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MAFF UK - DUPLICATE DIET STUDY OF VEGETARIANS - NITRATE ANALYSES

REVISED JANUARY 2000 - THIS VERSION REPLACES THAT ORIGINALLY ISSUED

Index to MAFF UK Food Surveillance Information Sheets, 2000

Summary

A duplicate diet study of vegetarians has been carried out to determine dietary exposures to inherent natural toxicants, 12 metals and other chemical elements, and nitrate. This information will be used to assess the risks to health to vegetarians by comparison with dietary exposures estimated for the general UK population and the appropriate recommended intakes.

Nitrate analyses were the first to be completed and are reported here. Results for metals and other elements are reported in Food Surveillance Information Sheet No. 193 in January 2000.¹ Results for natural toxicants will be reported once they are finished.

The individuals taking part in the study were asked to collect an exact duplicate of everything they ate over a week and the samples were analysed for nitrate. Dietary exposures were calculated from the concentrations of nitrate in the duplicate diet samples, the weights of the samples and the weights of the individuals participating in the study. Thus they should be exact records of dietary exposures to nitrate.

Dietary exposures of nitrate of the participants in the duplicate diet study of vegetarians were below the Acceptable Daily Intake (ADI), equivalent to 219 mg/day for a 60 kg person, recommended by the European Commission’s Scientific Committee for Food. The average (mean) dietary exposure of the vegetarians in this study was 83 mg/day and highest nitrate exposure was 209 mg/day. These are both below the ADI and are similar to exposures estimated for the general UK population (mean of 88 mg/day, upper range 136 mg/day) from the 1997 Total Diet Study. There are therefore no concerns for the health of vegetarians from nitrate in their diets.

Background

Vegetarians now make up a significant minority of the UK population. It has been estimated that more than 5 per cent of the UK population do not eat fish or meat.² Vegetarians are of interest as their dietary habits may cause them to have different exposures to certain chemicals (such as nutrients and contaminants) from food than the general population. They may therefore be exposed to different health risks than other UK consumers from some chemicals in food. JFSSG carried out this duplicate diet study to assess dietary exposures and associated risks to health from a range of chemical contaminants and inherent natural toxicants in vegetarian diets. This Food Surveillance Information Sheet reports the results of nitrate analyses of vegetarian duplicate diets. The nitrate analyses were the first to be completed.

Duplicate diet study of vegetarians

JFSSG carries out a number of dietary surveys to provide information on consumption of foods by the UK population and individuals.³ This information is used together with data on concentrations of chemicals in food to estimate dietary exposures and assess the risks to health. The two main dietary surveys used to provide such consumption data (i.e. The National Diet and Nutritional Survey Programme, and The National Food Survey) are based on the general UK population and do not provide information on groups with specific dietary habits.^4,5

Duplicate diet studies provide an exact account of consumption and dietary exposures of individuals. Participants in duplicate diet studies collect equivalent portions of all the foods (and drinks) they have consumed during the period of the study. The individual diets collected are then analysed for the contaminants and inherent natural toxicants of interest.³

Vegetarians may have a higher than average consumption of vegetables. They may therefore have higher dietary exposures of those chemicals which are present in vegetables. Plants naturally contain biologically active chemicals such as glucosinolates, phytoestrogens, glycoalkaloids, furocoumarins and nitrate. Chemicals such as glucosinolates, phytoestrogens, glycoalkaloids and furocoumarins are produced naturally by plants to respond to stress or as part of a plant’s defence mechanism and are known as ‘inherent natural toxicants’.⁶ Others such as lead and cadmium are contaminants and may be present in crops as the result of human activities such as certain agricultural practices and pollution from industrial processes and car exhausts.^7,8

Nitrate

Most foods contain nitrate. This can be present naturally, or may be present as a result of the use of fertilisers on crops,⁹ or from its use as a preservative.^10,11 The nitrate content of foods has been of interest for many years because of its possible health effects.¹² However, the chemistry of dietary nitrate is complex and there are a number of potentially detrimental and beneficial health effects. An example of a possible detrimental effect is the metabolism in the gut of dietary nitrate to potentially carcinogenic N-nitrosoamines.¹³ Conversely, the acidic conditions in the stomach cause the formation of nitric oxide which has recently been investigated for its role in the body’s defence against pathogenic bacteria.^{14 - 16}

The European Commission’s (EC) Scientific Committee for Food (SCF) has recommended an Acceptable Daily Intake (ADI) for the nitrate ion of 3.65 mg/kg body weight (equivalent to 219 mg/day for a 60 kg person).^9,17 The SCF ADI for nitrate has been endorsed by the UK Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment (a committee of independent experts which advises the UK Government).¹⁸ More information on how the ADI for nitrate was derived is given in the SCF reports, Food Surveillance Paper No. 32 and Food Surveillance Information Sheet No. 163.^{9,17
- 19}

JFSSG has carried out a number of surveys to determine nitrate concentrations in food.^{11,18
- 23} The results of these surveys have been used to assess the risks to UK consumers from nitrate in food by estimating dietary exposures and comparing them with the ADI. For example, dietary exposures of nitrate for adult consumers, estimated from the results of the 1997 UK Total Diet Study, were 88 mg/day for mean consumers and 136 mg/day for upper range (97.5 percentile) consumers.¹⁹ These estimates are both below the ADI.

Purpose

This study was carried out for two reasons. Firstly, it would provide current data on dietary exposures to nitrate by vegetarians for comparison with those estimated for the general population and the ADI. Secondly, the results may provide information to support the application of the derogation (exemption) from the maximum levels (limits) for nitrate set by European Commission Regulation (EC) No. 194/97 as amended by European Commission Regulation (EC) No. 864/99.^{24 - 27} The derogation applies only to lettuce and spinach grown and sold for consumption in individual Member States (the UK is one of a number of Member States applying this derogation). One of the conditions of European Commission Regulation (EC) No. 194/97 for application of the derogation is that nitrate concentrations in lettuce and spinach are not a public health risk. This can be assessed by comparing dietary exposures to nitrate of UK consumers of lettuce and spinach with the ADI. Previous assessments, which have been based on consumption data for the general population, are below the ADI.^28,29 It was hoped that the results of this study would provide further reassurance that there is no public health risk from nitrate in UK-grown lettuce and spinach.

Brand names

The duplicate diet study of vegetarians is excluded from the MAFF policy for the release of brand names when reporting the results of food chemical surveillance because there are no brand names to report.³⁰ All samples were composites of a number of foods and it is not possible to identify individual items.

Methodology

Study design

BMRB (British Market Research Bureau) International Limited recruited the participants of the vegetarian duplicate diet study and collected their diets. Participants were selected from 400 vegetarians who had previously taken part in a dietary diary study for MAFF.³¹ To be eligible to take part in the duplicate diet study, participants had to be vegetarians who consumed protein substitute products (e.g. tofu, textured vegetable protein, etc.), ate neither meat nor fish, and were non-smokers. All participants were financially compensated for the cost of the food for the duplicate diets and for their time in taking part.

Each participant was asked to collect an exact duplicate of all the foods eaten (including snacks and sweets, and foods/meals eaten outside the home) over a 7 day period. The only drinks collected were soya milk, herbal drinks and soup. Beverages such as tea and coffee were not collected although any soya milk added was. Participants were provided with household scales, foil trays and plastic cups in which to weigh food, and strong 2-litre plastic bottles in which to store the samples. A diary of the foods eaten (description of food, weight and time eaten, and if eaten outside the home), any dietary supplements or medicines taken, and whether a participant felt well or unwell were kept. The total weight of food recorded as eaten over 7 days was used as a check against the actual weight collected. The descriptions of the foods eaten were useful as a check against any unusual results and to ensure that participants were following vegetarian diets. It was also important to record the well-being of participants as this may have affected the types and amounts of foods consumed. Check visits were made by BMRB field workers on the second day of collection to ensure that the diary was being completed and the food properly gathered.

The vegetarian duplicate diet study was carried out in two phases to determine if there were any seasonal differences in dietary exposure to the contaminants of interest. These two phases were summer (26 August to 1 September 1997) and winter (9 to 15 February 1998). There were 49 participants in the summer phase and 54 in the winter phase, with 34 participants taking part in both phases. It should be noted that two samples from the summer phase were damaged in transit, resulting in 101 samples being analysed.

Sample collection and dispatch

Samples (each of which comprised a 7-day diet) were weighed on collection by a BMRB field worker at the end of each phase. A recall questionnaire was completed on sample collection to ascertain the participant’s weight and whether or not they had been ill during the collection week.

All summer-phase samples were sent, at the end of the collection week, to the Institute for Food Research, Norwich (IFR). However, on arrival at IFR it was noted that some samples had started to decompose. This may have affected the nitrate concentrations in these samples. To avoid this recurring in the winter phase, participants were asked to store their sample bottles in a freezer or refrigerator. However, as many participants did not have sufficient refrigerator or freezer space, a mid-week collection was introduced. This improved the condition of the samples arriving at IFR. Both summer and winter phase samples were, as far as possible, transported to IFR in cool boxes.

Further information on the design of the duplicate diet study of vegetarians and sample collection is given in the final report from BMRB which will be available for public access in MAFF’s library once all the results are reported.

Sample preparation

On arrival at IFR, samples were stored at -18^oC. All samples were weighed before being prepared for analysis by coarse blending and homogenisation. The homogenised samples were divided into sub-samples before being frozen at -18^oC until required for analysis.

Analysis

Samples were analysed for nitrate by CSL Food Science Laboratory, Norwich (CSL) using a method based on that of Dennis et al (1990)³² which was used to determine nitrate concentrations in samples from the 1994 UK Total Diet Study.²³

The analysis of nitrate in homogenised duplicate diet samples basically involved hot alkali extraction followed by clean-up by passing through a solid phase extraction (SPE) column prior to injection onto a Dionex AS11 anion exchange column (250 mm x 4 mm) with a guard column (50 mm x 4 mm) of the same packing material. Nitrate was separated by High Performance Liquid Chromatography (HPLC) with a mobile phase of 80:20 v/v water: 21 mM sodium hydroxide at a flow rate of 1 ml/minute. Detection was by UV at 214 nm.

Quality control

BMRB is ISO 9001 accredited and works to the ethical codes of the Market Research Society and ESOMAR (European Society). All BMRB field workers carry IQCS (Interviewer Quality Control Scheme) cards and operate to these standards.

CSL participate in and have achieved satisfactory z-scores in the nitrate rounds in FAPAS (Food Analysis Performance Assessment Scheme).

The quality control procedures used for nitrate analyses in this study were considered and agreed by the Working Party on Chemical Contaminants in Food’s Analytical Group.³³

To check inter-batch precision an in-house reference material (cabbage puree) was extracted and analysed with each batch of samples (i.e. 10 times in total). The mean nitrate concentration in this sample was found to be 72.9 mg/kg, RSD (relative standard deviation) = 12.1 per cent.

To check intra-batch precision a sample in each batch was analysed in duplicate. If the results for a duplicate analysis were different by more than 20 per cent, the batch was rejected.

Recovery was determined by spiking 10 portions of a duplicate diet sample with sodium nitrate solution before extraction and analysis. Unspiked portions of the same sample were extracted and analysed by the same procedure to allow blank correction. The mean recovery of the 10 spiked portions was 91.4 per cent with a range of 83.0 per cent to 107.2 per cent (RSD = 7.6 per cent). If the recovery for a batch was outside of the range 80 per cent to 120 per cent, the batch was rejected.

The limit of detection (LOD) was defined as 3 times the standard deviation of the reagent blank and was established as being 6 mg/kg wet weight.

Further information on the nitrate analyses of duplicate diet samples is given in CSL’s final report which will be available for public access in MAFF’s library once all the analyses are complete.

Under-reporting of foods consumed

The weights of many of the samples were lower than would be expected for the amounts of foods typically consumed by adults over a week to the extent that the energy content of some of the diets collected would have been insufficient to maintain body weight for an extended period. In other words, many of the duplicate diet samples did not reflect actual food consumption.

Duplicate diet studies are time consuming and intrusive and it is known that participants can change their diets for the duration of the study (e.g. eat more ready prepared foods than usual), fail to collect exact duplicate portions or under-report the amounts of foods eaten.³⁴ To determine which of the diets collected were representative of actual food consumption, the diary records completed by participants were independently assessed by the Nutrition Research Centre at South Bank University, London.³⁵ For each participant, mean daily energy intake (EI) was estimated using the nutrition computer programme (Comp-Eat 5, Nutrition Systems, London) and basal metabolic rate (BMR) estimated from standard equations using the information recorded on gender, age and weight.³⁶ To identify energy intakes too low to represent actual food consumption, an EI:BMR cut-off ratio of 1:1 was used. Thirty-five diets (16 summer and 19 winter) were identified as being representative of actual consumption using this cut-off. These 35 duplicate diet samples were used to calculate dietary exposures as described below.

Dietary exposures

Dietary exposures were calculated from the nitrate concentrations detected in individual duplicate diet samples, the weights of the samples and the weights of the participants. They are thus accurate records of the dietary exposures to nitrate of the 35 of the individuals who participated in the vegetarian duplicate diet study.

For those individual duplicate diet samples where nitrate concentrations were below the LOD of 6 mg/kg it has been assumed, for the purposes of calculating dietary exposures, that nitrate was present at a concentration equal to the LOD.

Results and Interpretation

Dietary exposures

The daily dietary exposures of participants in the study are summarised and compared with the ADI in Table 1. There was a wide variation in dietary exposures from food from the lowest exposure of 5.9 mg/day to the highest of 178 mg/day (Table 1). This variation reflects differences between the weights of participants and the amounts of food they ate, and nitrate concentrations in the samples. The mean dietary exposure of nitrate in summer was 56 mg/day and in winter 49 mg/day (Table 1).

To estimate total nitrate exposure, all sources of nitrate and nitrite in the diet should be taken into account wherever possible. Tap water and beer can make a significant contribution to total nitrate exposure. If it is assumed that the average consumption of water is 1 litre/day and the average nitrate concentration in tap water is within the range of 10 mg/l to 20 mg/l, then the additional contribution to the total nitrate exposure from this source will be 10 mg/day to 20 mg/day.^18,22 A MAFF survey of nitrate in beer carried out in 1988/89 found a mean nitrate concentration of 16 mg/kg.¹⁸ If it is assumed that the average consumption of beer is 0.7 l/day, the nitrate exposure from this source will be 11 mg/day.¹⁸

The total mean nitrate exposure of participants in the vegetarian duplicate diet study is 83 mg/day taking into account exposure from food (52 mg/day), water (20 mg/day) and beer (11 mg/day). The corresponding total exposure for the participant in the vegetarian duplicate diet study with the highest nitrate exposure is 209 mg/day (i.e. 178 mg/day from food, 20 mg/day from water and 11 mg/day from beer). Both these exposures are below the ADI for the nitrate ion which is equivalent to 219 mg/day for a 60 kg person and are similar to those estimated for mean (average) and upper range (97.5th percentile) UK consumers from the 1997 Total Diet Study which were 88 mg/day and 136 mg/day, respectively.¹⁹

Seasonal differences

Nitrate concentrations in vegetables are affected by season. Previous JFSSG surveys have shown that lettuce, spinach, potatoes, carrots, cauliflower and onions grown in the UK in the summer have lower nitrate concentrations than those produced in the winter months.^20,28,29 It would therefore be expected that dietary exposures of nitrate for consumers of these vegetables would be higher in the winter. However, there were no statistically significant differences between dietary exposures to nitrate in the summer and winter for participants in this study.

Actions

Results of the analysis of vegetarian duplicate diet samples for metals and other chemical elements (aluminium, arsenic, cadmium, chromium, cobalt, lead, mercury, nickel, selenium, tin and zinc) are reported in Food Surveillance Information Sheet No. 193.¹ Analyses for phytoestrogens (coumestrol, daidzein, daidzin, genistein, and genistin), furocoumarins, glycoalkaloids and total glucosinolates are currently being carried out. Once completed, the results of these analyses will be reported in the MAFF/Department of Health Food Safety Information Bulletin and an accompanying Food Surveillance Information Sheet.

A JFSSG survey of aluminium, arsenic, cadmium, chromium, cobalt, copper, lead, mercury, nickel, selenium, tin and zinc in vegetarian foods on sale in the UK was reported in the November 1998 edition of the Food Safety Information Bulletin and in Food Surveillance Information Sheet No. 166.^37,38

A JFSSG survey of nitrate concentrations in lettuce and spinach on sale in the UK were reported in Food Surveillance Information Sheet No. 177 in May 1999.³⁹

Conclusions

Dietary exposures to nitrate for vegetarians are very similar to those of other consumers and are below the ADI. There are no significant risks to health to consumers from nitrate in either the general UK diet or vegetarian diets.

References

Ministry of Agriculture, Fisheries and Food (2000). Duplicate Diet Study of Vegetarians - Dietary Exposures to 12 Metals and other Elements. Food Surveillance Information Sheet No. 193
Kelly, J. (1997). Mass market culture of vegetarianism. FT Food Business Sector Report. Issue 6 - July 1997, i-iv.
Ministry of Agriculture, Fisheries and Food (1994). The British Diet: Finding the Facts. Food Surveillance Paper No. 40. The Stationery Office, London.
Gregory, J., Foster, K., Tyler, H. and Wiseman, M. (1990). The Dietary and Nutritional Survey of British Adults. The Stationery Office, London.
Ministry of Agriculture, Fisheries and Food (1997). National Food Survey. The Stationery Office, London.
Ministry of Agriculture, Fisheries and Food (1996). Inherent Natural Toxicants in Food. Food Surveillance Paper No. 51. The Stationery Office, London.
Ministry of Agriculture, Fisheries and Food (1998). Lead, Arsenic and other Metals in Food. Food Surveillance Paper No. 52. The Stationery Office, London.
Ministry of Agriculture, Fisheries and Food (1998). Cadmium, Mercury and other Metals in Food. Food Surveillance Paper No. 53. The Stationery Office, London.
Commission of the European Communities Scientific Committee for Food (1995). Opinion on Nitrate and Nitrite, expressed on 22 September 1995. Annex 4 to Document III/5611/95.
The Preservatives in Food Regulations 1989 (S.I. 1989 No. 533). The Stationery Office, London.
Ministry of Agriculture, Fisheries and Food (1998). Survey of nitrite and nitrate in bacon and cured meat products. Food Surveillance Information Sheet No. 142.
Gangolli, S.D., van den Brandt, P.A., Feron, V.J., Janzowski, C., Koeman, J.H., Speijers, G.J.A., Spiegelhalder, B., Walker, R. and Wishnok, J.S. (1994). Assessment: nitrate, nitrite and N-nitrosocompounds. European Journal of Pharmacology, Environmental Toxicology and Pharmacology. Section 292, 1-38.
Sugimura, T., Fujimura, S. and Baba, T. (1970). Tumour production in the glandular stomach of the rat by N-methyl-N1-nitro-N-nitroguanidine. Cancer Research 30, 455-465.
Duncan, C., Dougall, H., Johnston, P., Green, S., Brogan, R., Leifert, C., Smith, L., Golden, M. and Benjamin, N. (1995). Chemical generation of nitric oxide in the mouth from the enterosalivary circulation of dietary nitrate. Nature Medicine 1, 546-551.
Dykhuizen, R.S., Frazer, R., Duncan, C., Smith, C.C., Golden, M., Benjamin, N. and Leifert, C. (1996). Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defence. Antimicrobial Agents and Chemotherapy 40, 1422-1425.
McKnight, G.M., Smith, L.M., Drummond, R.S., Duncan, C.W., Golden, M. and Benjamin, N. (1997). Chemical synthesis of nitric oxide in the stomach from dietary nitrate in humans. Gut 40, 211-214.
Commission of the European Communities Scientific Committee for Food (1992). Opinion on Nitrate and Nitrite, expressed on 19 October 1990 (26th series).
Ministry of Agriculture, Fisheries and Food (1992). Nitrate, Nitrite and N-Nitroso Compounds in Food: Second Report. Food Surveillance Paper No. 32. The Stationery Office, London.
Ministry of Agriculture, Fisheries and Food (1998). 1997 Total Diet Study - Nitrate and Nitrite. Food Surveillance Information Sheet No. 163.
Ministry of Agriculture, Fisheries and Food (1998). Nitrate in Vegetables. Food Surveillance Information Sheet No. 158.
Ministry of Agriculture, Fisheries and Food (1996). Nitrate in Vegetables. Food Surveillance Information Sheet No. 91.
Ministry of Agriculture, Fisheries and Food (1987). Nitrate, Nitrite and N-Nitroso Compounds in Food. Food Surveillance Paper No. 20. The Stationery Office, London.
Ministry of Agriculture, Fisheries and Food (1997). 1994 Total Diet Study - Nitrate and Nitrite. Food Surveillance Information Sheet No. 137.
European Commission (1997). Commission Regulation (EC) No. 194/97. Official Journal of the European Communities No. L31/48.
European Commission (1999). Commission Regulation (EC) No. 864/99. Official Journal of the European Communities No. L108/16.
The Contaminants in Food Regulations 1997 (S.I. [1997] No. 1499). The Stationery Office, London.
The Contaminants in Food (Amendment) Regulations 1999 (S.I. [1999] No. 1603). The Stationery Office, London
Ministry of Agriculture, Fisheries and Food (1997). 1996/97 UK Monitoring Programme for Nitrate in Lettuce and Spinach. Food Surveillance Information Sheet No. 121.
Ministry of Agriculture, Fisheries and Food (1998). 1997/98 UK Monitoring Programme for Nitrate in Lettuce and Spinach. Food Surveillance Information Sheet No. 154.
Ministry of Agriculture, Fisheries and Food/ Department of Health (1997). Food Safety Information Bulletin No. 88.
Ministry of Agriculture, Fisheries and Food/ Department of Health (1997). Food Safety Information Bulletin No. 82.
Dennis, M.J., Key, P.E., Papworth, T., Pointer, M. and Massey, R.C. (1990). The determination of nitrate and nitrite in cured meat by HPLC/UV. Food Additives and Contaminants 7(4), 455-461.
Ministry of Agriculture, Fisheries and Food (1998). Food Chemical Surveillance Annual Report 1997. MAFF Publications.
Macdiarmid, J. and Blindell, J. (1998). Assessing dietary intake: who, what and why of under-reporting. Nutrition Research Reviews 11, 321-253.
Davies, J., Lightowler, H. and Anderson, E. (1999). Estimation of energy and low energy reporters from a duplicate diet survey of vegetarians - a report for MAFF. South Bank University London, Nutrition Research Centre.
Schofield, W.N. (1985). Predicting basal metabolic rate, new standards and review of previous work. Human Nutrition: Clinical Nutrition 39c (Supplement 1), 5-41.
Ministry of Agriculture, Fisheries and Food/ Department of Health (1998). Food Safety Information Bulletin No. 102.
Ministry of Agriculture, Fisheries and Food (1998). Metals and Other Elements in Vegetarian Foods. Food Surveillance Information Sheet No. 166.
Ministry of Agriculture, Fisheries and Food (1999). Nitrate in Lettuce and Spinach. Food Surveillance Information Sheet No. 177.

Further information

Further information on the nitrate analyses of vegetarian duplicate diet study samples can be obtained from:

Dr Patrick Miller
MAFF, Joint Food Safety and Standards Group,
Food Contaminants Division
Room 238, Ergon House, c/o Nobel House
Smith Square
London SW1P 3JR.

Tel: +44 (0) 20 7 238 5751
Fax: +44 (0) 20 7 238 5331
E-mail: pf.miller@fsci.maff.gov.uk

Further copies of this and other Information Sheets can be obtained from:

MAFF, Joint Food Safety and Standards Group,
Publicity and Information Section
Room 303b, Ergon House, c/o Nobel House
Smith Square
London SW1P 3JR

Tel: +44 (0) 20 7 238 6223
Fax: +44 (0) 20 7 238 6330
Email: s.h.fssginfo@fssg.maff.gov.uk

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These pages were last updated on 30th December 1998