N05011: Speciation, bioavailability, bio-potency, and functional markers for D vitamers

Study Duration: June 1998 to March 1999

Contractor: Laboratory of the Government Chemist

There is considerable interest in the dietary intake of vitamin D, its role in human health and the bioavailability and biological potency of vitamin D and its metabolites. Vitamin D status has been shown to be linked to a number of human diseases but its precise role and biological action is still not fully defined. Vitamin D deficiency is still of concern. Those most at risk are the elderly, young children, pregnant mothers and sectors of the Asian community.

The amount of vitamin D ingested varies widely (from <1 microgram to around 10 micrograms per day) depending upon the foods consumed, which are either naturally rich sources of vitamin D (eg oily fish & eggs) or have been fortified (eg margarine). Children older than four years and adults less than 65 years old in the UK, are generally considered to meet vitamin D requirements without specific need for dietary intake due to endogenous synthesis from exposure of the skin to strong sunlight. Despite this, vitamin D deficiency symptoms are increasing amongst vulnerable groups of the population.

There is a need to re-assess dietary intake values as the vitamin D content of certain foods (eg some meat and fish) has recently been shown to be higher than previously thought. This may be partly due to the presence of 25 hydroxy vitamin D3, which is thought to have a higher biological activity in humans (between 2 & 5 times that of vitamin D3). Although it has been known for many years that this is the main circulating form in humans, until recently it has not been possible to determine this metabolite in foods.

Further investigation is required to establish the bioavailability and biopotency of vitamin D in humans. Methods that will specifically determine vitamin D and its metabolites in food and sera are required.

Rationale and Objectives

The project consisted of three work packages as follows:

• 1. To obtain data on the cholecalciferol (vitamin D₃) and 25-hydroxy cholecalciferol (25-OHD₃) content of imported, freshwater fish consumed by the Bangladeshi community in Whitechapel, London, so that the effect of dietary vitamin D intake could be correlated to earlier clinical study data.
• 2. To obtain information on the vitamin D₃ and 25-OHD₃ content of additional selected foods to supplement national food database data.
• 3.To develop and validate a method for the determination of 25-OHD₃ in serum.

The data obtained supplements data contained in national food tables, informs the assessment of dietary vitamin D intake on an 'at-risk' group of the population and provides a means of measuring vitamin D status in humans. Such information can be used to assess the bioavailability of vitamin D in future human feeding trials.

1. Samples of imported freshwater fish were purchased from local retail outlets in the Whitechapel area of East London. Bulked samples of each fish type were analysed for vitamin D3 and 25 OHD3 content using an HPLC procedure. Data obtained was reviewed by Dr B. Boucher of the Royal London Hospital and correlated with clinical data obtained from earlier studies where the dietary vitamin D intake was not determined.

2. A selection of other fish, eggs, and dairy products were analysed for vitamin D3 and 25 OHD3 using an HPLC procedure. At least five retail samples of each food were purchased from retail outlets. Samples were prepared for eating according to normal practice and were bulked and homogenised before analysis. Composite samples were prepared fore each food type.

3. A method using gas chromatography with mass-spectrometric detection was developed and validated. The method can be used to determine 25 OHD3 in human sera at normal concentrations.

• The imported freshwater fish consumed by the Bangladeshi population of East London was found to contain vitamin D3 at concentrations between 0.7 and 16.4 micrograms per 100 grams of food. Cooking did not significantly affect the vitamin D3 concentrations found. In general, the oily fish contained the most vitamin D3. 25 OHD3 was not found in the fish analysed.
• The oily fish were shown to be a source of vitamin D3 for the target population. Reduction in vitamin D content or in frequency of fish consumption was shown to be a determinant in spot blood glucose testing and in subject glycaemia. Consumption of oily fish was shown to improve vitamin D status and reduce glycaemia but did not achieve repletion. Vitamin D supplementation of apparently healthy adults in this population is likely to remain necessary unless changes in dietary habits are implemented.
• Vitamin D3 was found in all of the other foods tested, with the exception of roasted chicken. Concentrations between 0.03 and 9.23 micrograms per 100 grams of food were found with the highest concentrations in the oily fish. Small but significant concentrations of 25 OHD3 were found only in pilchards and mackerel.
• A gas chromatography-mass spectrometric(GC-MS) method for the determination of 25 OHD3 in human serum was developed and validated. The method has a limit of detection of 4.7 nanograms of 25OHD3 per millilitre of blood/serum. Recoveries from spiked samples ranged between 108 and 133%. Response was linear between 5 and 200 nanograms per millilitres. d7-cholesterol was used as an internal standard.

The most suitable internal standard would be a deuterated 25 OHD compound and deuterated vitamin D is also required for future human feeding trials. Attempts to source and to synthesise compounds of this type were largely unsuccessful and until such compounds become available, the study of vitamin D bioavailability will remain an analytical challenge.

What it means and why it's important

To determine the contribution of dietary intake on vitamin D status, it is necessary to ensure that all forms of vitamin D consumed are accurately measured and to have information about the bioavailability of the different forms. Such data can be used to supplement and update the national food database to ensure that accurate information is available to consumers and interested parties.

The role of vitamin D in humans is still not fully understood and further investigation is required. Deficiency diseases and symptoms are still of concern especially in vulnerable groups of the UK population. Vitamin D metabolites play a significant role in a number of biochemical processes, such as the regulation of serum calcium and phosphorus concentrations and are also required for insulin secretion. Deficiency can therefore be a factor in diseases of the bones (rickets, osteoporosis etc) and also in diseases related to blood glucose concentration, such as diabetes.

Certain groups of the population such as infants, pregnant women and the elderly are more at risk of deficiency. Some Asian sectors of the community are also at risk because they have low exposure to sunlight, limited dietary intake of vitamin D and may be genetically disposed in some instances to diseases such as diabetes. The data obtained enabled an assessment of the effect of dietary vitamin D intake on the vitamin D status and upon disease factors within an at-risk group of the population. This information will inform debate in this area and provides information to allow this group and other interested parties to make appropriate choices with regard to diet, supplementation and health.

To study bioavailability and bio-potency issues, it is necessary to be able to determine individual vitamin D metabolites. Vitamin D status is normally determined using the serum 25 OHD3 concentration, as the clearance rate of this metabolite is low. Methods to determine this metabolite in serum are required. Such methods should also be capable of measuring vitamin D3 and other metabolites if required. Gas chromatography with mass-spectrometric detection allows positive identification of natural concentrations of 25 OHD3 and other metabolites and suitable equipment for this technique is now generally available. The method developed will allow appropriate determinations to be carried out in support of future bioavailability studies.

Final report is available from the FSA Library and Information centre. To obtain a copy, please contact the Enquiry Desk, Dr. Elsie Widdowson Library and Information Services, Food Standards Agency (020 7276 8181/8182 or at library&info@foodstandards.gsi.gov.uk). Split into two reports for library purposes:

Part 1: The determination of vitamin D₃ and 25-Hydroxy vitamin D₃ in fish and other foods and the effect of dietary intake on an 'at-risk' population.

Part 2: Development of a method for the analysis of deuterated and non-deuterated vitamin D₃ and 25 hydroxy vitamin D₃ in serum.

Contact: Dr Alison Tedstone
Tel: 020 7276 8929
Email: alison.tedstone@foodstandards.gsi.gov.uk