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`Part A
`Biochemistry and Molecular Biology
`C. D. B. BRIDGES, Houston, Tex., U.S.A.
`
`Retinal Physiology, Cell Biology,
`Neurotransmitters, Morphology
`A. L. Bvzov, Moscow, U.S.S.R.
`J. G. HOLLYFIELD, Houston, Tex., U.S.A.
`A. KANEKO, Okazaki, Japan
`H. KOLB, Salt Lake City, Ut., U.S.A.
`D. VAN NORREN, Soesterberg, The Netherlands
`Central Nervous System Physiology and
`Morphology
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`S. S. EASTER, Ann Arbor, Mich., U.S.A.
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`H. SPEKREIJSE, Amsterdam, The Netherlands
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`Part B
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`Colour Perception
`R. M. BOYNTON, La Jolla, Calif., U.S.A.
`J. KRAUSKOPF, Murray Hill, N.J., U.S.A.
`H. G. SPERLING, Houston, Tex., U.S.A.
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`Physiological Optics, Acuities, Pattern Vision,
`Contrast Sensitivity, Infant Vision
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`J. J. KOENDERINK, Utrecht, The Netherlands
`R. SI-ZKULER, Evanston, Ill., U.S.A.
`G. WESTI-EEIMER, Berkeley, Calif., U.S.A.
`D. Y. TELLER, Seattle, Wash., U.S.A.
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`Binocular Vision: Depth & Motion Perception
`O. J. BRADDICK, Cambridge, U.K.
`R. FOX, Nashville, Tenn., U.S.A.
`Eye Movements, Strabismus, Amblyopia
`N. BARMACK, Portland, 0reg., U.S.A.
`H. COLLEWIJN, Rotterdam, The Netherlands
`A. SKAVENSKI, Boston, Mass., U.S.A.
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`r.’
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`1/[S1011 Res. Vol. 25, No. 11, pp. 1531—1535, 1935
`Printed in Great Britain. All rights reserved
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`Copyright © 1985 Pergamon Press Ltd
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`PRELIMINARY IDENTIFICATION OF THE HUMAN
`
`MACULAR PIGMENT
`
`RICHARD A. BONE, JOHN T. LANDRUM and SARA L. TARs1s
`Departments of Physics and Chemistry, Florida International University, Miami, FL 33199, U.S.A.
`
`(Received 20 March 1985; in revised form 6 June 1985)
`
`Abstract—The human macular pigment has'been' found to be composed of two chromatographically
`separable components, which are tentatively identified as lutein [(3R,3’R,6’R)-fi,s—Carotene-3,3’-diol] and
`zeaxanthin [(3R,3’R)-,8,/5‘-Carotene-3,3’-diol]. Chromatograms of retinal extracts, obtained by HPLC on
`three different stationary phases, were found to match those obtained with mixtures of lutein and
`zeaxanthin standards. Identical retention times were confirmed by coinjection of each of the isolated
`components with the appropriate standard. U.V.—visible spectra of the purified components were identical
`in all respects with those of lutein and zeaxanthin. Further support for our identification was obtained
`by the preparation and chromatographic comparison of derivatives of the macular pigment and of the
`standards.
`
`Macular pigment
`
`Lutein
`
`Zeaxanthin
`
`Carotenoids
`
`HPLC
`
`INTRODUCTION
`
`purpose of establishing a preliminary identification
`through the powerful analytical potential of high-
`performance liquid chromatography. This technique,
`applied to macular extracts and their chemical deriv-
`atives, and supported by spectroscopic studies, has
`revealed the composition of the macular pigment as
`a mixture of two carotenoids, most probably lutein
`and zeaxanthin.
`
`EXPERIMENTAL METHODS
`
`Extraction of the macular pigment
`
`Human eyes were obtained frozen from the Florida
`Lions Eye Bank and the National Diabetes Research
`Interchange. Retinas were dissected in 0.9% saline
`solution, care being taken to minimize exposure to
`u.v. and visible light. The macular tissue, identified by
`its yellow coloration, was removed from each retina.
`In the few cases where no yellow pigment was
`discernible, the retinas were discarded. The macular
`pigment was extracted by grinding the tissue in
`redistilled acetone at 0"C. The resulting yellow solu-
`tion was filtered (0.4 pm Millipore filter) and placed
`in a freezer at —-20°C for several hours. During this
`period, fractional crystallization of lipophilic com-
`pounds occurred, indicated by a white deposit ap-
`pearing on the container walls. The remaining solu-
`tion was
`transferred to another container and
`
`9‘
`
`‘ The existence of the yellow macular pigment, which
`characterizes the retinas of primates including man,
`has been known since the eighteenth century (Polyak,
`7 1941). Yet a definitive characterization of the species
`1 responsible is still lacking. Early attempts to identify
`the pigment have been summarized by Walls and
`‘ Mathews
`(1952),
`and
`the
`generally
`accepted
`identification of the pigment as lutein is based solely
`on its characteristic carotenoid absorption spectrum
`reported by Wald (1945, 1949). Since then, it has been
`. suggested (Brown and Wald, 1963) that a mixture of
`the cis and trans isomers of lutein might be involved,
`this producing a closer spectroscopic match with the
`pigment than the trans isomer alone.
`The macular pigment was shown by Wald (1949)
`to reduce the sensitivity of the macula region to blue
`light by behaving as a broad band filter. Con-
`sequently it influences color appearance, color match-
`ing and tests for color vision (Ruddock, 1972). An
`3 advantage to the visual system of its presence may be
`the improvement of visual acuity. This may be
`achieved through compensation for chromatic aber-
`ration in the eye’s refractive media and by reduction
`in the amount of atmospherically scattered blue light
`reaching the receptors. (Walls, "1967; Reading and
`Weale, 1974). While the identity of the pigment is not
`crucial to these hypotheses, it is more relevant to the
`postulated‘ function of the pigment of protecting
`retinal
`tissue
`against photosensitized
`reactions
`(Kirschfeld,
`l982; Bone and Landrum, 1984). Fur-
`ther, a definitive characterization would be of interest
`to biochemists and nutritionists concerned with the
`
`1
`
`thoroughly dried under a stream of pure nitrogen. On
`redissolving the pigment in ethanol, at 3-peaked spec-
`trum, characteristic of carotenoids, was obtained.
`Assuming an extinction coefficient of approximately
`1.3 X 1051 mol" crn”', typical of a wide range of
`metabolic and/or dietary origin of the pigment.
`carotenoids (Strain, 1938), an estimate was obtained
`of ~10 ng of pigment available, on average, from
`The present
`investigation was initiated for the
`1531
`
`V.R. 25/l |——A
`
`

`
` 1
`
`
`
`r..,,.,....4-.......,,,....,.,,._,,,..,.,,W__,,.....w....\.......,<W-
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`S.10:7il
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`-we’'
`
`
`
`1532
`
`RICHARD A. Born: et al.
`
`a.1
`
`b.1
`
`c.1
`
`10
`
`20
`
`30
`
`r--—r-"r-—'r
`0 5
`10 I5
`
`plat
`
`20
`10
`Time, min
`
`30
`
`I0 15
`5
`0
`Time,min
`
`0
`
`I0
`
`20
`Time , min
`
`30
`
`40
`
`Fig. 1. Chromatograms of the macular pigment components (upper diagrams) and lutein and zeaxanthin
`standards (lower diagrams) on 3 different stationary phases. (a) Column, reversed-phase 5 am ODS;
`eluent, 93% methanol, 7% water/acetonitrile (75:25, v/v); flow rate, 0.5 ml/min; sample volume, 20 pl.
`(b) Column, normal-phase 5 pm SiO2; eluent, 90% n-hexane, 10% acetone; flow rate,
`1 ml/min; sample
`volume, 20 it]. (c) Column, normal-phase 73% CaCO3, 15% MgO, 12% Ca(OH)2 (<25 um); elucnt, 55%
`n—hexane, 45% chloroform; flow rate, 2.5 ml/min; sample volume, 20111. In all cases, MP1 and lutein
`eluted with shorter retention times than MP2 and zeaxanthin.
`
`each retina. In each of the experiments described
`below, between 10 and 20 retinas were used.
`
`High -performance liquid chromatography (HPLC)
`
`HPLC was carried out on a Laboratory Data
`Control unit with dual pump and gradient capability,
`using 3mm (ID) x 25 cm columns. All eluting sol-
`vents were of HPLC quality but, nevertheless, were
`redistilled prior to use.
`Initial purification of the macular pigment was
`accomplished using a reversed-phase (5 am Tech-
`sphere ODS) column. Chromatograms consistently
`indicated the presence of two major components,
`MP1 and MP2,
`in similar, but somewhat varying
`proportions. Baseline separation was achieved [Fig.
`1(a.1)] and the two components could be collected
`individually. Further HPLC studies were conducted
`with a normal-phase column packed with 5 am silica
`gel (Techsphere) and a noncommercial, normal-phase
`column containing_73% 'CaCO3, 15% MgO and 12%
`Ca(OH)z (by weight): a combination known to pro-
`duce good separation of lutein and zeaxanthin when
`applied to thin layerchromatography (Hager and
`Meyer-Bertenrath, 1966).
`
`Isolation of carotenoid standards
`
`U.V.-visible spectra of the purified components
`MP1 and MP2 were found to be consistent with those
`
`reported for
`
`lutein and zeaxanthin,
`
`respectively
`
`(Strain, 1938). Also their HPLC retention times on
`the reversed-phase column were found to match
`closely those obtained for lutein and zeaxanthin by
`Braumann and Grimme (1981) when using similar
`elution conditions.
`
`Isolation of these pigments from spinach and, in
`the case of zeaxanthin from corn as well, was accom-
`
`plished by the following procedures. Methanol ex-
`tracts were treated with KOH to saponify the chloro-
`phylls, and the carotenoids were separated by solvent
`partition using diethyl ether (see, for example, Strain,
`1938). Initial purification and identification of the
`individual carotenoids was achieved by thin layer
`chromatography using the methods of Hager and
`Meyer—Bertenrath (1966). For large scale work, these
`methods were modified for
`liquid-column chro-
`matography. Final purification was by reversed-
`phase HPLC. Further support for the identification
`of the standards was provided by the observed consis-
`tency with literature data on HPLC retention times
`(Braumann and Grimme, 1981), u.v.-visible spectra
`(Strain, 1938) and, most importantly, mass spectra
`(Heller and Milne, 1978). Experimental details and
`results of the mass spectrometry are given in the
`Appendix.
`
`Derivatization
`
`Derivatives of the two macular pigments and of the
`standards were prepared successfully on the nano-
`
`

`
`Identification of human macular pigment
`
`1533
`
`Ho
`
`Lutein
`
`OH
`
`OH
`
`Ho
`
`’
`
`Zeaxanthin
`
`Fig. 2. Structures of lutein and zeaxanthin.
`
`gram scale by modifications of synthetic procedures
`reported in the literature for lutein and zeaxanthin.
`Acetate esters (Britton and Goodwin, 1971) were
`obtained in good yield by the addition of 0.1 ml of
`acetic anhydride to 0.2 ml pyridine solutions of each
`of the parent compounds. The reactions were allowed
`to proceed in the dark under a nitrogen atmosphere
`for 18 hr. Work-up was accomplished by the methods
`described by Britton and Goodwin (1971). Chro-
`matography Of the esterified pigments was carried out
`on the reversed-phase column, eluting with methanol
`at a flow rate of 1 ml/min.
`Lutein is distinguished from zeaxanthin by its
`single allylic hydroxyl group (Fig. 2). This may be
`exploited in the selective preparation of the allylic
`monomethyl ether of lutein by treatment of lutein
`with methanol and hydrochloric acid (Curl, 1956;
`Jensen and Hertzberg, 1966). Under the same condi-
`tions, zeaxanthin is unreactive. Solutions of MP1,
`MP2, lutein and zeaxanthin were dried under streams
`of pure nitrogen and the residues redissolved in
`5-10 pl of methanol/con. HCI (9:l by volume). The
`reactions were terminated after 7 min by the addition
`of saturated solutions of sodium bicarbonate. Chro-
`
`matography of the products was conducted on the
`reversed-phase column (Fig. 5).
`
`RESULTS AND DISCUSSION
`
`Chromatography of extracts of the macular pig-
`ment has proven that it consists of two distinct
`components, MP1 and MP2 (Fig. 1). Absorption
`spectra in the u.v.-visible (see Figs 3 and 4) were
`
`absorbance
`Relative
`
`450
`350
`Wavelength . nm
`
`550
`
`Fig. 3. Absorption spectra of the macular pigment com-
`P0nent MP1 (—-——-) and lutein (---). Solvent, 93% meth-
`‘H101, 7% water/acetonitrile (75:25, v/v), as used for HPLC
`elution. The III/II absorption ratio for each pigment is 0.91.
`
`'
`
`absorbance /
`Relative
`
`\_--..—
`T
`‘TM’?
`450
`350
`Wavelength , nm
`
`F
`
`I
`550
`
`Fig. 4. Absorption spectra of the macular pigment com-
`ponent MP2 (--) and zeaxanthin (---). Solvent, 93%
`methanol, 7% water/acetonitrile (75:25, v/V), as used for
`HPLC elution. The III/II absorption ratio for each pigment
`is 0.89.
`
`found to be totally consistent with those of lutein and
`zeaxanthin respectively. Among the characteristic
`features, which emphasize the consistency, are the
`wavelengths of the absorption maxima and minima,
`the III/II absorption ratios (absorption at the long
`wavelength maximum (111) to that at the main peak
`(11)) and _the definition of the short wavelength shoul-
`ders and long wavelength peaks. The previous sug-
`gestion (Brown and Wald, 1963), that both cis and
`trans lutein were present in the macular pigment, may
`now be ruled out as no cis bands were observed in the
`
`320-340 nm range (Vctter et al., 1971).
`Chromatography was performed on three difierent
`stationary phases (see Experimental Methods).
`In
`each case, the same retention times, within the limits
`of the equipment, were observed for MP1 and lutein
`as well as for MP2 and zeaxanthin (Fig. 1). Coin-
`jections of MP1 and lutein were carried out on all
`three chromatographic stationary phases, resulting in
`the elution of only single peaks. Similarly, MP2 and
`zeaxanthin coeluted as single peaks.
`initial
`Chromatograms
`obtained
`during
`purification of retinal extracts on the reversed-phase
`column indicated the presence of other weakly ab-
`sorbing compounds [Fig.
`l(a.l)]. The peak which
`appears at about 22 min retention time showed some
`increase in height with the length of time of tissue
`storage and, in one experiment, was sufificient for a
`crude absorption spectrum of the collected com-
`pound to be recorded. This was characterized by a
`broad maximum in the 400-420 nm range and may,
`therefore,
`represent one of the subsidiary, non-
`macular pigments (P410) found by Snodderly et al.
`(1984a). None of the other minor compounds which
`appear in the chromatogram of Fig.
`l(a.l) was
`sufficient for spectral analysis.
`Lutein and zeaxanthin are isomers having the
`structural formulas shown in Fig. 2. They differ only
`in the placement of one double bond. In lutein, one
`of the end group hydroxyl moieties is allylic while in
`zeaxanthin, neither hydroxyl is associated with the
`double bonds. Both hydroxyl groups in these two
`xanthophylls can be esterified to produce less polar
`
`

`
`
`
`occurring, giving rise to the two derivatives. This
`would be consistent with the reaction proceeding by
`a carbocation mechanism. MP1 likewise reacted to
`produce two chromatogram peaks at 30 and 32 min
`While the exact nature of the two components is Smi
`under investigation, the observation of identical reac,
`tivity of MP1 and lutein to produce indistinguishable
`products is compelling evidence that these two pig
`ments are indeed identical.
`
`Under the same experimental conditions, neither
`MP2 nor zeaxanthin reacted, the recovered products
`having identical retention times (16.4 min) to that of
`the untreated zeaxanthin. This result shows conclu-
`sively that the second component of the macular
`pigment cannot be a cis isomer of lutein, as Suggested
`in the earlier work (Brown and Wald,
`l963).
`
`CONCLUSIONS
`
`While a definitive identification of the human
`macular pigment must await mass spectrometry, our
`present studies suggest that it is composed of two
`pigments that are similar to lutein and zeaxanthin. If
`the macular pigment is derived from dietary sources,
`as it almost certainly must be (Malinow et a[., 1980),
`the relative abundances of lutein and zeaxanthin in
`the diet might be expected to be duplicated in the
`macular pigment. However, while lutein is vastly
`more abundant in green plants, our studies have led
`us to conclude that this is never the case with the
`macular pigment. In general,
`the two components
`were found to be present in similar amounts [see, for
`example, Fig. l(a.l)], neither exceeding the other by
`more than about 30%. Occasionally,
`though less
`frequently, zeaxanthin was in excess, yet
`the only
`common plant source where this is
`so is com
`(Weedon, 1971). We plan to study this anomaly and
`its possible metabolic implications further, initially by
`examining the relative abundance of the two car-
`otenoids in individual maculae.
`
`We have previously shown (Bone and Landrum,
`1984) that Haidinger’s polarization figure is consis-
`tent with incorporation of the macular pigment.
`assumed to be lutein, into the membranes of Henle
`fibers, the inner segments of cone receptor cells. This
`hypothesis is supported by the results of a recent
`microdensitometry study (Snodderly et al.,
`l984b).
`Owing
`to
`the
`close
`similarity of
`lutein and
`zeaxanthin, our explanation of the brushes is Still
`valid, and suggests that both carotenoids may be
`involved in the protection of these membranes
`against photosensitized reactions (Kirschfeld, 1982)-
`
`
`
`
`
`r4\“49‘)‘»-.uV">V/Yrs/4")‘-...w....-.x,4§,-re9w;.m~,-1"‘-_v«.\.,-¢~r*~M
`
`1534
`
`RICHARD A. BONE el al.
`
`chro-
`different
`distinctly
`having
`derivatives
`matographic properties when compared with the
`parent compounds.
`Upon reaction of MP1 with excess acetic anhy-
`dride, we obtained a derivative with a retention time
`on the reversed-phase HPLC approximately twice
`that of the parent compound, and matching precisely
`that of lutein diacetate prepared under identical
`conditions. Coinjection of the two derivatives re-
`sulted in a single chromatogram peak. Similarly the
`MP2 acetate derivative and zeaxanthin diacetate
`
`eluted as a single peak when coinjected, the retention
`time again being roughly double that of zeaxanthin.
`Yields from these reactions were sufficient for a
`
`comparison of u.v.-visible spectra. Complete consis-
`tency was observed between the acetate derivatives of
`the macular pigments and the derivatives of the
`corresponding standards. The spectra were, in fact,
`essentially indistinguishable from those of the stan-
`dards themselves (Figs 3 and 4).
`To further substantiate the assignment of MP1 and
`MP2 as lutein and zeaxanthin respectively, we sought
`data which would distinguish clearly between these
`two carotenoids. The allylic hydroxyl group of lutein
`provides the means by which such distinction may be
`made. Conversion of this group to a methyl ether has
`been reported to occur under conditions which do not
`alter zeaxanthin (see Experimental Methods). How-
`ever, reaction of lutein with HCl/methanol consis-
`tently produced two new compounds, rather than
`one, with retention times on the reversed-phase
`column of approximately 30 and 32 min, compared
`with 15.2 min for lutein itself (Fig. 5). This obser-
`vation suggests that rearrangement accompanies the
`reaction. Racemization at
`the 3’ carbon may be
`
`
`
`0
`
`10
`
`20
`Time, min
`
`30
`
`40
`
`Fig. 5. Chromatograms of monomethyl ethers of (a) MP1
`and (b) lutein. The single peaks at ~15 min are tentatively
`identified as unreacted parent compounds, based on coin-
`jection with the lutein standard; those at ~30 and 32 min,
`the reaction products. Column, reversed-phase 5 pm ODS;
`eluent, 93% methanol, 7% water/acetonitrile (75:25, v/v);
`flow rate, lml/min; sample volume, 20 #1.
`
`Acknowledgements-—We thank the Florida Lions Eye Bank,
`Inc. of Miami and the National Diabetes Research Inter-
`change for supplying the human eyes used in this study.
`Support was provided by grants from the National Insti-
`tutes of Health, No.
`1 R01 EY05452-01, and the Florida
`International University Foundation. R. A. Yost and E. D'
`Britten kindly performed the mass spectroscopy Of the
`
`

`
`
`
`Identification of ‘human macular pigment
`
`1535
`
`Camgenoid standards. We acknowledge the laboratory assis-
`tance of A. Gonzalez and wish to thank J. M. E. Quirke for
`his helpful comments.
`
`APPENDIX
`
`Mass spectra of lutein and zeaxanthin standards were run
`on a triple-stage quadrupole mass spectrometer (Finnigan
`MAT TSQ45, INCOS data system) scanning m/z 35-950 in
`33cc. Source conditions for electron impact ionization were
`70 eV electron energy, 0.3 mA emission current and 190°C.
`samples were volatilized from the solids probe at ~330°C
`during a programmed linear temperature gradient of 50°C
`per min.
`.
`.
`.
`The relative intensities of some characteristic peaks for
`the lutein standard were as follows (literature values (Heller
`and Milne, 1978) are in parentheses): m/z 568:28 (23); m/z
`550:4l (31); m/z 476:not detected (8); m/z 1192100 (70); m/z
`91:53 (100). For the zeaxanthin standard, the values were:
`m/z 5682100 (100); m/z 550:5 (12); m/z 476:43 (34); m/z
`[19:85 (92).
`
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