and calculated by Beer’s law. The molar extinction coefficient ε
at 450 nm of β-carotene in hexane is 125,000 L Â mol
À1
 cm
À1
,
zeaxanthin in ethanol 133,000 L Â mol
À1
 cm
À1 , and for
lutein in ethanol 145,000 L Â mol
À1
 cm
À1 .
15. We routinely carry out HPLC analyses with an Agilent 1260
Infinity Quaternary HPLC system (Santa Clara, CA, USA)
equipped with a pump (G1312C) with an integrated degasser
(G1322A), a thermostated column compartment (G1316A),
an autosampler (G1329B), a diode-array detector (G1315D),
and online analysis software (Chemstation). The analyses are
carried out at 25
C using a normal-phase Zorbax Sil (5 μm,
4.6 Â 150 mm) column (Agilent Technologies, Santa Clara,
CA) protected with a guard column with the same stationary
phase. As mobile phase we use hexanes ethyl acetate mixes at a
flow rate of 1.4 mL Â min
À1 . The composition of the mobile
phase depends on the polarity of the carotenoid and
apocarotenoids.
Acknowledgments
The research was supported in part by U.S. National Institute of
Health grants EY020551. The authors thank present and former
members of the von Lintig laboratory for their manifold contributions to establishing this method.
References
1. Harrison EH, Quadro L (2018) Apocarotenoids: emerging roles in mammals. Annu Rev
Nutr 38:153–172
2. von Lintig J (2012) Metabolism of carotenoids
and retinoids related to vision. J Biol Chem
287:1627–1634
3. Rhinn M, Dolle ´ P (2012) Retinoic acid signalling during development. Development (Cambridge England) 139:843–858
4. Ross AC (2012) Vitamin a and retinoic acid in
T cell-related immunity. Am J Clin Nutr
96:1166S–1172S
5. Tang XH, Gudas LJ (2011) Retinoids, retinoic
acid receptors, and cancer. Annu Rev Pathol
6:345–364
6. Giuliano G, Al-Babili S, von Lintig J (2003)
Carotenoid oxygenases: cleave it or leave
it. Trends Plant Sci 8:145–149
7. von Lintig J, Kiser PD, Golczak M, Palczewski
K (2010) The biochemical and structural basis
for trans-to-cis isomerization of retinoids in the
chemistry of vision. Trends Biochem Sci
35:400–410
8. Sui X, Golczak M, Zhang J, Kleinberg KA, von
Lintig J, Palczewski K, Kiser PD (2015) Utilization of Dioxygen by carotenoid cleavage
Oxygenases. J Biol Chem 290:30212–30223
9. Kiefer C, Hessel S, Lampert JM, Vogt K,
Lederer MO, Breithaupt DE, von Lintig J
(2001) Identification and characterization of a
mammalian enzyme catalyzing the asymmetric
oxidative cleavage of provitamin a. J Biol Chem
276:14110–14116
10. Redmond TM, Poliakov E, Yu S, Tsai JY, Lu Z,
Gentleman S (2005) Mutation of key residues
of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle. Proc Natl
Acad Sci 102:13658–13663
11. Redmond TM, Gentleman S, Duncan T, Yu S,
Wiggert B, Gantt E, Cunningham FX Jr
(2001) Identification, expression, and substrate specificity of a mammalian beta-carotene
15,15
0 -dioxygenase.
J
Biol
Chem
276:6560–6565
12. Gollapalli DR, Maiti P, Rando RR (2003)
RPE65 operates in the vertebrate visual cycle
Mammalian Carotenoid Cleavage Dioxygenases
87
at 450 nm of β-carotene in hexane is 125,000 L Â mol
À1
 cm
À1
,
zeaxanthin in ethanol 133,000 L Â mol
À1
 cm
À1 , and for
lutein in ethanol 145,000 L Â mol
À1
 cm
À1 .
15. We routinely carry out HPLC analyses with an Agilent 1260
Infinity Quaternary HPLC system (Santa Clara, CA, USA)
equipped with a pump (G1312C) with an integrated degasser
(G1322A), a thermostated column compartment (G1316A),
an autosampler (G1329B), a diode-array detector (G1315D),
and online analysis software (Chemstation). The analyses are
carried out at 25
C using a normal-phase Zorbax Sil (5 μm,
4.6 Â 150 mm) column (Agilent Technologies, Santa Clara,
CA) protected with a guard column with the same stationary
phase. As mobile phase we use hexanes ethyl acetate mixes at a
flow rate of 1.4 mL Â min
À1 . The composition of the mobile
phase depends on the polarity of the carotenoid and
apocarotenoids.
Acknowledgments
The research was supported in part by U.S. National Institute of
Health grants EY020551. The authors thank present and former
members of the von Lintig laboratory for their manifold contributions to establishing this method.
References
1. Harrison EH, Quadro L (2018) Apocarotenoids: emerging roles in mammals. Annu Rev
Nutr 38:153–172
2. von Lintig J (2012) Metabolism of carotenoids
and retinoids related to vision. J Biol Chem
287:1627–1634
3. Rhinn M, Dolle ´ P (2012) Retinoic acid signalling during development. Development (Cambridge England) 139:843–858
4. Ross AC (2012) Vitamin a and retinoic acid in
T cell-related immunity. Am J Clin Nutr
96:1166S–1172S
5. Tang XH, Gudas LJ (2011) Retinoids, retinoic
acid receptors, and cancer. Annu Rev Pathol
6:345–364
6. Giuliano G, Al-Babili S, von Lintig J (2003)
Carotenoid oxygenases: cleave it or leave
it. Trends Plant Sci 8:145–149
7. von Lintig J, Kiser PD, Golczak M, Palczewski
K (2010) The biochemical and structural basis
for trans-to-cis isomerization of retinoids in the
chemistry of vision. Trends Biochem Sci
35:400–410
8. Sui X, Golczak M, Zhang J, Kleinberg KA, von
Lintig J, Palczewski K, Kiser PD (2015) Utilization of Dioxygen by carotenoid cleavage
Oxygenases. J Biol Chem 290:30212–30223
9. Kiefer C, Hessel S, Lampert JM, Vogt K,
Lederer MO, Breithaupt DE, von Lintig J
(2001) Identification and characterization of a
mammalian enzyme catalyzing the asymmetric
oxidative cleavage of provitamin a. J Biol Chem
276:14110–14116
10. Redmond TM, Poliakov E, Yu S, Tsai JY, Lu Z,
Gentleman S (2005) Mutation of key residues
of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle. Proc Natl
Acad Sci 102:13658–13663
11. Redmond TM, Gentleman S, Duncan T, Yu S,
Wiggert B, Gantt E, Cunningham FX Jr
(2001) Identification, expression, and substrate specificity of a mammalian beta-carotene
15,15
0 -dioxygenase.
J
Biol
Chem
276:6560–6565
12. Gollapalli DR, Maiti P, Rando RR (2003)
RPE65 operates in the vertebrate visual cycle
Mammalian Carotenoid Cleavage Dioxygenases
87