Human Health


Multiplexed Detection of Nutritional Deficiency

In working with at-risk populations there are manifold dimensions in which health is suboptimal. Merely focusing upon one area of human health does not necessarily reduce the complexities involved. For example there are many ways in which individuals may suffer from malnutrition. Individuals may suffer from a combination of nutritional deficiencies such as protein, vitamin, and iron deficiencies. Simultaneous detection of multiple markers, or multiplex analysis, has been used to tremendous success with cytokines and other serum proteins and is greatly needed in detection of nutritional deficiencies. PATH, headquartered in Seattle, WA has spearheaded an effort to simultaneously measure multiple nutritional deficiencies from a single sample. The chosen markers were retinol binding protein to determine vitamin A status, ferritin and soluble transferrin to measure iron status, and C-reactive protein (CRP) to determine inflammation.

Vitamin A is required for the development and function of the cardiovascular system, immune system, respiratory system, and for visual function[1]. In the developing world 250 million preschool aged children are estimated to have vitamin A deficiencies with 3 million exhibiting clinical ocular signs of deficiency with 250,000-500,000 needlessly blinded[2]. While many are aware that vitamin A deficiency is the leading cause of preventable childhood blindness, vitamin A deficiency also contributes to mortality from diarrhea, measles, and other infections[2]. While the most commonly used method measuring serum vitamin A status utilizes an expensive and complicated HPLC method, another less expensive surrogate measurement of serum vitamin A status is the measurement of serum levels of retinol binding protein. Retinol binding protein has better stability, is less temperature sensitive, and is measureable by simpler and less expensive immunologic methods[3].

Iron deficiency is thought to affect somewhere between 15-66% of the world’s population [4]. In perinatal development, iron deficiency affects the function of organ systems such as heart, muscle, gastrointestinal tract and especially the brain where long term abnormalities are not reversed by iron supplementation[5]. The best parameters for the measurement of iron status are ferritin and soluble transferrin receptor and from both, body iron stores can be calculated[6].

In examining levels of these proteins it is important to know the inflammation status of the patient since retinol binding protein is a negative acute phase response protein, or levels of the protein come down with inflammation [3] and ferritin levels increase during acute and chronic phases of inflammation. The assessment of C-reactive protein (CRP) levels has been used to determine infection/inflammation status so as to avoid improper determination of nutritional status[7].

PATH contacted Quansys biosciences in Logan Utah to help develop a multiplex array for vitamin A and iron deficiencies. The Quansys array is based on a simple platform that utilizes antibodies placed onto the bottom of 96 or 384 well plates in small spots. Each spot is composed of capture or primary antibodies to particular proteins and a spot is distinguished from other spots by its location on a Cartesian coordinate system. The Quansys system utilized by the PATH array has a horseradish peroxidase chemiluminescent reporter mechanism. Rather than a luminometer that is used with singleplex chemiluminescent ELISAs, the Quansys multplex system utilizes cooled CCD or CMOS cameras with background subtraction features to detect the luminance originating with the individual spots. High resolution cameras are effective at distinguishing the multiple signals that originate from the multiple spots within the wells rather than detecting the average luminance from a single well. Software is then used to analyze the luminescent response of each spot which is proportional to the concentration of the particular protein being measured by the particular spot. The values of the spots of the unknown samples are then compared to the values of standard curves to quantify the unknown. This is simultaneously performed for each protein in the array. Each protein in the array is examined for cross reactivity and functionality alone as well as arrayed in a group.

The wide variation of serum levels of the differing proteins presented challenges to Quansys in the development of the array. For example CRP, retinol binding protein, and soluble transferrin receptor levels needed to be measured in the µg/ml range while ferritin required measurement in ng/ml. A sandwich ELISA is very sensitive and is often used in the detection of serum proteins that are normally present in low levels such as inflammatory cytokines. While sandwich ELISAs can be used for detection of proteins in higher concentrations the individual ELISA must be built around the high protein concentration, or alternatively multiple dilutions must be used to assure that the protein levels in the samples fall within the range of the standard curve. This difficulty was surmounted by combining competitive ELISAs (for CRP, retinol binding protein, and soluble transferrin receptor) and traditional sandwich ELISA for ferritin. The result is an array that allows for the simultaneous quantification of retinol binding protein, ferritin, soluble transferrin receptor, and CRP enabling the determination of vitamin A and iron deficiency as well as infection/inflammation from a single sample. In initial validation the array detected endogenous levels of each marker in multiple serum/plasma samples with coefficients of variation less than 15% (Table 1). The PATH array requires only 5 μl of each sample per test (5 μl sample in 45 μl buffer/well) greatly reducing the amount of sample needed and it is currently undergoing laboratory validation as a tool to assess dietary status in large population based studies.

  1. Mactier, H. and L.T. Weaver, Vitamin A and preterm infants: what we know, what we don’t know, and what we need to know. Arch Dis Child Fetal Neonatal Ed, 2005. 90(2): p. F103-8.
  2. Underwood, B.A. and P. Arthur, The contribution of vitamin A to public health. Faseb J, 1996. 10(9): p. 1040-8.
  3. Gamble, M.V., et al., Retinol binding protein as a surrogate measure for serum retinol: studies in vitamin A-deficient children from the Republic of the Marshall Islands. Am J Clin Nutr, 2001. 73(3): p. 594-601.
  4. Cook, J.D., C.H. Flowers, and B.S. Skikne, The quantitative assessment of body iron. Blood, 2003. 101(9): p. 3359-64.
  5. Rao, R. and M.K. Georgieff, Iron in fetal and neonatal nutrition. Semin Fetal Neonatal Med, 2007. 12(1): p. 54-63.
  6. Erhardt, J.G., et al., Combined measurement of ferritin, soluble transferrin receptor, retinol binding protein, and C-reactive protein by an inexpensive, sensitive, and simple sandwich enzyme-linked immunosorbent assay technique. J Nutr, 2004. 134(11): p. 3127-32.
  7. Tomkins, A., Assessing micronutrient status in the presence of inflammation. J Nutr, 2003. 133(5 Suppl 2): p. 1649S-1655S.

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