Determination of Vitamin A Total Body Stores in Children from Dried Serum Spots: Application in a Low- and Middle-Income Country Community Setting

Anthony Oxley, Reina Engle-Stone, Jody C Miller, M F Dolly Reario, Ame Stormer, Mario V Capanzana, Carl V D Cabanilla, Marjorie J Haskell, Georg Lietz, Anthony Oxley, Reina Engle-Stone, Jody C Miller, M F Dolly Reario, Ame Stormer, Mario V Capanzana, Carl V D Cabanilla, Marjorie J Haskell, Georg Lietz

Abstract

Background: The retinol isotope dilution (RID) method has been used to evaluate vitamin A (VA) status in healthy adults and children in low- and middle-income countries (LMIC) and to assess the efficacy of various VA interventions.

Objective: The study was designed to examine whether dried serum spots (DSS) can be applied to RID when conducting VA total body store (TBS) assessments in community settings.

Methods: Four days after an oral dose of 0.4 mg [13C10]retinyl acetate was administered to Filipino children (12-18 mo), a single blood draw was divided to isolate both serum and plasma. Serum (40 μL) was spotted and dried on Whatman 903 cards and shipped at ambient temperature whereas liquid plasma (LP) was frozen at -80°C and shipped on dry ice. The VA tracer to tracee ratio from DSS and LP was quantified by LC-MS/MS. Comparisons between DSS and LP paired samples (n = 72) were made for [13C10]retinol specific activity (SAp) by Pearson's correlation and for VA TBS by Bland-Altman analysis.

Results: The sum of 3 coextracted DSS were required to consistently detect [13C10]retinol above the LC-MS/MS limit of quantitation (LOQ). [13C10]retinol SAp from DSS was highly correlated with SAp from LP (r = 0.945; P < 0.01). A comparison of methods for TBS determination using Bland-Altman analysis indicated agreement with an intraindividual difference of 24.7 μmol (4.6%). Mean total liver reserve (TLR) values from DSS and LP were 1.7 μmol/g (± 0.6 SD) and 1.6 μmol/g (± 0.6 SD), respectively.

Conclusions: VA TBS can be determined from DSS thereby reducing the logistics and cost of maintaining a cold chain by shipping samples at ambient temperature and, thus, making the RID technique more feasible in LMIC community settings. This trial was registered at https://ichgcp.net/clinical-trials-registry/NCT03030339" title="See in ClinicalTrials.gov">NCT03030339.

Keywords: Philippines; children; dried blood spots; dried serum spots; liquid chromatography tandem mass spectrometry; retinol isotope dilution; stable isotopes; total body stores; vitamin A status.

© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.

Figures

FIGURE 1
FIGURE 1
LC-MS/MS chromatogram of a typical dried serum spot (DSS) extract sampled 4 d after oral ingestion of 0.4 mg [13C10]retinyl acetate. Selected reaction monitoring (SRM) of m/z 269→93 (A) shows the separation of endogenous [12C]retinol (5.05 min) and the [12C]retinyl acetate internal standard (5.82 min). The [13C10]retinol peak at m/z 279→100 (B), from ingested [13C10]retinyl acetate, sufficiently exceeds both the limit of detection (LOD) and the limit of quantitation (LOQ). Unconfirmed peaks within the m/z 269→93 channel were apparent at 4.81 min and 7.50 min. Ion signal intensity is defined as counts per second (cps).
FIGURE 2
FIGURE 2
Linear correlation between the specific activity (SAp) of [13C10]retinol from dried serum spots (DSS) and liquid plasma (LP) paired samples (n = 72) at 4 d postdose. Pearson's correlation coefficient (r) with significance level (P) and equation for the linear relation are displayed.
FIGURE 3
FIGURE 3
Bland–Altman comparison of total body store (TBS) determinations from dried serum spots (DSS) and liquid plasma (LP) paired samples (n = 72) at 4 d postdose. Solid line shows the mean difference with dashed lines displaying 95% limits of agreement (± 1.96 SD).

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