Food for thought: Protein indicators of appetite in saliva

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  • Published: Jun 1, 2012
  • Author: Steve Down
  • Channels: Proteomics & Genomics / Proteomics
thumbnail image: Food for thought: Protein indicators of appetite in saliva

How hungry are you?

A preliminary study on the response of the saliva proteome to food has identified two proteins which might be biomarkers for appetite, say researchers in the UK, with the potential to replace invasive blood tests and subjective measures.

We are all familiar with our own appetite and have no problem deciding when we want food or drink. But how can you do the same for someone else? The measurement of appetite is a difficult concept but has great importance in areas like obesity control and the claims of manufacturers that certain foods can control appetite and food intake.

One approach adopted by scientists in the UK was to search for indicators of appetite in saliva. Bernard Corfe and colleagues from the University of Sheffield and Cultech Limited, a manufacturer of food supplements based in Port Talbot, thought that a clinical approach would be more reproducible and might lead to specific compounds that would estimate the appetite.

Currently, some of the main methods for appetite measurement are highly subjective. Allowing subjects to eat freely until they are full, measuring the amount of food consumed, or keeping food diaries have all been used but they are poor measures. In addition, the food intake does not necessarily reflect the energy intake.

One different approach involved the measurement of gut hormones that are released into the bloodstream but this invasive procedure, which might itself affect the appetite, is seen as technically difficult to achieve. So, testing of saliva was thought to be a valid approach as it is produced by the oral cavity, is easy to collect, and has been proven to hold biomarkers of other diseases like diabetes.

Spit test

The researchers tested the saliva of 15 male volunteers in a randomised, controlled, crossover study. To begin with, the diurnal variations in the proteome were assessed for saliva collected after eating a standardised high fat diet, taking samples throughout the day from 8.55 to 17.00.

On the next day, saliva was taken at 8.45, 9.05 and 9.30, with food taken in between. At 8.50, the subjects consumed an emulsion consisting primarily of docosahexenoic acid (DHA) or oleic acid (OA) on the basis that long-chain fatty acids are known to be good at reducing appetite and affecting energy input. A control set took no emulsion. Then at 9.10, the subjects ate a low-fat breakfast comprising jam (jelly) sandwiches and orange juice.

A third study consisted of a four-way crossover experiment in which the subjects were given an emulsion of either one of the acids or a blend of oils contained in the Western diet and their saliva proteomes were compared with those eating nothing. This was combined with subjective appetite scoring and self-reported food intake.

From each set, the saliva samples were pooled and the proteins present were digested with trypsin and subjected to multiplex isotope labelling using the 8-plex iTRAQ reagents. The resulting labelled peptides were fractionated by hydrophilic interaction chromatography into 35 fractions which were analysed by HPLC-tandem mass spectrometry for protein identification and relative quantitation.

Proteins predict appetite, possibly

A diurnal pattern of the total salivary protein concentrations was observed consistently for the 15 subjects, although there were individual variations. This suggested that the sampling time should be taken into account for saliva studies, and led to the sampling regime that the researchers finally adopted.

The most abundant protein identified was amylase but the team decided not to remove this before protein identification. Nevertheless, a total of 118 proteins were identified including cystatin S and cystatin SA, which are involved in host defence mechanisms, and mucin 5B which is involved in lubrication.

When the saliva was sampled after taking DHA, reduced levels of thioredoxin and increased levels of serpin B4 were detected consistently for the different men compared with those taking OA or no food. These changes were confirmed by immunoblotting tests.

The third study, which incorporated appetite scoring, was designed to check for a relationship between protein concentrations and energy intake, using multiple regression analysis to assess the ability of the appetite scores and salivary protein concentration to predict energy intake. This revealed that DHA was associated with a lower energy intake than OA.

This preliminary work suggests that saliva might be the way forward for identifying proteins and/or peptides that are biomarkers of appetite, aided by the ease of sample collection. The two proteins thioredoxin and serpin B4 should be evaluated further to see whether they are genuine candidates.

Related Links

Journal of Proteomics 2012, 75, 2916-2923: "Evaluation of the salivary proteome as a surrogate tissue for systems biology approaches to understanding appetite"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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