Identifying micronutrient deficiencies with proteomics

“Micronutrient deficiencies are common in undernourished societies, yet they remain inadequately assessed due to the complexity and costs of existing assays. A new approach holds promise for identifying micronutrient status by identifying and quantifying not the nutrients themselves but indicators (biomarkers) linked to the nutrients’ metabolism and function. The large-scale experimental analysis of the entire complement of proteins produced by an organism under certain conditions (proteomics) may help to identify specific plasma protein biomarkers reflecting the supply situation with defined micronutrients. There is increasing evidence of a strong correlation between plasma concentrations of micronutrients and their proteomics-derived, cognate proteins. Quantitative pro-teomics, in which hundreds of plasma proteins can be identified and quantified in relative abundance in a single mass spectrometry experiment, may offer a basis for discovering proteins and protein clusters that reflect nutrient functions and predict micronutrient status. Using proteomics to estimate micronutrient deficiencies would rely on identifying plasma protein biomarkers that are sufficiently linked via binding or less directly linked through complex metabolic networks with nutrient distributions in populations.

To validate this concept, we measured concentrations of vitamin A (retinol), vitamin D (25-hydroxyvitamin D), vitamin E (alpha-tocopherol), copper and selenium by conventional assays in plasma samples of a cohort of 500 six- to eight-year-old Nepalese children, and estimated correlations to the relative abundance of their major plasma-bound proteins, measured by quantitative proteomics using protein purification and mass spectrometry. The prevalence of low-to-deficient status of the children was 8.8% (below 0.70 mmol/L) for retinol, 19.2% (below 50 nmol/L) for 25-hydroxyvitamin D, 17.6% (below 9.3 mmol/L) for alpha-tocopherol, 0% (below 10 mmol/L) for copper, and 13.6% (below 0.6 mmol/L) for selenium. We identified 4,705 proteins, 982 of them in more than 50 children. We observed the following correlations:

• With respect to vitamin A, we identified a strong correlation with certain amounts of RBP4, its cognate plasma protein (1). On release from hepatic stores, retinol circulates in an equimolar complex with RBP4 and a larger protein, which delivers vitamin A to peripheral tissues for cellular uptake.

• A comparable weak correlation was found for plasma 25-hydroxyvitamin D and the vitamin D-binding protein (VDBP), maybe because VDBP circulates in concentrations 100-fold more than those of 25-hydro-xyvitamin D, binds to other vitamin D metabolites and has many non-vitamin D-related functions (2). Our findings demonstrate a need to find other vitamin D-networked proteins to increase explained variance and strengthen the potential to predict vitamin D status.

• Vitamin E has no specific plasma carrier protein. Rather, following absorption, different forms of vitamin E are released into circulation associated with chylomicrons (lipoprotein particles), redistributed to other plasma lipoproteins and tissues, and delivered to the liver (3). Hepatic alpha-tocopherol reenters circulation initially associated with very-low-density lipoprotein (VLDL) prior to being redistributed to other low- to intermediate-density lipoproteins for transport to the periphery. Strong correlations were found between plasma alpha-tocopherol and apolipoproteins, especially with apo C-III, which is a principal component of VLDL.

• Although copper – a trace element ubiquitously involved in gene transcription, cellular respiration and enzyme activation – binds to numerous intracellular and extracellular proteins, up to 95% of its plasma content is bound to ceruloplasmin (Cp), a ferroxidase that regulates iron metabolism and homeostasis (4). A strong association was expected and found between plasma copper concentration and relative abundance of Cp.

• SEPP1, a glycoprotein expressed and secreted largely from the liver, comprises the major circulatory pro-tein that delivers selenium to tissues throughout the body (5). In our study, this strong association was confirmed.

These findings from a large population sample of Nepalese children suggest that quantitative plasma pro-teomics may provide a new basis for identifying functional biomarkers that will eventually improve our ability to assess micronutrient status and deficiencies in populations. We expect that micronutrients lacking bound plasma proteins may have less recognizable, but nonetheless valid, correlated protein partners, which we are currently exploring.”

Based on: Cole R. N. et al. The Plasma Proteome Identifies Expected and Novel Proteins Correlated with Micronutrient Status in Undernourished Nepalese Children. The Journal of Nutrition. Published online August 2013.

1. Quadro L. et al. Understanding the physiological role of retinol-binding protein in vitamin A metabolism using transgenic and knockout mouse models. Mol Aspects Med. 2003; 24:421–430.

2. Speeckaert M. et al. Biological and clinical aspects of the vitamin D binding protein (Gc-globulin) and its polymorphism. Clin Chim Acta. 2006; 372:33–42.

3. Morrissey P. A. and Kiely M. Vitamin E / physiology and health effects. In: Caballero B, Allen L, Prentice A, eds. Encyclopedia of human nutrition. 2nd ed. Amsterdam: Elsevier Academic Press; 2005. p. 389–398.

4. Hellman N. E. and Gitlin J. D. Ceruloplasmin metabolism and function. Annu Rev Nutr. 2002; 22:439–458.

5. Burk R. F. and Hill K. E. Selenoprotein P-expression, functions, and roles in mammals. Biochim Biophys Acta. 2009; 1790:1441–1447.