Journal of Food Composition and Analysis 24 (2011) 916922

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Journal of Food Composition and Analysis

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Original Article

Oxalate content in commercially produced cocoa and dark chocolate

Theresa Schroder a, Leo Vanhanen b, Geoffrey P. Savage b,*
a b

Department of Food Chemistry, University of Hohenheim, Stuttgart, Germany Food Group, Department of Wine, Food and Molecular Biosciences, Lincoln University, Canterbury, New Zealand

A R T I C L E I N F O

A B S T R A C T

Article history: Received 17 December 2010 Received in revised form 6 March 2011 Accepted 6 March 2011 Available online 11 March 2011 Keywords: Cocoa Chocolate Soluble and insoluble oxalate Theobroma Cacao L. Bioavailability Food analysis Food composition

Cocoa and dark chocolate have been promoted as health foods due to the high levels of antioxidants found in cocoa beans (Theobroma cacao L.) and their products but they also contain moderate to high levels of oxalates which can cause some health concerns. Fifteen samples of commercially available cocoa powder were collected from four different countries and the total and soluble oxalate content was analysed by HPLC chromatography. The total oxalate contents ranged from 650 to 783 mg/100 g dry matter (DM), mean 729 8.4 mg/100 g DM, while the soluble oxalate contents ranged from 360 to 567 mg/ 100 g DM, mean 469 15 mg/100 g DM. The total oxalate contents of 34 samples of dark chocolate collected from 13 different countries ranged from 155 to 485 mg/100 g DM, mean 254 12 mg/100 g DM while the soluble oxalate contents ranged from 157 to 351 mg/100 g DM, mean 216 10 mg/100 g DM. Oxalate bioavailability was determined by feeding 68.0 0.7 g of dark chocolate containing 232.0 2.3 mg total oxalate as a test meal to 14 volunteers. The mean availability of total oxalate in the chocolate measured from the increase in urinary oxalate output over the following 6 h was 1.82 0.27%. 2011 Elsevier Inc. All rights reserved.

1. Introduction In recent years, cocoa and dark chocolate have been promoted as health foods due to the high levels of antioxidants found in cocoa beans (Theobroma cacao L.) and their products (Jamison, 2004; Borchers et al., 2000; Rusconi and Conti, 2010), but they also contain moderate to high levels of oxalates (Zarembski and Hodgkinson, 1962; Weissberger et al., 1971; Kasidas and Rose, 1980; Brinkeley et al., 1981; Ogawa et al., 1984; Aremu et al., 1995), which can cause some health concerns. Oxalic acid is an antinutrient present in many plant foods where it is found in the highest concentrations in the seeds and leaves (Noonan and Savage, 1999). It is found either as the free acid, the soluble salt or bound to bivalent ions. The soluble salt can be absorbed by the human body and contributes to the oxalic acid concentration in the urine (Holmes et al., 1995) while insoluble oxalate is directly excreted in the faeces. Once the urine becomes supersaturated oxalate may crystallise as an insoluble salt, calcium oxalate, in the soft tissues, i.e. the kidneys, and form kidney stones (Massey, 2007). Some people either suffer from an increased endogenous oxalate production or have a higher oxalate absorption from food and, hence, a higher urinary oxalate output. As chocolate is considered as a high oxalate food (Williams and Wilson, 1990; Massey et al., 1993; Noonan and Savage, 1999;

* Corresponding author. Tel.: +64 3 3253 803; fax: +64 3 3253 851. E-mail address: savage@lincoln.ac.nz (G.P. Savage). 0889-1575/$ see front matter 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2011.03.008

Mendonc et al., 2003), The Oxalosis & Hyperoxaluria Foundation a (OHF, 2004) recommends that affected persons should avoid eating chocolate. Apart from forming kidney stones, oxalate can bind to minerals, such as calcium, magnesium and iron, rendering these less available for absorption. The contribution of oxalate from chocolate to urinary oxalate excretion also depends on oxalate bioavailability which is likely to be dependent on a number of factors including the proportion of soluble oxalate in the food and the composition of the food, for instance and the fat and bre content of the foods consumed at the same time as the chocolate. Some of previously used methods to determine the oxalate content in cocoa and chocolate are now considered antiquated and unreliable (Honow and Hesse, 2002). The previously used methods concentrate on total oxalate while the soluble oxalate content, which contributes to human urinary oxalate excretion, is disregarded. Furthermore, it was shown for other plants that the oxalate content can vary among different cultivars and during processing (Noonan and Savage, 1999). Aremu et al. (1995) found, for example, a decrease in the oxalate content of cocoa beans during fermentation, an essential step in processing the cocoa beans to initiate the development of a strong chocolate avour. Cocoa beans for most cocoa products and chocolate bars come mainly from two different cultivars of Theobroma cacao L. var. Criollo, which is grown in South and Central America and var. Forastero, which is grown in the Amazon region. So far, there has been no study that compares cocoa powders and chocolates with the cocoa beans originating from different cultivars.

T. Schroder et al. / Journal of Food Composition and Analysis 24 (2011) 916922 Table 1 Cocoa powder samples. Sample no. 1 2 3 4 5 6 7 8 9 10 11 Name of product (on the retail package) Kakao Cacao Cocoa Powder Bourneville Cocoa Cocoa Powder Quality Baking Cocoa Organic Cocoa Powder Cacao Powder Raw Cocoa Powder Cocoa Baking Cocoa Country of purchase Germany Netherlands New New New New Zealand Zealand Zealand Zealand Additives Potassium carbonate Flavour Potassium carbonate 1020% alkalized Country of origin of the cocoa beans Dominican Republic Ghana, Malaysia West Africa Malaysia South America Peru Malaysia Dominican Republic Cocoa bean variety Forastero, Trinitario All varieties Forastero Criollo, Forastero, Trinitatio Forastero Manufacturer Alnatura GmbH, Birkenbach, Germany Droste B.V., Vaassen, Holland

917

New Zealand New Zealand New Zealand New Zealand New Zealand

Potassium carbonate, sodium carbonate

ADM Cocoa Pte. Ltd., Singapore Cadbury Confectionery Ltd., Dunedin, NZ Nestle NZ Ltd., Newmarket, Auckland, NZ Pams Products Ltd., Mt. Roskill, Auckland, NZ Piko Wholefoods, Christchurch, NZ Piko Wholefoods, Christchurch, NZ Signature Range Brands, Mangere, Auckland, NZ SunValley Foods Ltd., Ellerslie Auckland, NZ Trade Aid Importers Ltd., Christchurch, NZ Woolworth Homebrand, Mangere, Auckland, NZ Axfood Sverige, Solna, Sweden Fazer Konfektyr AB, Sweden ICA AB, Solna, Sweden

12 13 14 15

Cocoa Powder Cocoa Powder Cacao Kakao

New Zealand Sweden Sweden Sweden Potassium carbonate Flavour Potassium carbonate, sodium carbonate

West Africa Dominican Republic

This study was undertaken to compare the oxalate content of representative samples of cocoa powders and dark chocolate bars from around the world and to perform a preliminary assessment of the oxalate bioavailability from one commercially produced dark chocolate. 2. Materials and methods 2.1. Sample material Representative samples of different cocoa powders and chocolate bars, all labelled as either dark chocolate or bitter chocolate (Tables 1 and 2) were purchased at either local health food shops or supermarkets in Australia, Belgium, Denmark, Ecuador, Germany, Italy, Malaysia, New Zealand (NZ), France, Sweden, Switzerland, the United Kingdom (UK) and the United States of America (USA) between April and July 2010. Baking cocoa powder was either purchased in plastic bags inside a box or paper bag (3, 5, 7, 8, 9, 12 and 13) or in re-sealable paperboard boxes (6 and 10). All of the chocolate bars were wrapped in proprietarybranded foil and paper. The cocoa powders were kept at room temperature in desiccators and the chocolate was stored at 4 8C. In preparation for analysis the chocolate bars were stored at 20 8C before grinding in a knife mill (Grindomix GM 200, Retsch; Haan, Germany). 2.2. Dry matter determination Each sample of cocoa or chocolate bar was sub-sampled and dry matter (DM) was determined by drying in an oven (Watvic, Watson Victor Ltd., NZ) set at 105 8C to a constant weight (AOAC, 1990). All determinations were performed in triplicate. 2.3. Extraction of total and soluble oxalic acid and analysis The measurement of total and soluble oxalate was performed following the method outlined by Savage et al. (2000). Triplicate samples of chocolate or cocoa powder were extracted with 50 ml nanopure water and incubated in a water bath at 80 8C for 15 min to extract soluble oxalates. Total oxalates were extracted using

50 ml 0.2 M HCl at 80 8C for 15 min. The extracts were cooled and ltered through 0.45 mm cellulose acetate lter (Sartorious, Gottingen, Germany) prior to HPLC analysis. Insoluble oxalate content was calculated by difference (Holloway et al., 1989). 2.4. Bioavailability assay Fourteen female volunteers aged from 21 to 35 were recruited from students at Lincoln University, Canterbury, NZ, to participate in the study. All participants were asked to maintain their normal diet throughout the experimental period, but to avoid high levels of foods and drinks known to contain high levels of total and soluble oxalate for the week proceeding and during the experimental period (a list was given to each participant). Each volunteer was also instructed not to have breakfast on the day the test meal was served. The volunteers were encouraged to drink half a cup of water every hour during the test period to ensure an adequate urine production. Participants ate a light lunch during the collection periods avoiding oxalate containing foods. Each participant consumed approximately 65 g (12 pieces) of Whittakers dark Ghana chocolate, containing 72% cocoa (J.H. Whittaker & Sons, Ltd., Porirua, NZ) as a test meal served as breakfast (9 am) after overnight fasting. Starting from the time when the test meal was consumed, total urine output was collected for 6 h in an individual plastic container. A urinary control sample was collected from each volunteer on a separate day in the week when the test meal was consumed. The volunteers ate a normal breakfast avoiding known high oxalate containing foods. The urine sample was collected for 6 h and was used as the reference blank for each person. 2.5. Urine analyses Total urine output was recorded and 10 ml of 35.4% HCl (Aristar, BDH Chemicals, Ltd., Poole, UK) was added. The samples were then mixed thoroughly to prevent the precipitation of oxalate as well as the conversion of ascorbic acid to oxalate and to inhibit microbial growth. The total oxalate content of the urine collected over 6 h was determined using a Trinity Oxalate Kit no 591 (Trinity Biotech Plc., Wicklow, Ireland). Oxalate was determined using an oxalate

918 Table 2 Information given on the chocolate bars. Sample no. New Zealand 1 2 Germany 3 4 5 Australia 6 7 8 Sweden 9 10 11 12 England 13 14 Name of product

T. Schroder et al. / Journal of Food Composition and Analysis 24 (2011) 916922

Cocoa content (%) 72 70

Ingredients

Country of origin of the cocoa beans

Manufacturer

Dark Ghana Dark Chocolate

Cocoa mass, cocoa butter, sugar, soya lecithin, avour Cocoa mass, sugar, cocoa butter, cocoa powder, avour, milk solids Cocoa mass, cocoa butter, sugar Cocoa mass, cocoa powder, cocoa butter, sugar Cocoa mass, sugar, soya lecithin

Whittakers, Porirua, New Zealand Cadbury, Dunedin, New Zealand

Grand Noir Edelbitter Excellence Bitter Schokolade Dunkle Schokolade edelbitter Cocoa Dark Chocolate Belgian Dark Chocolate Dark Chocolate

85 99 72

Ecuador

Gepa, Wuppertal, Germany Lindt, Aachen, Germany Sarotti, Berlin, Germany

70 70 70

Cocoa mass, sugar, cocoa butter, butter oil, soya lecithin, avour, milk solids Cocoa mass, sugar, cocoa butter, avour Cocoa liquor, cocoa butter, soya lecithin, vanilla Cocoa mass, sugar, cocoa butter, cocoa, butter fat, avour, soya lecithin Cocoa mass, cocoa butter, cocoa, sugar, avour, soya lecithin Cocoa mass, sugar, cocoa, canola lecithin, avour Cocoa mass, sugar, cocoa butter, cocoa Cocoa mass, sugar, cocoa butter, soya lecithin, vanilla Cocoa mass, raw cane sugar, cocoa butter, soya lecithin, vanilla extract, whole milk powder Cocoa mass, cocoa butter, cocoa powder, raw cane sugar, vanilla extract, whole milk powder, soya lecithin Cocoa mass, sugar, cocoa butter, cocoa powder, vanilla avour, soya lecithin Cocoa mass, cane sugar, cocoa butter, vanilla Cocoa mass, cane sugar, cocoa butter, vanilla Cocoa mass, cocoa, cocoa butter, cane sugar, bourbon vanilla Cocoa mass, cocoa, cocoa butter, sugar, butterfat, soya lecithin, vanilla extract Cocoa mass, sugar, cocoa butter, rapseed/ canola lecithin, vanilla Cocoa mass, sugar, cocoa butter, cocoa butter equivalent, cocoa powder, soya lecithin, vanilla Cocoa mass, cocoa butter, sugar, soya lecithin Cocoa liquor, cocoa butter, sugar, soya lecithin, algarrobina Chocolate liquor, evaporated cane juice, cocoa butter, soya lecithin, vanilla Cacao beans, sugar, vanilla beans Unsweetened chocolate Unsweetened chocolate, cocoa butter, sugar, milk fat, soya lecithin, vanilla, natural avour Unsweetened chocolate, sugar, cocoa butter, vanilla, soya lecithin

Nestle, Rhodes, NSW, Australia Coles, Tooranga, Victoria, Australia Kennedy&Wilson, Cold Stream, Victoria, Australia Marabou, Kraft Foods Sweden Marabou, Kraft Foods Sweden Hemkop, Solna, Sweden Aware, Solna, Sweden Divine, London, England Green&Blacks, London, England

Dark Chocolate Dark Chocolate Moerk Chokolad Dark Chocolate Dark Chocolate Dark Chocolate

70 86 72 70 70 70

Ghana Trinitario

15

Dark Chocolate

85

Madagascar

Green&Blacks, London, England

France 16 17 Italy 18 19 Switzerland 20 21 Denmark 22 Malaysia 23

Noir Chocolat Noir Cioccilato fondente extra Cioccilato fondente extra Noir puissant Noir extreme

76 99 70 70

Trinitario, Criollo Chuao

1848, Cadbury France, Paris, France Michel Cluizel, Paris, France Amedi Toscani, Pisa, Italy Amedi Churo, Pisa, Italy

85 80

Lindt, Kirchberg, Switzerland Cailler, Nestle, Vevey, Switzerland

Chocolate

72

Anthon Berg, Ballerup, Denmark

Dark Chocolate

55

Tudor, Selangor, Malaysia

Ecuador 24 25 USA 26 27 28 29

Dark Arriba Chocolate Chocolate

77 70

Ecuador (Arriba Nacional) Ecuador

Caoni, Quito, Ecuador Hacienda el Castillo, Guawaquil, Ecuador Newmans Own, Aptos, CA, USA Scharffenberger, Berkeley, CA, USA Ghiradelli, San Leandro, CA, USA Ghiradelli, San Leandro, CA, USA

Super Dark Chocolate Extra Dark Chocolate Unsweetened Chocolate Intense Dark

70 82 100 86

30 Belgium 31 32 33 34

Twilight Delight

72

Ghiradelli, San Leandro, CA, USA

Dark Papua Sao Tome Tanzania Ecuador

70 72 75 70

Papua New Guinea Sao Tome Tanzania Ecuador

Neuhaus, Neuhaus, Neuhaus, Neuhaus,

Vlezenbeek, Vlezenbeek, Vlezenbeek, Vlezenbeek,

Belgium Belgium Belgium Belgium

T. Schroder et al. / Journal of Food Composition and Analysis 24 (2011) 916922 Table 3 Mean dry matter (DM), total, soluble and insoluble oxalate content of the cocoa powders ( standard error). Sample 6 13 8 2 7 5 14 10 9 11 3 15 4 12 1 Mean Dry matter (%) 94 96 95 95 91 95 95 97 96 94 94 94 95 93 93 94 0.4 Total oxalate (mg/100 g DM) 783 1.9 768 1.0 754 3.0 743 3.9 743 0.5 743 1.5 738 2.0 736 3.8 728 4.3 722 6.7 720 9.2 716 4.7 700 5.4 687 7.7 650 3.6 729 8.4 Soluble oxalate (mg/100 g DM) 511 5.0 449 7.0 567 8.9 432 8.6 415 4.0 509 0.6 502 6.7 371 6.8 429 4.0 464 8.2 486 5.9 479 3.7 530 6.5 532 18 360 1.7 469 15

919

Insoluble oxalate (mg/100 g DM) 273 319 189 311 330 234 236 379 300 252 234 237 155 170 290 261 16

oxidase assay linked to a peroxidise reaction which yielded an indamine dye which was measured at 590 nm in an Abbot Architect C8200 Auto Analyser (Abbot Laboratories, IL, USA). 2.6. Bioavailability calculations The oxalate bioavailability was calculated using each participants 6-h urinary oxalate output after ingestion of the test meal; each participants reference blank 6 h urinary oxalate output was subtracted from the total output during the test day. The total urine collected was weighed and output (ml) calculated assuming a specic gravity of urine of 1.0 g/ml (Watson and Lefebvre, 2010). The values are presented as mean total oxalate intake and oxalate excreted and percentage of oxalate absorbed from total oxalate intake over the 6 h collection period. In addition the soluble oxalate intake and total oxalate excreted and percentage of soluble oxalate absorbed were also calculated for the same collection period. 2.7. Statistical analyses All calculations were performed using Excel1 2007 Version 12 and all determinations were carried out in triplicate and results presented as a mean SE. Statistical analyses of the total and soluble oxalate content of cocoa and chocolate were performed using Minitab version 15.1 (Minitab Ltd., Coventry, UK), mean differences were determined using a one-way ANOVA. 3. Results and discussion 3.1. Oxalate content in cocoa powder and chocolate The experiments were performed in triplicate and the results presented as the mean SE, on a dry matter basis. Mean dry matter and total, soluble and insoluble oxalate of the cocoa powders (mg/ 100 g) are shown in Table 3. The mean total oxalate content for all cocoa powders analysed, 729 8.4 mg/100 g DM, was higher than reported in previous studies by Weissberger et al. (1971) (240 430 mg/100 g DM) and Aremu and Abara (1992) (68.0164 mg/100 g DM). The mean soluble oxalate content for all cocoa powders analysed, was 469 15 mg/100 g DM, this is higher than reported by Aremu et al. (1995) for fermented cocoa beans (17.629.3 mg/100 g DM). Weissberger et al. (1971) performed their study by precipitating the oxalate as a salt, re-dissolving it and analysing it using gas chromatography. Aremu and Abara (1992) and Aremu et al. (1995) analysed the oxalate content using a colorimetric titration. The mean amount of insoluble oxalate in this study was 261 16 mg/100 g DM,

varying from 155 mg/100 g DM (sample no. 4) to 379 mg/100 g DM (sample no. 10). No relationship was found between the mean total oxalate content of the cocoa powder and manufacturing factors such as ingredient addition, the Dutching process (alkalization) or organic production of the samples, but there appeared to be some differences between the cultivars used to make the cocoa powder. Mean dry matter, total, soluble oxalate and insoluble oxalate contents of the 34 different chocolate bars analysed (mg/100 g DM) are shown in Table 4. Fig. 1 shows that the total and soluble oxalate contents of the chocolate bars were correlated to the cocoa content of the chocolate bars (total oxalate R2 = 0.602 and soluble oxalate R2 = 0.716). The chocolate bars analysed in this study were labelled as dark chocolate or bitter chocolate by the manufacturer and had an average cocoa content of 75.9 1.7%. The total oxalate contents ranged from 155 12 mg/100 g DM to 485 1.6 mg/100 g DM with an overall mean of 252 12 mg/100 g DM. The mean was higher than reported in previous works by Ogawa et al. (1984), Brinkeley et al. (1981), Kasidas and Rose (1980), and Zarembski and Hodgkinson (1962) who found values ranging from 123 to 176.4 mg/100 g DM. The soluble oxalate content ranged from 108 14 mg/100 g DM (sample no. 26) to 351 5.1 mg/100 g DM (sample no. 5). The overall mean was found to be 214 10 mg/100 g DM, which was much higher than previously reported (Zarembski and Hodgkinson, 1962; Kasidas and Rose, 1980; Brinkeley et al., 1981; Ogawa et al., 1984). The values for insoluble oxalate in the chocolate samples were low and ranged from not detectable to 140 mg/100 g DM. The total and soluble oxalate values reported in this study for both cocoa powder and chocolate bars are higher than previous studies. This may be a result of more efcient extraction and analysis methods it is also possible, that there may have been a change in the extraction and processing methods used to manufacture cocoa and chocolate. The extraction of oxalates from foods poses some problems. Honow and Hesse (2002) used hot acids to extract total oxalate, while Zarembski and Hodgkinson (1962) used cold acid extraction, which may lead to incomplete extraction and explain the lower values found in their later study (Zarembski and Hodgkinson, 1962). Kasidas and Rose (1980) used an enzymatical determination. Honow and Hesse (2002) suggest that this may also lead to incomplete extraction due to the generation of carbon dioxide which explains why the values for total oxalate in chocolate found by Kasidas and Rose (1980) (117 mg/100 g FW) are lower than the values observed in this study (254 12 mg/100 g DM). The Oxalosis & Hyperoxaluria Foundation (2004) considers an intake of

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T. Schroder et al. / Journal of Food Composition and Analysis 24 (2011) 916922

Table 4 Cocoa content,a mean dry matter (DM), total, soluble and insoluble oxalate content of chocolate bars ( standard error). Sample 4 17 1 28 31 34 3 20 2 32 27 21 33 16 10 29 11 25 19 8 18 22 14 6 15 7 13 9 30 12 26 23 24 Mean
a

Cocoa content (%) 99 99 72 100 70 70 85 85 70 72 82 80 75 76 86 86 72 70 70 70 70 72 70 70 85 70 70 70 72 70 70 55 77 75.9 1.7

Dry matter (%) 97 96 98 98 97 98 97 97 98 97 97 97 98 99 98 98 98 98 98 98 98 98 98 98 98 98 99 98 99 98 98 98 97 97.8 0.10

Total oxalate (mg/100 g DM) 485 1.6 381 5.7 360 1.4 322 6.6 310 1.1 302 7.0 300 1.0 297 4.8 279 3.4 270 7.5 260 6.9 257 7.7 257 7.4 254 1.2 240 1.9 236 2.7 233 1.2 224 7.5 224 2.7 222 4.0 221 1.7 214 3.9 211 2.4 207 2.5 201 7.4 199 2.0 198 4.1 194 9.4 191 1.7 189 2.8 169 5.7 157 0.3 155 12 254 12

Soluble oxalate (mg/100 g ) 345 4.6 306 8.2 269 4.7 284 12 267 9.5 177 6.7 223 4.3 299 3.2 184 9.3 240 2.6 197 4.5 234 7.0 189 5.5 212 1.3 233 5.6 205 11 224 6.0 186 3.8 190 3.8 180 7.2 188 4.0 174 1.6 180 10 182 4.8 181 8.6 196 14 164 3.8 168 6.7 203 3.7 214 0.5 108 14 124 0.9 157 5.8 216 10

Insoluble oxalate (mg/100 g DM) 140 75 91 38 34 125 77 95 30 63 23 68 42 7 31 9 38 34 42 33 40 31 25 20 3 34 26 61 33 39 5.9

Cocoa content stated by the manufacturer on the retail package.

<80 mg of oxalate/day as a low oxalate diet. This is equivalent to approximately 10 g cocoa powder or 30 g dark chocolate/day. Germans consume an average of 7 g and New Zealand an average of 0.6 g (FAOStat, 2007) of cocoa (pure and as products) per person per day. If it is assumed that Germans consume 7 g chocolate/day then they would be consuming 45.5 mg total oxalate from that source alone. Chocolate and cocoa powder are already considered high oxalate foods (Williams and Wilson, 1990; Massey et al.,

1993; Noonan and Savage, 1999; Mendonc et al., 2003; OHF, a 2004) and it is advised that patients at risk of forming kidney stones should avoid eating cocoa products. Cocoa and chocolate is often added to cakes and biscuits, which is often overlooked when oxalate intakes are considered. In addition, the bioavailability of oxalate in these products also needs to be considered as this will have an effect on the oxalate actually absorbed in the digestive tract.

Fig. 1. Total and soluble oxalate (mg/100 g dry matter standard error [SE]) compared to the stated cocoa content (%) of the dark chocolate bars.

T. Schroder et al. / Journal of Food Composition and Analysis 24 (2011) 916922

921

The values of total, soluble and insoluble oxalate found for chocolate (254 12 mg/100 g DM; 216 10 mg/100 g DM; 39 5.9 mg/100 g DM, respectively) were signicantly lower (p < 0.05) than those for the cocoa powders (729 8.4 mg/100 g DM; 469 15 mg/100 g DM; 216 16 mg/100 g DM, respectively). The oxalate content of dark chocolate was lower due to the dilution of cocoa with other ingredients used in the production of chocolate. The mean cocoa content of the analysed samples was 75.9 1.7%. However, extrapolated to 100% cocoa content (335 mg/100 g DM; 286 mg/100 g DM; 51.7 mg/100 g DM, respectively), the values are still lower than for pure cocoa powder. This suggests that there is some reduction of oxalate content during, mixing rening and conching used to make chocolate. The oxalate content in chocolate is determined by the amount of cocoa in the particular chocolate bar (Fig. 1) shows the total and respective soluble oxalate contents (mg/100 g DM SE) in comparison to the amount of cocoa (%). A mean was calculated for the oxalate contents of chocolate bars with the same cocoa content and the standard error was determined. Hence, if there is no standard error shown, as only one chocolate bar was analysed at each particular cocoa content. A relationship can be seen between the cocoa content and the total as well as the soluble oxalate content. The higher the cocoa content is, the higher is the oxalate content. This suggested that the oxalate found in chocolate originated from the cocoa beans. In addition, it can be recommended that people on low oxalate diets to eat chocolate with a low cocoa content. This cannot be said for all chocolate bars, as it can also be seen from Fig. 1 that some bars were higher in oxalate (total and soluble) even though they contain a low amount of cocoa. However, the chocolate that contained the lowest amount of cocoa (55%) contained signicantly lower (p < 0.05) total and soluble oxalate content than the chocolate bar containing the highest amount of cocoa (99 and 100%). The chocolate bars with a cocoa content between 70 and 86% contained a mean of total oxalate of between 232 6.9 mg/100 g DM (70% cocoa) and 268 14.9 mg/100 g DM (72% cocoa) and a mean amount of soluble oxalate of between 192 5.4 mg/100 g DM (75% cocoa) and 245 13.7 mg/100 g DM (72% cocoa), with the exception of sample no. 24 (Caoni Dark Arriba Chocolate, from Ecuador), which has a signicantly lower (p < 0.05) value for both, total and soluble, oxalate (155 12 mg/100 g DM and 157 5.8 mg/100 g DM, respectively). It is interesting to note that the chocolate sourced from the Nacional Arriba cultivar grown in Ecuador had lower levels of total and soluble oxalate contents when compared to all the other chocolate produced in other countries. 3.2. Bioavailability of oxalate from chocolate The results of this study demonstrates that the consumption of 68 g dark chocolate increased the urinary oxalate output in the following 6 h by a mean of 69% above the reference value when no oxalate was consumed (Table 5). Urinary oxalate analyses showed
Table 5 Mean intake, excretion and bioavailability of soluble and total oxalate following the consumption of 68 g of dark chocolatea by 14 female volunteers. Mean oxalate intake (mg/test meal) Mean urinary output (mg oxalate/6 h) Mean oxalate availability calculated from the intake of 68 0.7 g dark chocolate (%)

that the mean oxalate output of the 14 volunteers when no oxalate was consumed was 6.82 mg/6 h (range 3.8811.7 mg/6 h). The urinary output of oxalate after the ingestion of chocolate ranged from 5.56 to 16.7 mg/6 h with a mean output of 11.1 mg/6 h. The increase in total oxalate output ranged from 1.17 to 8.86 mg/6 h, with a mean of 4.24 0.65 mg/6 h. The mean rise of urinary total oxalate was 69.6 13% above the reference value, which is within the range of results presented in previous studies: 250% (Finch et al., 1981), 28% (Brinkeley et al., 1981), 213% (Nguyen et al., 1994) and 20% (Mendonca et al., 2003). However these studies were performed differently. Mendonca et al. (2003) and Finch et al. (1981) collected urine samples for 24 h, Nguyen et al. (1994) collected the urine for 3 h and Brinkeley et al. (1981) for 8 h. All of the studies were carried out using dark chocolate apart from Brinkeley et al. (1981), who used milk chocolate. The mean bioavailability, calculated as the percentage of the increase in total urinary oxalate compared to the intake of total oxalate from the chocolate, was 1.82 0.27%. It was higher than the total oxalate bioavailability from spinach, which was 0.75 0.48% (Brogren and Savage, 2003) but lower than the value obtained following feeding baked taro and stir-fried silverbeet, which were 2.66 0.49% and 2.41%, respectively (Martensson and Savage, 2008; Johansson, 2010). The mean soluble oxalate consumed by the volunteers was 172.8 1.7 mg which gave a mean soluble oxalate availability of 2.45 0.37%, which conrms that most of the insoluble oxalate in chocolate is not absorbed and is voided in the faeces. Liebman et al. (1999) reported that the presence of fat lowers the absorption of oxalate. Martensson and Savage (2008) conrmed this when showing that soluble oxalates are benecially reduced in taro (Colocasia esculenta) when consumed with additional plant oil or calcium. As chocolate contains around 40% of total lipids (National Nutrient Database for Standard Reference, 2010), this suggests that the oxalate absorption might be inhibited or possibly slowed down by the fat consumed at the same time. In contrast, Bailly et al. (2000) suggested that the amount of soluble oxalate absorbed was higher when the product was consumed with additional fat, due to the fatty acids binding to the calcium in the gastrointestinal tract and leaving it unavailable to form insoluble salts with oxalate. The results of this study conrmed that the dietary intake of oxalate following the consumption of 68 0.7 g dark chocolate affected the excretion of oxalate in the urine by increasing the oxalate output by a mean of 69.6 13% above the base value when no additional oxalate was consumed by the volunteers. The consumption of 68 g chocolate resulted in the consumption of 232.0 2.3 mg total oxalate, which is almost three times higher than the maximum intake (80 mg) of oxalate recommended by the OHF (2004) as a low oxalate diet. 4. Conclusions The total and soluble oxalate of cocoa powder and chocolate in this study was found to be higher than reported in previous studies (Zarembski and Hodgkinson, 1962; Weissberger et al., 1971; Kasidas and Rose, 1980; Brinkeley et al., 1981; Ogawa et al., 1984; Aremu and Abara, 1992 and Aremu et al. 1995). This suggested that the oxalic acid extraction and analysis used in earlier studies might not have been fully effective. This study supports the suggestion that cocoa and chocolate do contain high levels of oxalate. In the light of the relatively low oxalate bioavailability observed in this study it is unclear whether typical servings of cocoa or chocolate would lead to signicant increases in urinary oxalate output and associated health problems. Cocoa was found to contain more oxalate than chocolate. Chocolate bars containing higher levels of cocoa contained higher amounts of oxalates. However, here was no trend found between the oxalate content and the origin or cultivar

Reference blank, no chocolate eaten Soluble oxalate Total oxalate

a

6.8 0.6

172.8 1.7 232.0 2.3

11.1 0.8 11.1 0.8

2.45 0.37 1.82 0.27

Whittakers, Dark Gahana (Table 2, sample no 1).

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of the cocoa bean, methods of processing or country of purchase, for either the cocoa powder or the chocolate analysed in this experiment. Further experiments should consider the ingredients and origins of the cocoa powders and chocolate bars in greater detail. Furthermore, a study on the apparent change in the oxalate content following processing the cocoa bean to chocolate should be undertaken. Acknowledgements We would like to thank Jillian Donaggio, Mary-Ann Hunt and John Wallace at the Canterbury Health Laboratories, Christchurch Hospital, for assistance with the analyses of urine oxalate; as well as the volunteers who participated in the human study. References
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