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Oxalates in some Indian green leafy

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Article in International Journal of Food Sciences and Nutrition · June 2008

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International Journal of Food Sciences and Nutrition,
May 2008; 59(3): 246
260

Oxalates in some Indian green leafy vegetables

M. RADEK1 & G. P. SAVAGE2

1
Institut National Polytechnique de Lorraine, Ecole Nationale Supérieure d’Agronomie et des
Industries Alimentaires de Nancy, Vandoeuvre-lès-Nancy, France, and 2Food Group, Agriculture
and Life Sciences, Lincoln University, Canterbury, New Zealand

Abstract
The soluble and total oxalate contents of 11 leafy vegetables grown in India were determined.
Spinach, purple and green amaranth and colocasia contained high levels of total oxalates, which
ranged from 5,138.0937.6 mg/100 g dry matter up to 12,576.19107.9 mg/100 g dry matter.
Seven other leafy vegetables (curry, drumstick, shepu, fenugreek, coriander, radish and onion
stalks) contained only insoluble oxalate, which ranged from 209.095.0 mg/100 g dry matter to
2,774.9918.4 mg/100 g dry matter. In vitro digestion of the samples showed that the gastric
available oxalate was 10% lower than the values obtained from acid extraction and that
intestinal available oxalate was 20% lower than the values obtained following hot water
extraction. The percentage calcium bound in the insoluble oxalate fraction of the dried leafy
vegetables ranged from 3.3% to 86.7% of the total calcium. Addition of four different sources of
calcium (low fat milk, whole milk, calcium carbonate and calcium sulphate) resulted in a range
of 32
100% reductions of intestinal available oxalate in the mixture.

Keywords: Indian leafy vegetables, soluble and insoluble oxalates, gastric available oxalate,
intestinal available oxalate, calcium availability

Introduction
Leafy green vegetables occupy an important place in the diets of millions of people in
India. A wide range of vegetables can be grown on the Indian subcontinent as it has a
variety of natural habitats, climates and seasons (Singh et al. 2001). Curry leaves,
fenugreek, coriander, drumstick leaves, radish leaves, onion stalks, shepu, spinach,
green and purple amaranth and colocasia are some of the most common vegetables
easily grown and they are widely consumed throughout India. These leafy vegetables
are generally inexpensive foods rich in important nutrients such as carotenoids,
vitamins, ascorbic acid, riboflavin, folic acid, polyphenols, iron, calcium and other
minerals. However, antinutritional constituents such as oxalate can reduce the
bioavailability of some minerals, especially calcium.
Oxalates can be found widely in many plant families in relatively small amounts, but
some plant species can accumulate oxalate in a range of 3
15% (w/w) of their dry
weight (Zindler-Frank 1976; Libert and Franceschi 1987; Nakata 2003; Franceschi
and Nakata 2005). Depending on the species, oxalate can occur as soluble salts of
potassium and sodium (soluble oxalates) and as insoluble salts of calcium, magnesium

Correspondence: G. P. Savage, Food Group, Agriculture and Life Sciences, Lincoln University, Canterbury,
New Zealand. Tel: 64 3 3253 803. Fax: 64 33253 851. E-mail address: savage@lincoln.ac.nz

ISSN 0963-7486 print/ISSN 1465-3478 online # 2008 Informa UK Ltd

DOI: 10.1080/09637480701791176
 Oxalates in some Indian foods 247

and iron (insoluble oxalates) or as a combination of these two forms (Noonan and
Savage 1999). The insoluble oxalate fraction in plants is most commonly made of
calcium oxalate, and in some plants is formed into crystals (Nakata 2003). Oxalate has
been shown to have different roles in plants such as calcium regulation, ion balance,
plant protection, tissue support and heavy metal detoxification (Libert and Franceschi
1987; Franceschi and Nakata 2005). Accumulation of oxalate in plants leads to a
change of structure that renders the plant less appetising and toxic for animals
consuming the plant tissue because of renal failure (Franceschi and Loewus 1995).
Soluble oxalate affects the human body because it can bind with calcium and other
minerals. It forms strong chelates with dietary calcium, rendering the complex
unavailable for absorption and assimilation. Insoluble oxalate in plant tissues is
excreted in the faeces while free oxalate and the soluble oxalate are absorbed by the
body and must be eliminated as they have no metabolic use (Albihn and Savage
2000). Although the main source of oxalate in urine is thought to be the results of
endogenous metabolism (Jaeger and Robertson 2004), it has been shown that kidney
stone formation may be increased by additional oxalate provided by the diet (Noonan
and Savage 1999; Siener et al. 2003). Calcium oxalate is one of the most common
kidney stone constituents (Savage 2002). Recent studies by Holmes and Assimos
(2004) have shown that, on average, one-half of the oxalate excreted in the urine is
derived from the diet, depending on the relative amounts of calcium and oxalate
ingested. These results contradict previous observations that dietary oxalate only
contributes up to 20% of the urinary oxalate excreted.
Calcium must be in a soluble form, generally ionized (Ca2), at least in the upper
small intestine or bound to a soluble organic molecule before it can cross the intestinal
wall (Guéguen and Pointillart 2000). This soluble calcium can also combine with
soluble oxalates in the small intestine, making both the calcium and the oxalate
unavailable for absorption. The consumption of foods containing high levels of soluble
oxalate may have a considerable effect on the amount of calcium absorbed. In most
countries dietary calcium is supplied by the consumption of dairy products and
vegetables with high calcium contents. In many poor countries, the intake of calcium
may be considerably compromised and this may affect the development of the foetus
(Harville et al. 2004). Low calcium intake can then be a danger for the mother’s bones
(Franz and Seelig 1997) and this may affect the level of calcium in their breast milk
(Ortega et al. 1998), further endangering the growing baby.
The Mumbai Maternal Nutrition Project uses food, rather than pharmaceutical
supplements, to increase women’s intake of a wide range of vitamins and minerals,
and thus improve their micronutrient status, prior to conception and during
pregnancy. The women taking part in this project are mainly vegetarian, and of
low income. Women of child-bearing age in India are frequently deficient in a
number of micronutrients, and there is evidence that this impairs their own health
(causing anaemia, susceptibility to infection, and possibly pre-eclampsia), contri-
butes to adverse pregnancy outcomes (congenital malformations, low birth weight
and pre-term delivery), and results in low micronutrient status in the newborn.
Green leafy vegetables are rich sources of micronutrients including b-carotene,
ascorbic acid, riboflavin, folic acid as well as calcium, iron, magnesium and zinc
(Gupta et al. 2005). Participants in the Mumbai Maternal Nutrition Project are
offered one ‘snack’ daily, made from dry powdered green leafy vegetables, fruit and
milk, incorporated into a deep-fried samosa and other types of ‘fast-food’ snack as a
248 M. Radek & G. P. Savage

way to increase their intakes of a number of essential nutrients. Gupta et al. (2006)
have shown that the bioavailability of iron and calcium in some Indian grown green
leafy vegetables is low, and these authors suggested that the oxalic content of some of
these leafy vegetables appeared to reduce the availability of iron and calcium in these
vegetables.
As calcium and oxalate were an issue in this project, calcium from external sources
was added in order to attempt to bind all of the soluble oxalate in each of the dried
foods. Calcium carbonate and calcium sulphate were added as they are cheap
inorganic sources of calcium and they will not affect the texture or flavour of the
samples at the low levels that need to be added to these foods. Whole milk and low fat
milk are known to be good sources of available calcium but these two sources of
calcium are more expensive to add to the diet. The plant foods analysed in this study
will be used to make samosas that will be given to the pregnant women as a dietary
supplement.

Materials and methods

Sample materials
The raw materials for this study (Table I) were purchased from wholesale markets in
Mumbai, India. They were harvested in rural districts up to 200 km from the city
markets, and were purchased approximately 24 h after harvesting. Once purchased,
the food materials were washed, air-dried at ambient temperature and then ground
into a powder, by a commercial supplier (Tech-Know Ltd, Mumbai, India). All the
extractions were carried out on these dry materials.

Chemical extraction: total and soluble oxalates

Soluble and total oxalate contents of 1 g each sample of dried food were extracted and
measured, as described in detail by Savage et al. (2000). Insoluble oxalate content
(calcium oxalate) was calculated by difference (Holloway et al. 1989). Each sample was
analysed in quadruplicate and all data are presented as a mean of mg oxalate/100 g dry
matter (DM) (9standard error).

Table I. English, Indian, Latin names and dry matter (%) of the 11 raw food materials.

English name Plant part Indian name Scientific name % DM

Onion Stalks Kanda Pyaaz Allium cepa 5.46

Purple amaranth Leaves Chauli, chavleri Amaranthus cruentus 16.72
Green amaranth Leaves Chauli, chavleri Amaranthus viridis 19.29
Taro Leaves Arbi Colocasia esculenta 12.03
Coriander Leaves Dhania patta Coriandrum sativum 7.91
Drumstick Leaves Seeng Moringa oleifera 15.56
Curry Leaves Kari patta Murraya koenigii 17.76
Dill Leaves Shepu Peucedanum graveolens 5.57
Radish Leaves Mooli Raphanus sativus 5.87
Spinach Leaves Palak Spinacia oleracea 6.11
Fenugreek Leaves Methi Trigonella foenum-graecum 6.45
 Oxalates in some Indian foods 249

In vitro extraction: gastric and intestinal soluble oxalates

The in vitro digestion method was based around the addition of four key solutions*a
saliva solution, gastric juice, duodenal juice and bile solution (Versantvoort et al.
2004)*and the method attempts to simulate the events that occur in vivo during
human digestion under fed conditions. The dried food samples were incubated
in a shaking water bath together with 6 ml saliva solution (378C) for 5 min. Gastric
juice solution (12 ml) was added and the sample was incubated in a shaking
water bath (378C) for 2 h. To the intestinal samples, duodenal juice solution (12 ml)
and bile solution (6 ml) was added and the sample was incubated in a shaking water
bath (378C) for another 2 h. After cooling to room temperature the samples
were quantitatively transferred to 250 ml volumetric flasks. The gastric extractions
were made up to volume with 0.2 M HCl, and the intestinal extraction with nanopure
water (Nanopure II; Barnstead International, Dubuque, Iowa, USA). Forty millilitres
of each sample was centrifuged at 3,500 rpm for 15 min at 48C (Varifuge 3.0R;
Heraeus, Centrifuge, Thermo Fisher Scientific Inc. Waltham, MA, USA). The
supernatant was filtered through a 0.45 mm cellulose nitrate filter (Sartorius AG,
Goettingen, Germany) prior to analysis. The soluble oxalates in each of the
extractions were measured using high-performance liquid chromatography (Savage
et al. 2000).

Recovery study
The recoveries of oxalic acid for the chemical and in vitro methods were studied by
adding 10 mg oxalic acid to 2 g dried colocasia leaf samples. The samples were
extracted and analysed using the chemical and in vitro methods, and were compared
with the samples with no oxalic acid added. The study was carried out in
quadruplicate. The mean recovery of adding oxalic acid from the acid and water
extraction methods was 103.390.4% and 102.190.4%, respectively, and the mean
recovery for the gastric and intestinal extraction methods was 103.990.03% and
97.190.04%, respectively.

Addition of different sources of calcium assay

The amount of calcium that could theoretically bind all of the intestinal available
oxalate in each of the food samples (spinach, purple amaranth, green amaranth and
colocasia) that contained intestinal available oxalate was calculated, and four different
sources of calcium were added and mixed directly to the dried samples in the digestion
flasks: low-fat milk powder (Pam’s Instant Skim Milk Powder, 0.5% fat; Pam’s
Products Ltd., Mt Roskill, Auckland, New Zealand), whole milk powder (Anchor
Instant milk powder 3.4% fat; New Zealand Dairy Foods Ltd., Takanini, Auckland,
New Zealand), calcium carbonate (BDH, Poole, UK) and calcium sulphate (Fisons,
Loughborough, UK). The resulting mixture of dried food samples with additional
calcium following incubation and extraction were analysed for intestinal available
oxalates. The solubilized oxalates in each of the extractions were measured using high-
performance liquid chromatography (Savage et al. 2000).
250 M. Radek & G. P. Savage

Calcium analysis
The total calcium contents of the 11 samples were analysed using an HNO3/HClO4
digestion: Inductively coupled plasma-optical emission (ICP-OES) determination on
a Perkin-Elmer 2100 machine (Perkin-Elmer Life. Analkytical Services Inc, Waltham,
MA, USA). The analysis was performed by a private laboratory (Ruakura Research
Centre, Hamilton, New Zealand).

Statistical analysis
Minitab version 14 (Minitab Ltd, Coventry, UK) was used to perform analysis of
variance on the data, and Genstat 8.2 (VSN International Ltd. Hemel Hempstead,
UK) was used to perform linear regression to compare the chemical and in vitro
methods.

Results
Extraction of oxalates
The total, soluble and insoluble oxalate contents of the 11 foods are presented in
Table II, together with the values for gastric and intestinal available oxalates. Each
mean value is the result of four separate extractions and determinations for each
sample. Each sample was statistically analysed to compare the total oxalic acid and the
gastric available oxalate values and to compare the soluble oxalic acid and the
intestinal available oxalate values.
The results in Table II have been divided into two groups based on the chemical
analysis. The upper group*spinach, purple and green amaranth and colocasia*
contained soluble oxalate (which varied from 25.2% to 94.6% of the total oxalates
for colocasia leaves and spinach leaves, respectively), while the other group of plant
materials had undetectable levels of soluble oxalate in their tissue. Another important
feature of the upper group of samples was the high level of total oxalates they
contained, which ranged from 5,138.0937.6 mg/100 g DM up to 12,576.19107.9
mg/100 g DM for colocasia leaves and spinach leaves, respectively.
The seven other samples contained only insoluble oxalate, which ranged from a very
low value of 209.095.0 mg/100 g DM up to 2,774.9918.4 mg/100 g DM for radish
leaves and curry leaves, respectively. In this group of foods, drumstick leaves and curry
leaves showed quite similar values of 2,754.0943.5 and 2,774.9918.4 mg/100 g DM,
while coriander leaves, fenugreek leaves and onion stalks also showed quite similar
values of 513.299.6, 527.5912.5 and 532.891.2 mg/100 g DM.
The results for the in vitro analysis, which gave values for gastric and intestinal
oxalates, are also presented in Table II. No intestinal available oxalate could be
detected in seven of the samples, whereas colocasia, purple and green amaranth and
spinach leaves have values for intestinal available oxalate from 925.2910.0 mg/100 g
DM up to 9,495.19147.2 mg/100 g DM for colocasia leaves and spinach leaves,
respectively. Regression analysis of the gastric available oxalate against the total
oxalate values showed that there was a good fit (R2 0.951). Statistical analysis
showed that the y intercept is not significant (P 0.999) but that the slope is
significant (P B0.001) which means that the gastric available oxalate values were, on
average, about 10% lower than those for total oxalate obtained by the acid extraction.
Table II. Mean9standard error oxalate content of 11 different food samples extracted using either chemical (acid and water extraction) or in vitro extraction method
(mg oxalate/100 g DM).

Chemical extraction (mg/100 g DM) In vitro extraction (mg/100 g DM)

Soluble oxalate
Foodstuff Total oxalate (% of total oxalate) Insoluble oxalate Gastric available oxalate Intestinal available oxalate

Spinach leaves 12,576.29107.9 11,899.89181.2 (94.6%) 676.3992.6 12,301.5943.3NS 9,495.19147.2***

Green amaranth leaves 10,056.3926.2 4,674.7992.8 (46.5%) 5,381.7982.7 8,421.5976.5*** 3,769.6969.1***
Purple amaranth leaves 8,106.0952.0 3,558.0980.6 (43.9%) 4,548.09115.2 5,493.7913.0*** 3,176.9942.0*
Colocasia leaves 5,138.0937.6 1,294.0921.4 (25.2%) 3,844.1945.6 4,615.2987.0** 925.2910.0***
Curry leaves 2,774.9918.4 nd 2,774.9918.4 725.4915.6*** nd
Drumstick leaves 2,754.0943.5 nd 2754.0943.5 1,151.0918.4*** nd
Shepu leaves 935.499.4 nd 935.499.4 1,032.696.8*** nd
Onion stalks 532.891.2 nd 532.891.2 505.596.8* nd
Fenugreek leaves 527.5912.5 nd 527.5912.5 653.0910.4** nd

Oxalates in some Indian foods 251

Coriander leaves 513.299.6 nd 513.299.6 400.799.6** nd
Radish leaves 209.095.0 nd 209.095.0 242.591.8** nd

nd, not detected.

Gastric available oxalate compared with total oxalate and intestinal available oxalate compared with soluble oxalate; significance: NS not significant; *P B0.05;
**P B0.01; ***PB0.001.
252 M. Radek & G. P. Savage

Regression analysis of the intestinal available oxalate against the soluble oxalate values
showed a good fit between the values (R2 0.997). Statistical analysis showed that
the y intercept was not significant (P 0.999) but that the slope was significant
(PB0.001), which means that the intestinal available oxalate values were, on average,
about 20% lower than those for soluble oxalate obtained by the water extraction.

Oxalates and calcium

Table III presents shows the total oxalate and total calcium and the molecular ratio
of the total oxalate over the total calcium contents. The table also shows the
calculated calcium content of the insoluble oxalate fraction of each sample assuming
that this fraction was only made up of calcium oxalate (but note that oxalate can
also bind to iron, zinc, manganese, etc., and these may also form part of the
insoluble oxalate fraction) as well as the percentage of the total calcium bound in the
calcium oxalate.
Noonan and Savage (1999) suggested that foodstuffs can be divided into three
groups depending on the ratio total oxalate/total calcium in mEq. This ratio is
interesting because it shows the effect on calcium availability from this food and
other foods consumed at the same time. Spinach was the only sample classified into
group 1, with a ratio of 4.58. Purple and green amaranth leaves were classified into
group 2, with 1.22 and 1.62 ratios. The eight other samples fell into group 3, with
ratios ranging from 0.03 up to 0.98 for radish leaves and colocasia leaves,
respectively.
Another way of classifying the samples is to consider the calcium bound in calcium
oxalate/total calcium as a percentage (Table IV). This ratio makes it possible to
calculate the amount of total calcium in a foodstuff that is available. It is then possible
to classify foods into three different groups using this new ratio: high ratio, 50%;
medium ratio, 20
50%; low ratio, B20% (Table IV).
Spinach, which belongs to group 1 with a ratio of 4.58 using Noonan and
Savage’s (1999) criteria, now falls into the medium ratio group whereas green
amaranth, purple amaranth and colocasia leaves fall into the high unavailable
calcium ratio group with very high proportions of calcium bound to oxalate, ranging
from 68.3% to 86.7%. In the medium unavailable calcium ratio group, the levels
varied between 22.4% for shepu leaves and 40.3% for curry leaves. The range for
the low unavailable calcium ratio group was from 3.3% for radish leaves up to
18.4% for coriander leaves.

Addition of calcium sources

Spinach, green and purple amaranth and colocasia leaves contained appreciable
levels of soluble oxalates, and if these foods were consumed in large amounts in the
diet it would result in the absorption of considerable amounts of soluble oxalates
into the body. As there is no metabolic use for oxalate in the body, it has to be
excreted by the kidneys*increasing the possibility of kidney stone formation. As
soluble oxalate has the potential to combine with calcium to form insoluble calcium
oxalate, the addition of calcium from milk or other common inorganic sources of
calcium may reduce the soluble oxalates available for absorption in the small
intestine.
Table III. Total oxalate (mg/100 g DM), total calcium (mg/100 g DM), total oxalate/total calcium (mEq) ratio, calculated calcium bound to oxalate (mg/100 g DM) and
percentage of the total calcium bound in oxalate for the 11 samples.

Total oxalate Total calcium Total oxalate/total Calculated calcium bound Calcium bound in
Foodstuff (mg/100 g DM) (mg/100 g DM) calcium (mEq) to oxalatea (mg/100 g DM) oxalate/total calcium (%)

Group 1
Spinach leaves 12,576.19107.9 1,220 4.58 300.6941.1 24.6
Group 2
Green amaranth leaves 10,056.1926.2 2,760 1.62 2,391.9936.8 86.7
Purple amaranth leaves 8,106.0952.0 2,960 1.22 2,021.3951.2 68.3
Group 3
Colocasia leaves 5,138.0937.6 2,320 0.98 1,708.5920.2 73.6
Curry leaves 2,774.9918.4 3,060 0.40 1,233.398.2 40.3
Drumstick leaves 2,754.0943.5 3,260 0.38 1,224.0919.3 37.5
Shepu leaves 935.499.4 1,860 0.22 415.794.2 22.4

Oxalates in some Indian foods 253

Onion stalks 532.891.2 2,010 0.12 236.890.6 11.8
Fenugreek leaves 527.5912.5 1,410 0.17 234.495.5 16.6
Coriander leaves 513.299.6 1,240 0.18 228.194.3 18.4
Radish leaves 209.095.0 2,850 0.03 92.992.2 3.3

Samples divided into three groups (group 1, 2.0; group 2, 1.0
2.0; group 3, B1.0) according to Noonan and Savage (1999). aAssumed all insoluble oxalate is calcium
oxalate but note that oxalate can also bind to iron, zinc, manganese, etc.
254 M. Radek & G. P. Savage
Table IV. Total calcium (mg/100 g DM), ratio of calcium bound in calcium oxalate/total calcium (%) and
available calcium (mg/100 g DM) for the 11 different food samples.

Foodstuff and unavailable Total calcium Calcium bound in calcium Available calcium
calcium ratio grouping (mg/100 g DM) oxalate/total calcium (%) (mg/100 g DM)

High ratio, 50%

Green amaranth leaves 2,760 86.7 367.1
Colocasia leaves 2,320 73.6 612.5
Purple amaranth leaves 2,960 68.3 938.3
Medium ratio, 20
50%
Curry leaves 3,060 40.3 1,826.8
Drumstick leaves 3,260 37.5 2,036.0
Spinach leaves 1,220 24.6 919.9
Shepu leaves 1,860 22.4 1,443.4
Low ratio, B20%
Coriander leaves 1,240 18.4 1,011.8
Fenugreek leaves 1,410 16.6 1,175.9
Onion stalks 2,010 11.8 1,772.8
Radish leaves 2,850 3.3 2,756.0

Additions of calcium from whole milk, low-fat milk, calcium carbonate and calcium
sulphate were made to the four foods that contained high levels of soluble oxalate:
spinach, green and purple amaranth and colocasia leaves. Sufficient calcium was
added from the four calcium sources to combine with all the intestinal available
oxalate in each sample. The remaining intestinal available oxalate values are presented
in Table V.
Statistical analysis showed that there was a significant reduction (P B0.001) in
the intestinal available oxalate of the calcium supplemented samples compared with
the original samples. Spinach showed high percentage reduction of intestinal available
oxalate when low-fat milk or whole milk were added (69% and 79.3%, respectively)
but the reduction was lower when inorganic sources of calcium were added (47.3% for
calcium carbonate and 31.7% for calcium sulphate). Green amaranth leaves showed a
59.8% of reduction when whole milk was added, but in the presence of low-fat milk
the reduction was only 38.7%. Calcium carbonate gave a 63.6% reduction, but only a
43.3% reduction with calcium sulphate. The percentage reduction of intestinal
available oxalate for purple amaranth leaves was similar for all the sources of calcium
(from 42.2% to 50.9%) and the mean reduction over the four experiments was 46.9%.
In contrast, colocasia leaves showed different results depending on the source of
calcium added. Low-fat milk and calcium carbonate gave reduced intestinal available
oxalate by 100%, whereas whole milk and calcium sulphate gave very low reductions
of only 26% and 15.5%, respectively.

Discussion
Analysis of all 11 plant food samples showed that oxalates can be found in each of the
samples. The values ranged from very high for spinach, green and purple amaranth
and colocasia leaves to low for coriander and radish leaves. Only the food samples that
contained high levels of total oxalates contained soluble oxalates, these ranged from
95% of the total oxalates for spinach leaves to 25% for colocasia leaves.
Table V. Mean9standard error intestinal available oxalate content without and with the addition of four different sources of calcium for the four samples with soluble
oxalate (mg oxalate/100 g DM).

Intestinal available oxalate (mg/100 g DM)

Without a source of Low-fat milk added Whole milk added Calcium carbonate added Calcium sulphate added
Foodstuff calcium added (% reduction) (% reduction) (% reduction) (% reduction)

Spinach 9,495.19147.2 2,946.2954.3*** (69.0%) 1,964.0935.0*** (79.3%) 5,005.8933.2*** (47.3%) 6,487.19140.5*** (31.7%)
Green amaranth 3,769.6969.1 2,311.5911.5*** (38.7%) 1,514.4930.3*** (59.8%) 1,373.0931.8*** (63.6%) 2,135910.5*** (43.3%)
Purple amaranth 3,176.9942.0 1,558.896.7*** (50.9%) 1,704.5913.4*** (46.3%) 1,641.2917.0*** (48.3%) 1,837.7958.7*** (42.2%)
Colocasia 925.2910.0 nd*** (100%) 784.793.7*** (15.5%) nd*** ( 100%) 686.796.4*** (26.0%)

Oxalates in some Indian foods 255

nd, not detected. Significant compared with intestinal available oxalate without a source of calcium added: ***PB0.001.
256 M. Radek & G. P. Savage

Extraction of oxalates
Brogren and Savage (2003) reported that raw spinach total oxalate content varies from
330 to 992 mg/100 g wet matter (WM). In the present study the total oxalate value for
spinach leaves was 768 mg/100 g WM, which is within the range of the values
previously reported. The values of total oxalate for different varieties of amaranth
ranged from 1,355 mg/100g WM up to 1,939 mg/100 g WM (Pingle and Ramasastri
1978; Noonan and Savage 1999) and from 3,760 mg/100 g DM for red amaranth up
to 5,000 mg/100 g DM for dried leaves of amaranth (Hels et al. 2004). The amaranth
species (purple and green) analysed in this study showed levels of total oxalate of
8,106 and 10,056 mg/100 g DM or of 1,355 and 1,940 mg/100 g WM. Those values
are comparable with the ones reported in the literature. Studies on colocasia report
values of total oxalate to range from 443 to 589 mg/100 g WM in young and older taro
leaves (Oscarsson and Savage 2007) and from 73 to 171 mg WM in the cormels of
Japanese taro (Catherwood et al. 2007). The value of 619 mg/100 g DM determined
in the present study is at the higher end of range of the values previously reported in
taro leaves. There are no reported oxalate values for the seven other food samples
analysed in this study.
Comparison of the chemical and in vitro methods showed that the in vitro method
gave lower results for both gastric and intestinal available oxalate compared with total
and soluble oxalates. It was expected that gastric available values should have been the
same as the total oxalate values given by the acid extraction, but they were 10% lower.
This may be explained by the fact that the acid treatment was carried out at 808C
whereas the gastric digestion was a milder treatment at 378C. During the gastric
digestion almost all of the oxalate (90% according to the gastric available values) is
dissolved and in the free oxalate form as the pH is around 1.3, but when oxalate goes
through the small intestine it has the potential to combine with minerals and, more
especially, calcium to form insoluble oxalate as the pH is around 8.1. A part of the
total calcium of the samples is available as it is not bound to oxalate (13.3%, 26.4%,
31.7% and 73.4% for green amaranth, colocasia, purple amaranth and spinach leaves,
respectively) and during the intestinal digestion the oxalate dissolved during the
gastric digestion has the possibility of binding more calcium than initially in the plant.
This could explain why the values for the intestinal available oxalate were 20% lower
than the soluble oxalate values given by water extraction. Mårtensson and Savage
(2007) analysed raw and cooked taro leaves and also found lower values for gastric
and intestinal available oxalate compared to the total and soluble oxalate extracted
with acid and water.

Potential effect of oxalate on calcium availability

The insoluble oxalate fraction of the food samples can bind many minerals, and more
especially calcium, rending them unavailable. It was interesting then to study the
potential calcium availability of the plant foods assuming that the insoluble oxalate
was only calcium oxalate as this is the major oxalate salt in the plant tissue. The
samples in this study were then classified according to the Noonan and Savage (1999)
molecular ratio (total oxalate/total calcium) and according to the ratio giving the
percentage of the total calcium bound in calcium oxalate.
The chemical analysis confirmed that spinach contains a high level of total oxalate
(2,576.19107.9 mg/100 g DM) but the insoluble oxalate fraction represents only
 Oxalates in some Indian foods 257

5.4% of the total oxalate value (676.3992.6 mg/100 g DM). It has then been shown
that this insoluble fraction binds only 24.6% of the total calcium. Purple amaranth,
colocasia and green amaranth seemed to have more interesting levels of total calcium
than spinach (2,960, 2,320 and 2,760 mg/100 g DM compared with 1,860 mg/100 g
DM, respectively), but 68.3%, 73.6% and 86.7% of these total calcium contents were
contained in the insoluble oxalate, rendering it unavailable. The amount of calcium
available is still quite similar for purple amaranth and spinach (938.3 and 919.9 mg/
100 g DM, respectively) but is lower for colocasia and green amaranth compared with
spinach (612.5 and 367.1 mg/100 g DM, respectively).
The result for the calcium availability in spinach is interesting but the problem of
the soluble oxalate remains because of its potential to lower the calcium availability
from other foods consumed at the same time, and so it is still an issue for kidney stone
formation. Previous studies reported that spinach belongs to group 1 with values
between 4.27 (Noonan and Savage 1999) and 4.73 (Brogren and Savage 2003). This
experiment showed a ratio of 4.58, which confirm that spinach belongs to group 1.
Amaranthus polygonoides and Amaranthus tricolor have also been studied by Noonan
and Savage (1999) and showed ratio of 1.18 and 1.07, respectively. The two
amaranths that have been analysed are different cultivars but showed quite similar
ratios of 1.62 for green amaranth and 1.22 for purple amaranth, which confirm that
plants belonging to the Amaranthaceae family and to the Amaranthus genus should be
classified in group 2. Colocasia leaves had a ratio of 0.98 and were classified in
group 3, which differed from a previous study that gave ratios of 1.8 for young leaves
and 1.1 for the old leaves that placed colocasia in group 2 (Oscarsson and Savage
2007). However, the ratios of 0.98 and 1.1 are very similar and close to 1, which place
colocasia between group 1 and group 2.
For the food samples in the low unavailable calcium ratio group, radish leaves and
onion stalks are interesting from the point of view of calcium and oxalate levels. Both
samples contain a high level of total calcium (2,850 and 2,010 mg/100 g DM,
respectively) and only small proportions (3.3% and 11.8%, respectively) of this
calcium is bound to oxalate, giving high amounts of available calcium (2,756.0 and
1,772.8 mg/100 g DM, respectively). Drumstick and curry leaves are two other
interesting foods. The proportion of total calcium bound to oxalate is higher (37.5%
and 40.3%, respectively) but the amount of available calcium remaining is still high
(2,036 and 1,826.8 mg/100 g DM, respectively) due to the fact that the levels of total
calcium in these two plants are very high. Moreover, those four samples did not
contain detectable levels of soluble oxalate, which means that they will not contribute
to kidney stone formation if eaten in larger amounts in the diet or interfere with
calcium from other foods.

Effects of adding different sources of calcium on intestinal available oxalate

Spinach, green and purple amaranth and colocasia showed high levels of soluble
oxalate that could present a risk of kidney stone formation as there no metabolic use of
oxalate by the body and soluble oxalate has to be excreted by the kidney. Soluble
oxalate has the possibility of binding to calcium; therefore four different sources of
calcium (low-fat milk, whole milk, calcium carbonate and calcium sulphate) were
added to the raw samples to see the effect on intestinal available oxalate.
258 M. Radek & G. P. Savage

The response of each sample to the addition of the four different sources of calcium
was different; therefore, each sample has to be considered as an independent system.
The addition of an inorganic source of calcium to spinach resulted in lower reduction
of intestinal calcium available (47.3% with calcium carbonate and 31.7% with calcium
sulphate) than with addition of either low-fat or whole milk (75%). Green amaranth
showed the best reduction when whole milk and calcium carbonate were added
(60%) than for low-fat milk and calcium sulphate (40%). Purple amaranth was the
sample reacting in the most logical and consistent way, with a mean reduction of
47% for all four treatments. The intestinal available oxalate was totally reduced in
colocasia when low-fat milk and calcium carbonate were added, whereas the
reductions were very low when adding whole milk (15.5%) and calcium sulphate
(26%).
The amount of calcium added was, theoretically, supposed to bind all of the soluble
oxalate in the samples but the results showed that some soluble oxalate still remained.
These results are quite difficult to interpret but it is more likely that the composition of
each sample has an effect on the capacity of soluble oxalate to bind calcium. It would
have been expected that the inorganic sources of calcium would give better reductions
than the milk, but, overall, calcium sulphate had the least effect on all the treatments.
However, the addition of four different sources of calcium to these four foods with
high levels of soluble oxalate resulted in a significant reduction of the intestinal
available oxalate in the food samples. This confirms previous studies that showed
adding a source of calcium high-soluble-oxalate-containing foods led to a reduction of
the soluble oxalate value (Pingle and Ramasastri 1978; Brogren and Savage 2003;
Savage et al. 2003; Mårtensson and Savage 2007).
Nevertheless, it has to be remembered that the four sources of calcium were added
to the raw and dehydrated plant food samples but the mixture used to make the
samosas will be cooked. It has been shown in several studies that cooking plant foods
containing high levels of oxalate had a reducing effect on the oxalate content (Chai
and Liebman 2005; Judprasong et al. 2005; Savage and Catherwood 2007). It would
therefore be interesting when the samosas will be formulated to do some extractions to
determine the oxalate content of the final product. The cooking process may, however,
contribute to the loss of significant amounts of some of the important nutrients.

Conclusions
This study confirmed previous oxalate values reported for some food plants such as
spinach, amaranth and colocasia leaves, and it provides new values for the total oxalate
content of the seven other samples that have not been studied previously. The level of
oxalate available for absorption in the intestinal environment was shown to be lower
than available in the original plant materials. During intestinal digestion soluble
oxalate has the possibility to bind more minerals, especially calcium in the plant tissue.
These in vitro data give a better indication of how much oxalate is actually available for
intestinal absorption as they simulate as closely as possible the processes involved in
human gastric and intestinal digestion of ingested food.
The calculation of different ratios of calcium availability in the samples gave
interesting results and showed that for some foodstuffs such as radish leaves, onion
stalks, drumstick and curry leaves the oxalate content would not be an issue in
lowering the calcium availability from other foods consumed at the same time. In
 Oxalates in some Indian foods 259

contrast, green amaranth and colocasia contain high levels of total calcium but, as a
high percentage of this calcium was bound to oxalate, these foods contain low levels of
available calcium. Overall, radish leaves seem to be the best plant food from the
calcium and oxalate point of view. However, the dry matter content of radish leaves is
quite low (2.56%) so this plant food may not be the most useful for the preparation of
a food supplement.
The addition of a source of calcium to plant foods containing high levels of soluble
oxalate significantly reduced the intestinal available oxalate content of these foods. If
spinach, green amaranth, purple amaranth or colocasia were used to make the
samosas it would then be interesting to consider adding a source of calcium to lower
the amount of soluble oxalate and to avoid the potential reduction in availability of
calcium from other dietary constituents. Adding calcium carbonate or calcium
sulphate would be less expensive than adding milk powder, and the texture or flavour
of the samosas would not be affected as the amounts added would be very low;
however, adding milk powder would have other nutritional advantages.

Acknowledgements
The authors wish to thank Dr Ramesh Potdar, Jeevak Hospital, Dadar, Mumbai,
India, and Professor Caroline Fall, MRC Environmental Epidemiology Unit,
University of Southampton, UK, co-principal investigators of the Mumbai Maternal
Nutrition Project, for making samples of dried vegetables available from their study.
The Centre for the Study of Social Change, Bandra, Mumbai, where the project is
located, and the main funders of the Mumbai Maternal Nutrition Project, The
Wellcome Trust (UK), Parthenon Trust, and ICICI Bank Ltd, Mumbai are also
thanked for their assistance.

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This paper was first published online on iFirst on 1 August 2008.