20] Spinach is known to be a very high oxalate food, with reported values as high as 1145 mg/100 g of wet weight.[21]

Oxalate can enter into the formation of both soluble and insoluble salts with minerals, in particular the insoluble chelate complex with dietary calcium. The insoluble crystals of calcium oxalate can accumulate in the renal glomeruli and can lead to the formation of kidney stones and other renal disorders.[22] Approximately 75% of all kidney stones are primarily composed of calcium oxalate. The amount of soluble oxalate is important as it appears to be more bio-available than the insoluble oxalate.[23,24] The consumption of foods containing high levels of soluble oxalates may have a considerable effect on the absorption of calcium and are regarded as a high risk factor for individuals with hyperoxaluria.[25] Total urinary oxalate is always the sum of dietary oxalate absorbed as such and endogenously synthesized oxalate within the body from different precursors. Recent studies have shown that on average, one half of the oxalate excreted in the urine is derived from the diet, depending upon the relative amounts of calcium and oxalate ingested.[26] It is therefore important to have data on the oxalate contents of locally available foods.

Vegetables are usually consumed both as raw and/or after cooking, whereas the legumes are primarily consumed after cooking or processing. A number of studies have been conducted in various parts of the world to determine the oxalate contents of local foods and the effects of cooking and processing on it. Cooking foods with water, in particular boiling has been reported to produce variable effects on their oxalate contents, primarily due to loss of soluble oxalate in cooking water.[9,12,16,21,27] Negligible data is however available on the oxalate contents of commonly consumed vegetables and beans in Pakistan. The present study was therefore conducted to evaluate the effect of boiling on the oxalate contents of some commonly consumed vegetables and beans in Pakistan.

MATERIALS AND METHODS

Samples

Representative samples (in triplicate) of six different types of plant foods, spinach (Spinacia oleracea), beet root (Beta vulgaris), carrot (Daucus carota), white bean (Phaseolus vulgaris), red bean or kidney bean (Phaseolus vulgaris), and soybean (Glycine max), were randomly collected from the local markets of Faisalabad.

Sample Preparation

After removing the inedible portions, all the vegetables were chopped into small pieces. The chopped edible portions of vegetables were boiled in distilled water for 12–15 min until the vegetables reached the soft consistency. The water was discarded after boiling of food. The beans were soaked overnight in water and the water was drained off. The beans were then boiled in distilled water for about 2 h until they reached the soft consistency. The water was drained off after boiling the beans. Boiled vegetables and beans were dried to a constant weight at 80°C in an oven for 48 h. After drying both the raw and boiled vegetables and beans were ground into powder in a Retsch Mill laboratory (Retsch AS200, Germany) until the particle size no longer be further reduced.

Extraction of Total and Soluble Oxalates

Total and soluble oxalates were extracted according to the method as described by Ohkawa.[28] Five grams of ground sample was mixed with 5 ml of distilled water to make slurry. It was then homogenized with either 50 ml of 2 M HCl or 50 ml of distilled water for the extraction of total and soluble oxalates respectively. The mixtures were centrifuged at 5000 rpm for 20 min and the supernatants were transferred to 100 ml volumetric flasks and made up to volume with distilled water.

Sample Analysis

Total and soluble oxalates in vegetables and beans were analyzed by the reverse phase HPLC method as described below. All the chemicals used for the analysis were of analytical grade and obtained from Merck or Sigma-Aldrich, Germany.

Preparation of Standards

The standards of pure oxalic acid were prepared in 1–40 μg/mL concentrations. The standards were prepared from 40 mg/mL of stock solution of pure oxalic acid. The samples and standards were filtered through a 0.45 μm acrylic disc into 1 ml HPLC auto-sample vials.

Equipment Conditions

HPLC of extracted oxalic acid was carried out on a 250 × 4.6 mm reverse phase column (Supelco). Mobile phase was 0.25% dehydrogenate phosphate and 0.0025 M tetra- butyl ammonium hydrogen sulphate buffered at pH 2.0 with ortho-phosphoric acid. The equipment consisted of dual piston HPLC pump (Model LC-10A, Shimadzu, Japan) with a UV detector set at 210 nm. The samples (5 μL) were injected in duplicate in the column and eluted at a flow rate of 1 ml/min The oxalic acid peaks were eluted at 10 min (Fig.1 and 2). Each extract was analyzed for oxalate in triplicate.

Figure 1 Peaks of HPLC. (a) Peaks of standards of oxalic acid and (b) peaks of oxalic acid in food samples.

Figure 2 Peak area of the standards of oxalic acid during HPLC analysis.

Statistical Analysis

Data collected was subjected to statistical analysis using one way analysis of variance (ANOVA) and the means were compared by paired t-test.[29] Data was analyzed using MINITAB statistical software package Class LC-10.

RESULTS AND DISCUSSION

Mean values for total, soluble and insoluble oxalate contents of raw and boiled vegetables and beans are given in Table 1. The total oxalate contents were found to be relatively higher in spinach and soybean, moderate in white bean and red bean, while low in carrots and beet root. The highest total oxalate values (978 ± 5 mg/100 g of fresh weight, FW) were observed in spinach followed by soybean (497 ± 22 mg/100 g FW). Red and white beans contained moderate quantities of total oxalates (158 ± 16 and 113 ± 15 mg/100 g FW, respectively).

Table 1 Total, soluble and insoluble oxalate contents of raw and boiled vegetables and beans (mg/100 g of wet weight).*

Vegetables/Beans	Total oxalate			Soluble oxalate			Insoluble oxalate
	Raw	Boiled	% loss	Raw	Boiled	% loss	Raw	Boiled	% loss
Spinach	978 ± 5 ^a	477 ± 8 ^b	51.2	543 ± 2 ^a	184 ± 5 ^b	66.1	435 ± 3 ^a	293 ± 3 ^b	32.6
Carrot	49 ± 7 ^a	26 ± 9 ^b	46.9	28 ± 4 ^a	14 ± 5 ^b	50.0	21 ± 3 ^a	12 ± 4 ^b	42.8
Beet root	67 ± 12 ^a	52 ± 14 ^b	22.4	45 ± 7 ^a	38 ± 8 ^b	15.6	22 ± 5 ^a	15 ± 6 ^b	31.8
White bean	158 ± 16 ^a	47 ± 17 ^b	70.3	52 ± 8 ^a	12 ± 9 ^b	76.9	106 ± 8 ^a	34 ± 8 ^b	67.9
Red bean	113 ± 15 ^a	72 ± 17 ^b	36.3	37 ± 8 ^a	22 ± 9 ^b	40.5	76 ± 6 ^a	50 ± 8 ^b	34.2
Soybean	497 ± 22 ^a	224 ± 25 ^b	54.9	155 ± 13 ^a	64 ± 15 ^b	58.7	343 ± 8 ^a	162 ± 10 ^b	52.8
*n = 3 Extractions were done in triplicate for samples of raw and boiled vegetables and beans. Wet weight refers to the original store-purchased weight for raw and cooked weight (boiled, drained and dried) vegetables and beans.
a, b = Different alphabets in the same row for the data in coloumns on the total, soluble and insoluble oxalate contents of raw and boiled vegetables and beans differ signicantly (P < 0.05).

The lowest values (49 ± 7 mg/100 g FW) were observed in carrots. Spinach is known to be a high oxalate food, with reported values as high as 1145 mg/100 g of wet weight.[21] Spinach contains about 55% soluble oxalates and 45% insoluble mostly calcium oxalate.[30] The total oxalate values reported by Savage et al.[12] for the raw beetroot and carrots were 45.6 mg/100 g and 35.6 mg/100 g, respectively. The values observed in this study for beet root and carrots are however a little higher than the reported values. On the other hand Massey et al.[31] reported higher total oxalate values (670 mg/100 g) in soybean as compared to the values observed in our study. The oxalate content of foods can vary depending on the variety, growth, season, soil conditions, time of harvest and many other factors.[15–18,32] Differences observed in the reported values and the values determined in this study could be attributed to these factors.

Cooking of foods by boiling significantly (P < 0.05) reduced their oxalate contents (Table 1). The losses of soluble oxalates in vegetables ranged from 16–67%. The highest loss of soluble oxalate was observed in spinach (67%). Similarly the losses of soluble oxalates in different beans ranged from 40 to 76%. The highest loss (76%) was observed in white beans followed by soybean (59%) and red beans (40%). Cooking foods with water has been reported to affect their oxalate contents primarily due to loss of soluble oxalate in cooking water. Chai and Liebman[21] reported that boiling vegetables (spinach and carrots) decreased their total oxalate contents from 30 to 87%. Similar results were reported by Jaworska[16] and Savage et al.[12] Judprasong et al.[27] reported that cooking of different Thai vegetables by boiling significantly (P < 0.05) reduced their total and soluble oxalate contents and the percentage losses in different foods ranged from 18 to 76%. Cooking has been shown to have variable effects on different vegetables.[28] Boiling spinach for one minute only can remove about 10% of insoluble and 47% of soluble oxalate salts. Boiling of cormels of Japanese taro (Colocasia esculenta L. Schott) reduced the level of soluble oxalate in the cooked tissues below the detectable levels.[33] The results obtained in our study are in line with these findings. Cooking can however affect the sensory quality and caused significant losses in the ascorbic acid and beta-carotene contents of green leafy vegetables.[34] Quinteros et al.[35] reported that all the cooking procedures reduced the soluble oxalate contents in pulses, but microwave and industrial procedures were found more effective than the traditional domestic cooking. Thermal processing has also been shown to reduce the anti-nutritional factors (HCN, oxalate, phytate and tannins) in the horse-eye beans [Mucuna urens (L.) Medik][36] and in Phaseolus vulgaris.[37]

Spinach has been regarded as a high-risk food for individuals susceptible to kidney stone formation due to its capability in increasing the excretion of urinary oxalates.[38] The consumption of boiled spinach may lower risk as significant amounts of soluble oxalate are leached out during boiling (67% loss). Brogren and Savage[39] observed that the ingestion of spinach with dietary sources of calcium significantly lowered the oxalate bioavailability as compared to ingestion of spinach alone. The method used for cooking vegetables and the presence of other dietary components such as calcium may contribute to reduce the oxalate absorption. Thus kidney stone patients who form calcium oxalate-containing stones must be advised to limit their intake of oxalate rich foods with simultaneous consumption of calcium rich foods or supplements to reduce the oxalate absorption.[22] The average Western diet supplies about 50–150 mg oxalate/day, whereas the vegetarian diets can contribute somewhat higher.[40] The American Dietetic Association recommends that kidney stone patients should restrict their dietary oxalate to less than 40 to 50 mg per day.[41] The results of this study will be of great use for kidney stone patients as no such data was reported previously on the oxalate contents of local vegetables and beans grown in Pakistan. The amount of soluble oxalates in a food item is also important because soluble oxalate is reported to be more bioavailable than insoluble oxalate. The ability of various oxalate-containing foods to increase urinary oxalate excretion and pre-disposition to stone formation depends on both its oxalate content and bioavailability.[22] Further studies are needed to determine the oxalate bioavailability from these foods to make effective dietary recommendations in particular for people with hyperoxaluria.

CONCLUSION

Variable levels of oxalate were observed in different types of vegetables and beans tested in this study. Boiling significantly reduced both the total and soluble oxalate contents of these foods. Spinach and soybean were found to contain high levels of oxalate. Cooking should be employed as an effective strategy for reducing the dietary intake of oxalates from these foods in particular for individuals predisposed to the development of kidney stones.

ACKNOWLEDGEMENTS

The authors are grateful to Dr. Iqrar Ahmed Khan (Director General, PAEC, Pakistan) and his staff for their valuable cooperation and help at Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad, during sample analysis.