Oxalate Content of Egyptian Grown Fruits and Vegetables and Daily Common Herbs

Egyptian dieticians typically rely on foreign databases to find out oxalate content of food due to unavailability of local databases. The soil, fertilizers, climate and cultivars are often very different. Therefore, the purpose of this study is to establish a local database of oxalate content in Egyptian grown fruits and vegetables and selected daily common herbs. The current study analysed the total and the soluble oxalate in 37 Egyptian grown fruits, vegetables and 9 commonly used herbs. Two methods were used for screening the Egyptian foods for oxalate concentration; the first method was AOAC 1999 and the second was enzymatic method. Total oxalate varied greatly among the vegetables examined, ranging from 4 to 917 mg/100 g F.W. Total oxalate of analysed fruits ranged from 9 to 50 mg/100 g F.W. There is a strong correlation found between the two methods used. Vegetables were classified into 4 categories; low oxalate concentration containing less than 10 mg of oxalic acid /100 g F.W., such as cabbage, courgette, cucumbers, garlic, spring onions and turnip. Moderate oxalate concentration vegetables containing 10-25 mg/100 g F.W., such as aubergine, field bean, corn, peppers and watercress. High oxalate concentration vegetables containing 26-99 mg/100g F.W., such as fūl, green beans, celery, mallow, okra and sweet potatoes. Very high oxalate concentration containing 100-900 mg/100g F.W. such as Swiss chard, molokhia, purslane and vine leaves (fresh). Extensive amounts of total oxalate (201-4014 mg/100 g D.W.) were found in daily common herbs such as caraway seed, green cardamom, cinnamon, coriander seeds, cumin, curry powder, ginger and turmeric powder.


Introduction
Oxalic acid (ethanedioic acid), the name comes from the plant Oxalis (wood sorrel) from which it was first isolated (Liebman, 2002), occurs ubiquitously in nature, sometimes as a free acid, but more commonly as soluble potassium, sodium or ammonium oxalate or as insoluble calcium oxalate.Biosynthesis of oxalate occurs in members of all five kingdoms.Oxalate is associated with metabolic disorders and infectious diseases (Holmes & Assimos, 1998;Nakagawa et al., 1999).
Oxalic acid and its salts occur as an end product of metabolism in a number of plants.When these plants are eaten they may have an adverse effect on humans and animals because oxalate binds calcium and other minerals and may cause stone formation in the urinary tract when the acid is excreted in the urine (Noonan & Savage, 1999).Oxalate-producing plants, which include many crop plants, accumulate oxalate in the range of 3%-80% (w/w) of their dry weight (Libert & Franceschi, 1987).The oxalate production pathways the cleavage of isocitrate, hydrolysis of oxaloacetate, glycolate/glyoxylate oxidation, and/or oxidative cleavage of L-ascorbic acid (Hodgkinson, 1977).Of these pathways, the cleavage of ascorbic acid appears to be the most common (Yang & Loewus, 1975;Nuss & Loewus, 1978;Li & Franceschi, 1990;Keates et al., 2000).
The diversity of calcium oxalate crystal shapes and sizes, as well as their prevalence and spatial distribution, have led to a number of hypotheses regarding crystal function in plants.The proposed functions include roles in ion balance, in plant defense, in tissue support, in detoxification, and in light gathering and reflection (Franceschi & Horner, 1980).Recently, (Nakata & McConn, 2000) hypothesized the roles of calcium oxalate formed in plants in supporting tissue structure and in regulating excess tissue calcium.The oxalate content of food such as spinach can vary considerably between plants of the same species, due to differences in climate, soil quality, state of ripeness, or even which part of the plant is analyzed.Variations also may be caused by the different methods used for measuring oxalate in food (Eheart & Massey, 1962).
Insoluble oxalate crystals formed in the gut are not absorbed and are carried out with the feces, thus reducing the bioavailability and absorption of calcium and iron in diets (Bataille & Fournier, 2001).Human urine always contains small levels of calcium oxalate (Oke, 1969) that may be deposited in the kidneys of certain people as a common form of kidney stones (Massey et al., 1993).
In Egypt we have not enough data about how much oxalate in local grown fruits and vegetables or execrated oxalate in urine that comes from endogenous metabolic synthesis.On the other hand, healthy adults, with U.S. or European type western diets, 90% of oxalic acid excreted in the urine comes from endogenous metabolic synthesis (Massey et al., 1993).However, establishing a local database of oxalate content in Egyptian grown fruits and vegetables and daily common herbs may help Egyptian patients with hyperoxaluria to make more accurate dietary modifications.

Experimental Procedure
Thirty seven fruits and vegetables were analysed, the selection of which was based on those listed in the USDA Table 1984 with some modification to suit Egyptian food habits.The individual products were purchased from the same batch within the supermarket.On the day of purchase, where applicable, the edible parts of the fruits or vegetables were washed, dried, cut finely and weighed.For the purposes of this study three replicate samples of each product were used.In the case of products such as, cauliflower, and onions, the products were of a sufficient size and weight to allow for individual items to be used as single replicate.In the case of apricots, three individual fruits were taken as one replicate due to the small size, chopped up and a composite sample prepared from which the required sample weight was taken.Three replicates of each product were analysed in the dried state.Herbs were treated similarly apart from washing process.Following sampling and preparation as described above, three sub-samples of each ground product were prepared for the analysis.All results were expressed as mg oxalate/100 fresh weight (F.W.) of each fruit or vegetable or mg oxalate/100 dry weight (D.W.) for daily common herbs.

Drying Food Samples
A fruit or vegetable was dried at 28 °C from 2-7 days, depending on the type of food in a vacuum dryer (Remplissage evacuation, Arthermo Gessate MI, temperature and density guide, Italy).For example, spinach leaves and upper part of stalk of spinach were dried at 28 °C, for 3h then broken into small pieces by hand, and mixed spinach and left in the vacuum dryer for about 27 hours until dried.Moisture and dry matter were determined in all foods (Infrared Moisture Determination Balance FD-610-Japan).
*N= normal level-E elevated level.

Statistical Analysis
The results are presented as mean of three determinations ± standard error (± SEM).Pearson correlation coefficients (r) were computed to assess the strength of the association between oxalate levels measured by AOAC 1999 and enzymatic methods.Oxalate values were analysed in transformed scale (log 10 (X) for simple linear regression Wessa (2012).

Results
The total and soluble oxalate content of the selected grown Egyptian fruits and vegetables and common herbs are presented in Table 2 to Table 8.However, the amount of insoluble oxalate can be evaluated as the difference between the total and the soluble oxalate (Rahman et al., 2008).It is noted that fruits and vegetables contain varying amounts of total oxalate.The outcome of Pearson correlation in this study was strong correlation that equal 1 between the two methods used for determination of oxalate in all foods examined.Linear regression analysis of the total oxalate concentration in vegetables revealed a linear correlation (r 2 = 1.006) between the two methods (Figure 2).A similar correlation between the two methods occurred for the fruit samples (r 2 = 0.8739), (Figure 3) and (r 2 = 0.9969) (Figure 4) for herbs.*standard error of the mean, each method was tested 9 times.

Recovery Studies
Known amounts of oxalate were added to 10 food samples tested before and known their amounts of oxalate, in addition to testing the reagent recoveries in order to obtain percentage of oxalate recovery.The recovery of added oxalate ranged from 93-107% for samples and 100.5% for reagent recoveries.

Discussion
Since absorbed dietary oxalate can make a significant contribution to urinary oxalate levels therefore the Egyptian consumers and dieticians always rely on oxalate content of food but from oxalate analyzers that are different circumstances because the unavailability of a local database.Consequently, the aim of this work was to establish a local database of oxalate included the common foods in Egypt such as fūl, molokhia, baladi corn, vine leaves, mallows, mangoes and guava.The total food analysed in this study were 37 fruits and vegetables and 9 daily common herbs.The AOAC 1999 and Trinity biotech kit for oxalate in urine were used to evaluate the total and soluble oxalate.Advantages of selected methods; AOAC 1999 and enzymatic kit, were that they were time efficient and had relatively fewer steps.Results indicate that oxalate kit accuracy and precision was higher than AOAC and given higher values of oxalate than AOAC 1999.Enzymatic method is adequate to the analysis of oxalate contents in the examined foods, and the inter-day precision of the method expressed as standard error of mean was good (2.1), with an accuracy of the recovery of added oxalate ranged from 93-107% for samples and 100.5% for reagent recoveries.
In Egypt, consumption of vegetables is an important part of food habits, therefore grown vegetables were screened for total and water soluble oxalate that were divided into four groups: (i) Low oxalate concentration vegetables that were less than 10 mg/100 g F.W., include cabbage, cauliflower, courgette, cucumbers, garlic, onions, spring onions, peas and turnip, which are the most vegetables consumed in Egypt and would have a beneficial effect on patients with calcium oxalate stones.
Several studies were conflicting with regard to the reported oxalate level of strawberries with total oxalate levels ranging from 2.9 mg/100 g (Hönow & Hesse, 2002) to 23.4 mg/100g (Ogawa, Takahashi, & Kitagawa, 1984).In El-Wahsh et al., 2012, strawberries were reported to contain 6.0 mg and 2.5 mg of total and soluble oxalate/100 g, respectively.In the present study, amount of total oxalate ranged from 23-25 mg/100 g F.W. The variation in oxalate values in different sources of plants can be affected by factors such as soil quality, climate or different state of fruit ripeness (Libert & Franceschi, 1987).In addition, differences could also be due to dissimilarity in preparation of the samples and analytical techniques.
In Egypt, sweet potato is a common food eaten as a dessert while potatoes are staple food served as a main dish.The oxalate content of sweet potato and potatoes were found to be 48 and 27 mg/100g F.W., respectively, this is similar to the results of a Taiwanese study (48.6 mg/100 g.) (Tsai et al., 2005).fūl (Vicia faba) is a staple food in Egypt, especially for low income people, with the amount of soluble oxalate ranges between 40-44 mg/100 g D.W. (total oxalate 55-57 mg/100 g D.W.) there is a possibility that through soaking and cooking oxalates are lost, but care should be paid to the spices that are added to the ful, specifically cumin which has mega amounts of oxalate that found to be (1505-1500 mg/100g D.W.) .
Molokhia is a leafy green vegetable, another traditional food in Egypt, served as a thick green soup beside the main dish that is usually a meat source.Molokhia belongs to very high oxalate concentration group (450-457 mg/100 g F.W.) about 31 % of it have water soluble oxalate, coriander seed is used to spice traditional molokhia that is found to contain 995-1005 mg/100 g D.M. Spinach and mallow are traditional vegetables in Egypt that are eaten in winter similarly to molokhia in terms of adding spices.Mangoes are summer fruit in Egypt that are eaten as whole or as juices and contains from 8-12 mg/100 g fresh weights.
Oxalate concentration of some common herbs (mg/100 g D.W.) of a selected common herbs include green cardamom, coriander seeds, caraway seeds, cinnamon, cumin, curry powder, ginger, nutmeg and turmeric powder that also important values for Egyptian cuisine.The data presented in Table 8.Indicates to the concentration of oxalate in some common herbs used in the Egyptian cuisine.
Noticeably most these herbs contain extensive amounts of oxalate that exceeds 1000 mg / 100 g dry weight that may consumers, patients and dieticians focus on high and very high concentrations of vegetables apart from herbs that added to food.High consumption of oxalate rich foods can lead to secondary hyperoxaluria, a major risk factor for calcium oxalate stone formation, and even acute renal failure in the case of excessive dietary oxalate intake (Simpson et al., 1999).About 75% of all kidney stones are composed primarily of calcium oxalate (Williams & Wandzilak, 1989) and hyperoxaluria is a primary risk factor for this disorder (Goldfarb, 1988;Robertson & 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 & Wandzilak, 1989).Restriction of dietary oxalate intake has been proposed to prevent the formation of calcium oxalate kidney stones.

Conclusion
This study analysed traditional foods to establish a local database of oxalate content in 37 Egyptian grown fruits and vegetables and 9 selected daily common herbs.Two methods were used for screening the Egyptian foods for oxalate concentration.This study was analysed the traditional foods in Egypt such as fūl, molokhia, baladi corn, vine leaves, mallows, mangoes and guava.Total oxalate varied greatly among the vegetables examined, ranged from 4 to 917 (mg/100 g F.W).Total oxalate of analysed fruits ranged from 9 to 50 (mg/100 g F. W).Extensive amounts of total oxalate were found in daily common herbs 201-4014 (mg/100 g D.W.).We hope the data obtained herein can help dieticians, patients and urologists to instruct the public on urolithiasis prevention.

Figure 1 .
Figure 1.The chemical structure of different forms of oxalic acid

Figure 2 .
Figure 2. Simple linear regression analysis of the total oxalate between AOAC 1999 and enzymatic methods for vegetables; the values used were the log transformation

Table 1 .
The amount of reagents used for blank the standard and the sample

Table 2 .
Effect of using different methods on the total oxalate concentration and precision of the method (mg/100g)

Table 4 .
Moderate oxalate concentration group (10-25 mg oxalic acid /100 g fresh weight) Fw: fresh weight; insoluble oxalate such as calcium oxalate can be calculated from subtraction total oxalate from soluble oxalate

Table 7 .
Concentration of oxalate (mg/100g) in a selected Egyptian grown fruits

Table 8 .
Oxalate concentration of some common herbs (mg/100g D.W.) of daily selected common herbs