Abstract
Vitamin A (retinol) is a nutrient that is essential for developmental regulation but toxic in large amounts. Previous genetic studies have revealed that alcohol dehydrogenase Adh1 is required for efficient clearance of excess retinol to prevent toxicity, thus demonstrating that the mechanism involves oxidation of excess retinol to retinoic acid (RA). Whereas Adh1 plays a dominant role in the first step of the clearance pathway (oxidation of retinol to retinaldehyde), it is unknown what controls the second step (oxidation of retinaldehyde to RA). We now present genetic evidence that aldehyde dehydrogenase Aldh1a1, also known as retinaldehyde dehydrogenase Raldh1, plays a dominant role in the second step of retinol clearance in adult mice. Serum RA levels following a 50 mg/kg dose of retinol were reduced 72% in Raldh1-/- mice and 82% in Adh1-/- mice. This represented reductions in RA synthesis of 77-78% for each mutant after corrections for altered RA degradation in each. After retinol dosing, serum retinaldehyde was increased 2.5-fold in Raldh1-/- mice (indicating defective retinaldehyde clearance) and decreased 3-fold in Adh1-/- mice (indicating defective retinaldehyde synthesis). Serum retinol clearance following retinol administration was decreased 7% in Raldh1-/- mice and 69% in Adh1-/- mice. LD50 studies indicated a small increase in retinol toxicity in Raldh1-/- mice and a large increase in Adh1-/- mice. These observations demonstrate that Raldh1 functions downstream of Adh1 in the oxidative metabolism of excess retinol and that toxicity correlates primarily with accumulating retinol rather than retinaldehyde.
Highlights
Retinoic acid (RA)1 is a metabolic derivative of vitamin A that regulates developmental pathways in chordate animals [1]
We have obtained no evidence as of yet that Raldh1 is essential for development, contrasting with the results obtained from null mutants of Raldh2, which result in embryonic lethality at mid-gestation [31, 32]
A comparison of area under the curve (AUC) values for wild-type mice and the two mutant strains demonstrated that Raldh1 deficiency results in a 72% reduction in serum retinoic acid (RA) detectable during the 4-h period following retinol administration, whereas Adh1 deficiency results in an 82% reduction in serum RA (Table I)
Summary
Animals—Raldh1Ϫ⁄Ϫ mice were generated as described previously using a gene replacement targeting vector that removed exon 11 from the Raldh (Aldh1a1) gene [41]. Quantitation of Serum Retinoic Acid and Retinol—Retinoids were administered orally essentially as described previously [45]. All-transretinol or all-trans-RA (Sigma) were dissolved in acetone-Tween 20water (0.25:5:4.75 v/v/v) and were administered by oral injection at a dose of 50 mg/kg for retinol or 10 mg/kg for RA. Retinoids were extracted from 0.2 ml of serum under darkened conditions, and all-trans-retinol and all-trans-RA were quantitated by reversed-phase HPLC using a MICROSORB-MVTM 100 C18 column as described previously [43]. Quantitation of Serum Retinaldehyde—All-trans-retinol was dissolved in acetone-Tween 20-water (0.25:5:4.75 v/v/v) and administered by oral injection at a dose of 100 mg/kg. A standard solution of all-trans-retinaldehyde oximes (syn- and antioximes) was prepared by treatment of all-trans-retinaldehyde (Sigma) with 1 M hydroxylamine (pH 6.8) as above This standard solution was used to identify the peaks and obtain the calibration curves. Statistical significance was determined for raw data using the unpaired Student’s t test (Statistica version 5.0)
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