Deuterium enrichment of vitamin A at the C20 position slows the formation of detrimental vitamin A dimers in wild-type rodents

Abstract

Degenerative eye diseases are the most common causes of untreatable blindness. Accumulation of lipofuscin (granular deposits) in the retinal pigment epithelium (RPE) is a hallmark of major degenerative eye diseases such as Stargardt disease, Best disease, and age-related macular degeneration. The intrinsic reactivity of vitamin A leads to its dimerization and to the formation of pigments such as A2E, and is believed to play a key role in the formation of ocular lipofuscin. We sought a clinically pragmatic method to slow vitamin A dimerization as a means to elucidate the pathogenesis of macular degenerations and to develop a therapeutic intervention. We prepared vitamin A enriched with the stable isotope deuterium at carbon twenty (C20-D(3)-vitamin A). Results showed that dimerization of deuterium-enriched vitamin A was considerably slower than that of vitamin A at natural abundance as measured in vitro. Administration of C20-D(3)-vitamin A to wild-type rodents with no obvious genetic defects in vitamin A processing, slowed A2E biosynthesis. This study elucidates the mechanism of A2E biosynthesis and suggests that administration of C20-D(3)-vitamin A may be a viable, long-term approach to retard vitamin A dimerization and by extension, may slow lipofuscin deposition and the progression of common degenerative eye diseases.

Figures

FIGURE 1.

Replacing C20 hydrogen atoms of all-trans-retinaldehyde with deuteriums slows the formation of A2E. A, HPLC profiles of reaction mixtures containing C20-D3-retinaldehyde or retinaldehyde, plus ethanolamine and acetic acid after 30 h. B, expanded section of panel A. Gray line: retinaldehyde reaction; Black line: C20-D3-retinaldehyde reaction. C, plots of A2E formation over time for the reactions in panel A (kH/kD = 7 ± 1; from an average of three runs).

FIGURE 2.

Replacing C20 hydrogen atoms of all-trans-retinaldehyde with deuteriums slows the formation of ATR-dimer. A, HPLC profiles recorded after 1 h of reaction mixtures containing proline and either C20-D3-all-trans-retinaldehyde or all-trans-retinaldehyde to form ATR-dimer. B, plot of ATR-dimer formation over time for the reactions in panel A.

FIGURE 3.

APCI MS of a reaction mixture of retinaldehyde and C20-D3-retinaldehyde with ethanolamine after (A) 2 min and (B) 2 weeks. Mass to charge ratios are shown and ion intensities are shown as relative percents. The peak at m/z 328 corresponds to the Schiff base at natural abundance and the peak at m/z 331 corresponding to the deuterated Schiff base.

FIGURE 4.

Administration of C20-D3-vitamin A to wild-type mice slows A2E synthesis in vivo. A, representative HPLC trace of extracted retinoids from eyecups of mice administered C20-D3-vitamin A. B and C, UV-Vis absorbance spectra of the peaks at B ∼10.8, representative of A2E and C, ∼11.8 min, representative of iso-A2E. D, average A2E levels in animals given either vitamin A at natural abundance or C20-D3-vitamin A. Five animals per group were used. p: p value.

FIGURE 5.

Comparison of A2E in age-matched animals given different inhibitors of A2E biosynthesis. All groups were raised on a standard rodent diet for the first 50 days. The 100-day-old controls were raised on a standard diet throughout. While the Fenretinide, TDH, vitamin A, and C20-D3-vitamin A groups were given high doses of the amount of vitamin A for the remaining 50 days, except that the last mentioned group was given vitamin A in the form of C20-D3-vitamin A. Three animals per group were used.

FIGURE 6.

Liver vitamin A in response to dietary C20-D3-vitamin A. Representative ACPI mass spectra of liver extracts of animals on a diet containing 100,000 IU of C20-D3-vitamin A/kg of diet for (A) 2 and (B) 4 weeks. The peaks labeled “269” and “272” represent fragments of vitamin A and C20-D3-vitamin A, respectively.

FIGURE 7.

Biosynthesis of A2E and ATR-dimer from all-trans-retinaldehyde. Hydrogen atoms are presented at the C20 position of all-trans-retinaldehyde and on its Schiff base. Inset: possible transition states (TS) leading to the cis- or trans-enamines.

FIGURE 8.

Inhibition of A2E biosynthesis by C20-D3-vitamin A in wild-type animals. PE, phosphatidylethanolamine. The circular arrows represent the vitamin A cycle; two proteins are displayed. Upon photon recognition in the outer segment of the neural retina (rod cell shown), retinaldehyde is released from the opsin protein. A portion of this retinaldehyde condenses with PE to form retinaldehyde-PE, leading to the formation of vitamin A dimers. Deuteration of the C20-H bonds of vitamin A inhibits dimerization allowing more time for retinaldehyde-PE to be processed by ABCA4.