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Letters |
1 Institute of Biological Chemistry and Nutrition, University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart, Germany
2 Division of Nutrition, Institute of Food, Nutrition and Family Sciences, University of Zimbabwe, PO Box MP 167, Mount Pleasant, Harare, Zimbabwe
3 Craft Technologies 4344 Frank Price Church Rd., Wilson, NC 27893
aAuthor for correspondence.
To the Editor:
Vitamin A deficiency (VAD) is one of the most devastating dietary deficiencies worldwide. It causes significant increases in childhood and maternal morbidity and mortality among the poor of the nonindustrialized world. Several methods are available to assess VAD. The determination of retinol in blood is one of the most frequently used methods, but it has several disadvantages. Serum retinol is decreased only in severe VAD when liver stores are nearly exhausted. In addition, infection depresses the retinol concentration in blood, possibly leading to misclassification of individuals.
Because the majority of vitamin A in the body is stored in the liver, tests have been developed to measure vitamin A stores that tend to provide more reliable information about vitamin A status. The two most common tests of this nature are the relative dose response test (RDR) and the modified RDR test (MRDR). Of these, the most practical method for field collection is the MRDR. This test has the advantage of requiring only one blood sample (1). An equivalent of 5.3 µmol of didehydroretinol is given in the morning and a blood sample is taken 5 h later. Retinol and didehydroretinol concentrations in the blood are measured by HPLC, and the ratio of didehydroretinol to retinol is calculated. A percentage >6% is indicative of VAD.
The MRDR is an excellent test, but it has been underutilized, in part because of the poor chromatographic separation of didehydroretinol from retinol and the arduous sample preparation. The objectives of this study were therefore to (a) improve the chromatographic separation of retinol from didehydroretinol and (b) speed up the sample preparation process to allow higher sample throughput. The new procedure has been tested in a large HIV/vitamin A study in Zimbabwe using heel-prick samples in infants 6–12 months of age.
A 100-µL serum sample was denatured by mixing with 100 µL of ethanol containing butylated hydroxytoluene (5 g/L) and retinol acetate (0.5 µmol/L) as an internal standard. Hexane (400 µL) was then added, and the tubes were capped. The entire rack of tubes was vigorously mixed by hand for 1 min, using a vertical shaking motion. After centrifugation, 300 µL of the supernatant was evaporated to dryness, and the residue was dissolved in 100 µL of mobile phase (acetonitrile–water, 85:15 by volume). We injected 80 µL of this solution into the HPLC. The analytes were separated on a Thermo Hypersil Prism RP column [150 x 3 mm (i.d.); 3 µm bead size] at a flow rate of 1.5 mL/min, with detection at 350 nm. For economic reasons, the mobile phase was recycled for 
300 injections.
Typical serum chromatograms obtained with the new and traditional methods are shown in Fig. 1
. In the traditional method, the separation is performed with a Waters Resolve C18 column [150 x 4.6 mm (i.d.)] and methanol–water (90:10 by volume) as mobile phase. When we switched to the Thermo Hypersil Prism RP column and acetonitrile–water as mobile phase, the peaks were sharper and well separated, and the run time was shortened. As a result, the CV and sensitivity of this measurement are improved. The day-to-day CV was 3.7% (n = 5 days) for a sample with high didehydroretinol 0.056 µmol/L and 5.9% (n = 5 days) for a sample with low didehydroretinol (0.03 µmol/L). Our previous CV values and literature values were typically 10% (2).
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The new method has several advantages. Because only 100 µL of serum is used in this method, it is applicable to small samples, such as those from finger or heel pricks. Until now, all published methods specified at least 250 µL of serum. The detection limit for didehydroretinol in relation to retinol for a typical plasma sample with 0.7 µmol/L retinol is 1%, which is far below the cutoff value of 6%.
For large studies, the traditional method is time-consuming and delays information dissemination and intervention. To increase the throughput in these studies, some improvements have been made. The proposed protocol requires only one hexane extraction, rather than the three hexane extractions used in the traditional method. This reduces the workload substantially and has only a minor influence on the sensitivity of the measurement. The ratio between didehydroretinol and retinol is the critical value in the MRDR; we examined the residue after the first extraction and found no difference in the ratio. The second improvement is the use of one-piece disposable plastic autosampler vials with a conical sample compartment (Merck Eurolab). Because these hold up to 300 µL of solvent and have a wide opening on the top, hexane can be evaporated directly from these vials, saving one transfer step. With these modifications and a simple inexpensive mechanical evaporator unit with 68 lines (Evap-O-Rac System; Cole Parmer), it is possible to prepare 68 samples in one batch in 4 h. Consequently, throughput can be increased from 20 samples a day to >60.
In conclusion, the improved MRDR-HPLC method is more reliable and faster. With these modifications, the MRDR may find wider application for the detection of VAD.
References
The following articles in journals at HighWire Press have cited this article:
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  R. Ribel-Madsen, M. Friedrichsen, A. Vaag, and P. Poulsen Retinol-Binding Protein 4 in Twins: Regulatory Mechanisms and Impact of Circulating and Tissue Expression Levels on Insulin Secretion and Action Diabetes, January 1, 2009; 58(1): 54 - 60. [Abstract] [Full Text] [PDF]
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 A. R. Valentine and S. A. Tanumihardjo Adjustments to the Modified Relative Dose Response (MRDR) Test for Assessment of Vitamin A Status Minimize the Blood Volume Used in Piglets J. Nutr., May 1, 2004; 134(5): 1186 - 1192. [Abstract] [Full Text] [PDF]
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