The Journal of Nutrition Vol. 128 No. 4 April 1998, pp. 783-783

LETTER TO THE EDITOR:

Response to the Letter to the Editor from Rosales

	LETTER

Dear Dr. Visek:

Dr. Rosales (1997) draws attention to an important point concerning the molar ratio of retinol to retinol binding protein (RBP) in our recent paper (Willumsen et al. 1997). When our RBP results are recalculated as µmol/L, they give rise to retinol/RBP ratios (e.g., 1.72 for control children) that are not only unlikely for healthy children but also impossible according to established concepts of plasma retinol transport. This has caused us some concern, and we have investigated the matter further.

The high retinol/RBP ratios could result from laboratory errors in either of the biochemical measures or, as suggested by Dr. Rosales, from true low RBP values in this population. The quality control serum for our retinol HPLC assay was standardised against the standard used by Great Ormond Street Hospital, London, which belongs to the St. Helier Hospital external quality assurance scheme for fat-soluble vitamins. Because of this reliable standardization and our fairly normal retinol results from the South African children in our study, we believe the problem does not lie with retinol measurement.

We experienced greater difficulty with our RBP ELISA assay because of the unavailability of an appropriate standard. Our preference for an ELISA, rather than the more usual radial immunodiffusion (RID) assay was based on cost, precision and ease of reading assay results for the large number (200 expected) of serum samples. In addition, we needed the more sensitive ELISA for our urine RBP measurement. We experimented with several commercial standards and found poor agreement among calibration values. Values (mg/L) measured in our assay, using Behring OWXI 13N as the standard, for various other standard sera at calculated concentrations of 25 mg/L are shown below.

The Behring OWXI 13 nephelometry standard gave the most reasonable molar retinol/RBP ratios for three laboratory quality control sera from normal adults: 1.43, 1.05 and 0.66 (mean = 1.05, n = 4 duplicates for each). RBP values for these same three sera, measured against standard curves of the other commercial standards, gave surprisingly low ratios; for example, the next closest values to 1:1, using the Behring OWXK 13 standard, were 0.68, 0.44 and 0.91; mean = 0.67. We wondered whether the discrepancy resulted from differences between serum and urine RBP because the antibodies we used (Dako, High Wycombe, UK) had been raised in rabbits against purified urinary RBP and the purified urine standard gave the most discrepant values.

We note that other laboratories seem to have experienced problems with standardising RBP assays. For example, de Pee et al. (1997) noted the mean retinol/RBP ratio for a group of normal Indonesian women was 0.53, for which they had no particular explanation. Ratios of greater than 1:1 have also been obtained in healthy American infants (Neuzil et al. 1994) and Australian preschoolers (Karr et al. 1997).

Possibly we should have selected a standard that produced low retinol/RBP ratios instead of selecting one giving ratios closest to 1:1. The assay and standard we used, although reproducible (interassay CV = 11.3%), may have given values lower than the true values. We apologize for any confusion this may have caused. In addition, RBP values for the South African children in the study were slightly lower than might have been expected. We have no explanation for this but note that the normal serum RBP range for children of this age is not well defined (Karr et al. 1997). We believe our observations about the relative time course and magnitude of changes in retinol and RBP after an inflammatory stimulus remain valid for two reasons. First, odd RBP values seemed to result largely from technical problems. Second, although we did not present the data as such in our paper because results for cases at the 3-mo time point after recovery were so similar to controls it would have been redundant, we compared case children with themselves over time, in addition to with controls.

Given the increasing interest in vitamin A nutrition and measurement of RBP, it seems important that closer attention is paid to standardization of RBP assays, analogous to the widespread use of an international standard for retinol.

Suzanne Filteau
Juana Willumsen
Centre for International Child Health
Institute of Child Health
30 Guilford Street,
London WC1N 1EH, UK

	FOOTNOTES

Manuscript received 12 September 1997. Revision accepted 10 November 1997.

	LITERATURE CITED

• de Pee S., Yuniar Y., West C. E., Muhilal Evaluation of biochemical indicators of vitamin A status in breast-feeding and non-breast-feeding Indonesian women. Am. J. Clin. Nutr. 1997; 66:160-167 [Abstract/Free Full Text]
• Karr M., Kira M., Causer J., Earl J., Alperstein G., Wood F., Fett M. J., Coakley J. Plasma and serum micronutrient concentrations in preschool children. Acta Paediatr. 1997; 86:677-682 [Medline]
• Neuzil K. M., Gruber W. C., Chytil F., Stahlman M. T., Engelhardt B., Graham B. S. Serum vitamin A levels in respiratory syncytial virus infection. J. Pediatr. 1994; 124:433-436 [Medline]
• Rosales, F. J. (1998) Comments on the paper by Willumsen et al. J. Nutr.128: 782.
• Willumsen J. F., Simmank K., Filteau S. M., Wagstaff L. A., Tomkins A. M. Toxic damage to the respiratory epithelium induces acute phase changes in vitamin A metabolism without depleting retinol stores of South African children. J. Nutr. 1997; 127:1339-1343 [Abstract/Free Full Text]

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