Schools of Biomolecular Sciences and of 'Biological and. Earth Sciences, L iverpool John Moores University,. Liverpool L3 3AF, U.K.. Considerable evidence ...
Biochemical Society Transactions ( 1 996) 24
Carotenoid profiles o f human plasma lipoproteins GORDON M. LOWE, RODNEY F. BILTON, ANDREW J. YOUNG', JOHN M. GRAHAM, TERRY C. FORD, and DAVID BILLINGTON Schools of Biomolecular Sciences and of 'Biological and Earth Sciences, L iverpool John Moores University, Liverpool L3 3AF, U.K. Considerable evidence exists to suggest that carotenoids are involved in the prevention of arteriosclerosis by acting as antioxidants. Carotenoids are transported in blood bound largely to low density lipoproteins (LDL). In order to investigate their antioxidant role it is necessary to prepare lipoproteins with minimal oxidative damage to the carotenoids. Traditionally this has been achieved by ultracentrifugation using KBr gradients [l] including antioxidants such as Trolox or butylated hydroxytoluene [2,3]. However, the preparation of LDL by this method can take up to 48 h and the salt must be removed by dialysis or column chromatography prior to undertaking any oxidation studies. This may result in disruption of the stucture of LDL and further oxidative damage. In an effort to overcome these problems we have used Iodixanol, (available as a 60% (w/v) solution known as Optiprep"', Nycomed Pharma, Norway) to form selfgenerating gradients which can separate LDL from plasma within 3.5 h of collection. Since Optiprep"' is inert, oxidation studies can be performed on LDL without its removal. Slight modification of the gradient allows LDL to be subfractionated such that the distribution of carotenoids can be investigated. A simple gradient for the isolation of LDL was prepared by adding 8 ml of plasma to 2 ml of Optiprep"' to give a final concentration of 12.5% Iodixanol. This was mixed by gentle inversion, and layered on top of 0.5 ml of Optiprep"' in a 11.2 ml Beckman Optiseal"' tube. The tubes were topped u p with saline and centrifuged at 350,OOOg and 16°C for 3 h. LDL and HDL gave brightly coloured bands approximately 2 and 4 cm respectively from the top of the tube and were removed either with a Pasteur pipette or by upward displacement with Maxidens"' (Nycomed Pharma, Norway). A two-step gradient was used to subfractionate LDL. Plasma was made 12.5% (v/v) with respect to Iodixanol and 5 ml was layered under 5 m1 of 6% (v/v) Iodixanol (1 vol of Optiprep" t 1 vol of plasma). Tubes were underlayered with 0.5 ml of Optiprep"', filled with saline and centrifuged at 350,OOOg and 16°C for 3h. The upper part of the gradient was fractionated into seven 1 ml aliquots by upward displacement. The top two fractions were identified as VLDL and fractions 3-7 as LDL . Carotenoids were extracted from 1 ml samples by the addition of 1 ml of ethanol, followed by 1.5 ml of diethyl ether and 1.5 ml of hexane. The organic layer was removed and dried at 35°C under a stream of N,. The extract was then resuspended in 200 p1 of tetrahydrofuran:ethanol (1/9, v/v) and 20 pl was injected onto a reverse phase ODs2 Spherisorb column. The column was eluted at a flow rate of 1 ml/min with acetonitrile: tetrahydrofuran:methanol (65:22:13) containing 0.25% (w/v) a m n i u m acetate. Carotenoids were detected at 460 nm and were identified by comparison to the retention times of known standards and by their u 1 traviolet\v i s i ble spectra. Oxidation of LDL was performed by exposing LDL at a final protein concentration of 50 fig/ml in PBS to 5 pt4 CuSO, at 37". Aliqouts were removed every 30 min, and TBARS formation was assessed [ 4 ] . When LDL and HDL were prepared using a simple Iodixanol gradient the majority o f carotenoids were shown to reside in the LDL fraction. Only lutein and traces of lycopene were found in the HDL fraction. By this method 40% of each of lycopene and 6-carotene
17 1 S
Table 1. Percentage distribution of four carotenoids in plasma LDL subfractions. Fraction nuber
Density (q/#l)
6Carotene
Lycopene
6-Cryptoxanthin
Lutein
3
1.029
1.8f1.8
1.3f2.3
2.lf2.4
4.lf2.1
*
Values are means f S.E. for 5 healthy individuals. In
present in whole plasma were recovered from the LDL fraction. When the LDL fraction was oxidised in the presence of 5 pt4 CuSO,, a lag time of 93 f 4.6 min (n=3) was recorded. Subfractionation of LDL using the two-step gradient produced a higher total yield of carotenoids (54%). probably due to a more thorough recovery of LDL. Lycopene, B-carotene and B-cryptoxanthin showed a very similar distribution and were preferentially located in the less-dense LDL subfractions (Table 1). This is not surprising as they are similar hydrocarbon structures. In contrast, lutein is a more polar compound with hydroxyl groups on each ring moeity, and was preferentially located in the smaller, more-dense LDL subfractions (Table 1). LDL subfractions of higher density (p = 1.057 g/ml) showed a shorter oxidation lag time upon stimulation with Cu" (20.3 f 2.1 min, n=5) compared to the lower density (p = 1.036 g/ml), carotenoid-rich subfractions (193 f 29 min, n=5). This confirms previous reports [3,5] that smaller, more-dense LDL particles are more susceptible to oxidation. As a result individuals with higher levels of this LDL subfraction maybe at a greater risk of developing arteriosclerosis. In conclusion, the use of Optiprep"' has enabled the rapid isolation of intact LDL with little or no oxidation of associated carotenoids. By using a twostep gradient system, subfractions of LDL can be recovered which reveal differences in the distribution of carotenoids. These differences may explain why the more dense smaller particles have a shorter lag-time when oxidised with Cu". We thank the World Cancer Fund for supporting this work. 1. Clevidence, B.A. & Bieri, J.G. (1993) Methods Enzymo7. 214, 33-46 2 . Edelstein, C & Scanu, A.M. (1986) Methods Enzymo7.
128, 151-155 3. Dejager, S., Brukert, E. & Chapman, J. (1993) J. Lipid Res. 34, 295-308 4. Mao, S.J.T., Yates, M.T. & Jackson, R.L. (1994) Methods Enzymo7. 234, 505-513 5. Rajman, I, Maxwell, S., Cramb, R. & Kendall, M. (1994) Quart. J. Med. 87, 709-720
