Keratomalacia

Two German siblings were found to suffer from night blindness and mild retinal dystrophy but no other clinical symptoms of vitamin A deficiency. Even though they had no detectable plasma retinal-binding protein (RBP) and their plasma retinol was exceedingly low, they showed normal physiologic functions and growth. Their RBP gene was found to harbor two point mutations. Their postprandial plasma levels of retinyl esters were normal, and it is likely that they derived their tissue retinol from retinyl esters.

One could argue that the recognition of the hormonal role of vitamin A in the genomic regulation of differentiation and homeostasis (the "Retinoid Revolution"1) really began with the discovery of retinol-binding protein (RBP), the protein that transports retinol from the liver to the vitamin A target tissues.2 RBP is synthesized in the liver on instruction from a single gene. Retinyl esters stored in the liver are hydrolyzed to retinol; the latter combines with RBP in a 1:1 molar ratio, and the complex is then secreted into the bloodstream. The retinol-RBP complex combines with the plasma protein transthyretin (TTR) to be carried to the tissues, where the retinol enters the cells. There it is oxidized to its functional form, retinoic acid.

RBP secretion from the liver depends on the availability of retinol: under conditions of vitamin A deficiency, RBP continues to be synthesized. However, it is not secreted and accumulates in the liver.3 Protein-calorie malnourished children secrete less retinol-RBP into their circulation than well-sourished children, presumably because a lack of the necessary amino acids results in a lowered rate of RBP synthesis.4 The constant synthesis of RBP and its release as retinol-RBP maintains the homeostasis of the blood level of retinol at 1.5-3.0 (mu)mol/L (in the human adult), so long as stored retinyl esters are available.

In vitamin A deficiency, plasma retinol-RBP declines to very low levels. Clinical symptoms appear when the level reaches 0.35 (mu)mol/L.5 Night blindness is an early symptom in humans, followed by defects in the cornea (e.g., keratomalacia), and ending in blindness.

Reports of plasma RBP depletion have appeared in cases of familial amyloidotic neuropathy caused by a genetic defect in TTR.6,7 Waits et al.8 described an inherited mutation in TTR that lowers the production of TTR and is accompanied by low RBP levels. Much like the phenotype of the artificial TTR mutation in TTR-knockout mice,9 this phenotype presents no symptoms.

A recent report by Biesalski et al.10 describes a case of a mutation in the RBP gene in humans. Two sisters (in Germany), ages 14 and 17, were found to suffer from night blindness. Eye examination11 showed no signs of xerophthalmia and normal levels of retinol in the tear fluid. Dark adaptation thresholds were raised and visual acuity was reduced with atrophy ofthe posterior segments of the pigment epithelium. No other abnormalities were detected. Growth and physiologic functions were all normal. Vitamin A intake, revealed by a retrospective nutrition survey, was normal. Blood analyses showed their RBP levels to be below the limits of detection (

A noninvasive liver storage test (relative dose response12) revealed no depletion of vitamin A storage. A fat-absorption test, which included 31.5 (mu)mol retinyl palmitate, demonstrated that absorption of fat and vitamin A were not affected: Postprandial retinyl esters increased by factors of 11.8 in one sibling and 3.2 in the other; fat absorption as well as plasma levels of beta-carotene, a-tocopherol, and zinc were normal.

DNA was isolated from the siblings' whole blood. Oligonucleotide primers were designed for the TTR gene exon 3 and the RBP genes exon 3 and exon 4 and amplified by the polymerase chain reaction (PCR). The DNAs were sequenced automatically. Because a recent report8 showed that a human missense mutation in exon 3 in the TTR gene caused lowered plasma RBP, their TTR gene was analyzed; however, no mutation was found.

On the other hand, their RBP gene showed two point mutations: exon 3, T->A; exon 4, G->A, resulting in Ile for Ar at position 41 and Gly for Asp at position 74, present on different alleles. The girls' mother, who had a low normal retinol level and whose plasma RBP was 50% of the average normal concentration, had the mutation only on exon 3. Their father's RBP gene was not available but must have contained the exon 4 mutation. Clearly the girls' virtual lack of RBP was caused by each harboring both mutations on different alleles.

The fact that the mother, who was healthy in spite of the mutation on exon 3 of the RBP gene and was therefore synthesizing RBP with only one allele, had 50% of the normal level of plasma RBP leads to the conclusion that the rate of secretion of RBP from the liver is controlled by the rate of synthesis of RBP. This conclusion agrees with the early finding4 that lack of adequate dietary protein results in a decline in plasma RBP, presumably owing to a decline in the rate of RBP synthesis.

The question then arises: How did the girls' tissues obtain their retinol since (apart from the eyes) none of the tissues or their functions were in any way impaired? Most likely retinyl esters, which were present in plasma and occurred at normal levels postprandially, served the function of supplying retinol to target tissues. A number of reports support the view that retinyl esters circulating in chylomicrons can be taken up and stored extrahepatically.13,14 Gerlach et al.15 showed that an unphysiologic retinyl ester (retinyl margarinate), when injected into rats, was taken up directly by tissues, bypassing the liver, and was hydrolyzed to retinol and re-esterified to retinyl palmitate to some degree in extrahepatic tissues.

How can the human body adapt so readily to the absence of RBP? The answer may have an evolutionary basis. Shidoji and Muto16 found that most vertebrates had RBP in their blood with structures similar to human RBP (molecular weight 21 kD). Fish carried retinol bound to a protein (molecular weight 16 kD), which was unable to combine with TTR. The earliest vertebrate on the evolutionary scale, the boneless lamprey, however, supplied its tissues with retinyl esters bound to lipoprotein, presumably like the two sisters did. Possibly in case of a gene defect, an organism, to substitute for the lost process, can fall back on an earlier, less efficient process that has been genetically preserved from an earlier evolutionary period. Thus, the German girls have sacrificed their night vision and visual acuity but have otherwise lived by resurrecting the genetically preserved process first used by the lamprey. Their vitamin A transport system could be regarded as a "fossil." Perhaps the many cases recently reported in the literature of apparently healthy and fertile phenotypes in mice lacking a gene (knockout mice), such as the myoglobin knockout mouse," survive by substituting a preserved, evolutionarily earlier process for the one coded by the mutated gene.

1. Bollag W. The retinoid revolution. FASEB J 1996; 10:938-9

2. Kanai M, Raz A, Goodman DS. Retinol-binding protein: the transport protein for vitamin A in human plasma. J Clin Invest 1968;47:2025-44

3. Muto Y, Smith JE, Milch PO, Goodman DS. Regulation of retinol-binding protein metabolism by vitamin A status in the rat. J Biol Chem 1979;247:254250

4. Smith FR, Goodman DS, Zaklama MS, et al. Serum vitamin A, retinol-binding protein and prealbumin concentrations in protein-calorie malnutrition. Am J Clin Nutr 1973;26:973-81

5. Underwood BA. Methods for assessment of vitamin A status. J Nutr 1990;120(11 [suppl]):1459-63

6. Westermark P, Pitkanen P, Benson L, et al. Serum prealbumin and retinol-binding protein in the prealbumin-related senile and familial forms of systemic amyloidosis. Lab Invest 1985;52:314-8

7. Shoji S, Nakagawa S. Serum prealbumin and retinol-binding protein concentrations in Japanese-type familial amyloid polyneuropathy.

European Neurology 1988;28:191-3

8. Waits RP, Yamada T, Uemichi T, Benson MD. Low plasma concentrations of retinol-binding protein in individuals with mutations affecting position 84 of the transthyretin molecule. Clin Chem 1995;41: 1288-91

9. Episkopou V, Maeda S, Nishiguchi S, et al. Disruption of the transthyretin gene results in mice with depressed levels of plasma retinol and thyroid hormone. Proc Natl Acad Sci U S A 1993;90:2375-9

10. Biesalski HK, Frank J, Beck SC, et al. Biochemical but not clinical vitamin A deficiency due to mutations in the gene for retinol-binding protein. Am J Clin Nutr 1999;69:931-6

11. Seelinger MW, Biesalski HK, Wissinger B, et al. Phenotype in retinol deficiency due to a hereditary defect in the retinol binding protein synthesis. Invest Opthalmol Vis Sci 1999;40:3-11

12. Loerch JD, Underwood BA, Lewis KC. Response of plasma levels of vitamin A to a dose of vitamin A as

an indicator of hepatic vitamin A reserves in rats. J Nutr 1979;109:778-86

13. Blomhoff R, Skrede B, Norum KR. Uptake of chylomicron remnant retinyl ester via the low density lipoprotein receptor: implications for the role of vitamin A as a possible prevention for some forms of cancer. J Intern Med 1990;228:207-10

14. Nagy NE, Holven KB, Roos N, et al. Storage of vitamin A in extrahepatic stellate cells in normal rats. J Lipid Res 1997;38:645-58

15. Gerlach T, Biesalski HK, Weiser H, et al. Vitamin A in parenteral nutrition: uptake and distribution of retinyl esters after intravenous application. Am J Clin Nutr 1989;50:1029-38

16. Shidoji Y, Muto Y. Vitamin A transport in plasma of the non-mammalian vertebrates: isolation and partial characterization of piscine retinol-binding protein. J Lipid Res 1977;18:679-91

17. Garry DJ, Ordway GA, Lorenz JN, et al. Mice without myoglobin. Nature 1998;395:905-6

This review was prepared by George Wolf, D.Phil., Department of Nutritional Sciences, University of California, Berkeley, CA 94720-3104, USA. Reprint requests should be addressed to the Nutrition Reviews Editorial Office, 711 Washington Street, Boston, MA 02111, USA.

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