A growing body of evidence indicates that carotenoids protect the retina against damage by light and against risk of severe vision loss from age-related macular degeneration (reviewed by Snodderly 1995). The macula lutea (Latin for yellow spot) acquires its color because the retina accumulates two carotenoids, the dihydroxy xanthophylls lutein and zeaxanthin (Bone et al. 1985). Other carotenoids that are plentiful in the blood, including -carotene, are found in the macula only in trace amounts (Handelman et al. 1988 and 1992).

The distribution of carotenoids in the retina is very specific. The highest density occurs at the center of the fovea (Bone et al. 1988, Snodderly et al. 1984a and 1984b), where zeaxanthin is the dominant carotenoid (Bone et al. 1988, Snodderly et al. 1991). However, the concentration of zeaxanthin drops off rapidly with distance so that lutein becomes the dominant carotenoid outside the foveal center (Bone et al. 1988, Snodderly et al. 1991). The preferential accumulation of zeaxanthin in the foveal center is particularly intriguing in view of the fact that zeaxanthin is present in much lower concentrations than lutein in the blood (Krinsky et al. 1990, Snodderly et al. 1990). There are two possible mechanisms by which this situation might arise, and they are not mutually exclusive. Either zeaxanthin is taken up more avidly by the central retina, or the retina may be able to convert the more abundant lutein into a particular stereoisomer of zeaxanthin, meso-zeaxanthin (Bone et al. 1993).

Little is known about the absorption of zeaxanthin from the diet, although short-term dietary supplementation of humans with pure zeaxanthin has been shown to be effective in increasing plasma concentrations (Khachik et al. 1995). One question that does not appear to have been addressed is whether adding zeaxanthin to the diet could inhibit absorption of other carotenoids, as is thought to happen when -carotene is provided as a supplement (Kostic et al. 1995, Micozzi et al. 1992).

In this study, we examined the influence of dietary manipulation on plasma carotenoids of squirrel monkeys (Saimiri sciureus), New World monkeys that have a macula lutea similar in many respects to that of humans.

MATERIALS AND METHODS

To maximize the scientific information obtained from these animals, several experiments were conducted concurrently. Two monkeys (S27 and S28) had no other experimental interventions during the period of this study. Three monkeys (S29, S30, and S31) had single vitrectomies; the vitreous body was surgically removed from one eye. The space created was filled with silicone oil in two cases, and with a physiological salt solution in the third. The second eye was not disturbed. The similarity of results from the vitrectomized animals to those of the other animals indicates that the surgery did not affect our data. All procedures conformed to the ARVO Resolution on the Use of Animals in Research.

To establish baseline plasma carotenoid concentrations before depletion, three blood samples were taken from five squirrel monkeys fed the basal nonpurified diet supplemented with low carotenoid fruits. The batches of nonpurified diet used had total carotenoid concentrations of 24-30 mg/kg. During the 6-wk depletion period, monkeys were fed the low carotenoid diet containing only 2 mg carotenoids/kg, supplemented with the same low carotenoid fruits. Starting 2 wk after the low-carotenoid diet was begun, three blood samples were taken at 2-wk intervals to monitor changes in plasma carotenoids.

Dietary manipulation of the remaining monkeys (S27 and S28) was resumed, and plasma carotenoids were depleted again, followed by successful supplementation with carotenoids in gelatin beadlets. For supplementation, separate preparations of zeaxanthin and -carotene in gelatin beadlets were obtained from Hoffmann-La Roche, courtesy of Dr. Peter Sorter in Nutley, NJ. Zeaxanthin can have multiple stereoisomers, but the beadlets contained 99% 3R, 3R (W. Schalch, Hoffmann-La Roche, Basel, Switzerland, personal communication) which is the same isomer found in plasma and in usual dietary sources (Bone et al. 1993). In preparation for feeding, the beadlets were weighed in metal weighing pans and handled with metal spatulas to prevent dispersion by static electricity. Beadlets containing zeaxanthin and -carotene (10 mg of each preparation) were then loaded into the interior of a small (500 mg) white marshmallow, so that both carotenoids were consumed simultaneously.

To monitor precisely the intake of the monkeys, dry biscuits of the basal nonpurified diet were available for 4 hr/d, and at the end of that period any biscuits not consumed were removed. Carotenoid concentrations in the main batch of basal diet (used from week 64 through week 87) were measured at four different times, and the later basal diet batches were measured twice (for week 88 through week 96) or once (for week 97 through week 99).

Marshmallows containing the carotenoid supplements were fed to the monkeys before daily access to food, and the monkeys eagerly and completely consumed them. Two to four marshmallows were sufficient to deliver the required supplemental amounts of carotenoids. To maintain a constant marshmallow intake, eight marshmallows were given each day, and the number containing carotenoids was varied to achieve the desired level of carotenoid supplementation.

To verify the effects of wide-range manipulation, we also obtained plasma data from the sixth monkey, S33. Prior to our experiments, this animal had been fed a semipurified diet having little or no carotenoids for several years (exact number unknown) for nutritional study of plasma lipids. This constituted a depletion period which was followed by a period of feeding the basal nonpurified diet and a supplementation period. The semipurified diet had a higher fat concentration (80 g/kg diet, fat derived entirely from corn oil) than our basal nonpurified diet (50 g/kg). Because this animal was a relatively good absorber of carotenoids, his previous prolonged period of higher fat intake may be relevant.

The various dietary regimens used to control carotenoid intake were well tolerated by the monkeys. Body weights were stable and did not change by more than 2-3% from the mean value at any time.

Carotenoids were quantified by HPLC as previously described (Krinsky et al. 1990, Snodderly et al. 1990), with slight modifications to facilitate measurement of geometrical isomers. A calibrated standard was used so that measurements in the range of nmol/L of each carotenoid could be made. For determination of individual isomers, peak heights were measured manually to minimize interference from overlapping peaks, and values were adjusted according to the elution time and the extinction coefficient for each isomer (Krinsky et al. 1990, Snodderly et al. 1990). All-trans-zeaxanthin and 9-cis-zeaxanthin were measured by their absorbance at 450 nm. For 9-cis-zeaxanthin, the molar extinction coefficient was taken to be 130,000 L/mol·cm, as inferred from data on 9-cis--carotene (Tsukida et al. 1982), which has the same chromophore. The 13-cis isomers of lutein and zeaxanthin were measured by peak heights at 325 nm. For 13-cis-zeaxanthin, this helped to minimize occasional interference from a chemical artifact as described below.

To aid in the quantification of the 13-cis isomers, and to eliminate artifacts, we used chromatograms recorded simultaneously at visible (450 nm) and at ultraviolet (325 nm) wavelengths. Figure 1B illustrates the temporal segment of the chromatograms containing the cis isomers. We found that some extractions (Fig. 1B, basal diet) contained a contaminant (arrow) that eluted just after the 13-cis-zeaxanthin peak and could not be separated from it in the 450 nm chromatogram. This contaminant appeared to be present in some of the stock chemicals, because it was also seen when a pure lutein standard was run on the same day. However, the contaminant did not absorb in the UV, so its contribution was eliminated by quantifying 13-cis-zeaxanthin from the UV trace.

The 9-cis isomers are present in small amounts. In some chromatograms (basal diet), a small, separate peak of 9-cis-lutein was evident, but when a large amount of zeaxanthin was present, especially during supplementation, the 9-cis-lutein could be incorporated into the all-trans-zeaxanthin peak as a slight change in the slope of the trailing edge (lower traces). Under these circumstances, 9-cis-lutein could not be measured and would be recorded as 0, which resulted in a small error in the total amount of lutein present. Confounding of the measurement of all-trans-zeaxanthin was minimized, however, by using height of the all-trans-zeaxanthin peak, not area, for quantitation.

Because saponification can cause some cis-trans isomerization, separate portions of the nonpurified dry diets were extracted with methanol followed by methanol:acetonitrile:tetrahydrofuran (5:4:1) and analyzed by HPLC to quantify the geometrical isomers of lutein and zeaxanthin. Because we did not expect complete recovery with this relatively gentle procedure, the quantities of all-trans-zeaxanthin and the cis-isomers of lutein and zeaxanthin were measured in the nonsaponified extracts as ratios of the all-trans-lutein in the sample. The isomeric composition of the zeaxanthin was also determined to be predominantly all-trans, with 9.5% as the 13-cis isomer.

The -carotene beadlet preparation was extracted with a mixture of methanol, tetrahydrofuran and methylene chloride, followed by HPLC analysis. It contained approximately 85% all-trans- and 13% 13-cis--carotene. In addition, a small peak accounting for about 2% of the total eluted before the all-trans--carotene; this fraction had the retention time and the absorbance spectrum of -carotene.

RESULTS

Our main focus is the dihydroxy xanthophylls, lutein and zeaxanthin, that are selectively accumulated by the retina to form the macular pigment (e.g. Bone et al. 1988, Handelman et al. 1992, Snodderly et al. 1991). The region of the chromatogram surrounding the main lutein and zeaxanthin peaks (Fig. 1B) also includes several smaller peaks. Those preceding the all-trans-lutein peak are carotenoid oxidation products (Khachik et al. 1991a and 1992a) that are not considered in this study. We have identified the small peaks after all-trans-lutein and all-trans-zeaxanthin as the 9-cis and the 13-cis isomers of these carotenoids (Krinsky et al. 1990, Snodderly et al. 1990, confirmed by Khachik et al. 1992b). For the first experiment, only the all-trans carotenoids were measured from the peak integration routine of the HPLC instrument, and the smaller contributions of the cis isomers were ignored.

Depletion of plasma carotenoids

This depletion experiment demonstrated that plasma carotenoids of squirrel monkeys respond rapidly to dietary changes. It was also valuable because it identified the two monkeys (S27 and S28) that were available for further study as the highest and lowest members of the group. The differences between the two were consistent, and their individual values did not overlap. Furthermore, the mean plasma all-trans-zeaxanthin concentrations for these two were virtually identical to the means calculated for all five monkeys (solid line).

Effects of wide-range manipulation of dietary carotenoids

To provide as stable a baseline as possible, a large batch of the nonpurified basal diet was frozen at the beginning of the sequence and stored at 20°C. Unfortunately, this batch was exhausted by the time of the high supplement (Sup+), and other batches of the basal diet had to be used starting with week 88. The new batches had higher carotenoid concentration, which is reflected in the step in the diet curve for total lutein (Ltot) at week 88 (Fig. 3A). During the high supplement period, the influence of the basal nonpurified diet on total intake of zeaxanthin was minor because zeaxanthin intake was dominated by the supplement, and the effect on dietary intake of the change in zeaxanthin in the basal diet is only apparent in the final basal period.

The intake of zeaxanthin indicated on the graph ignores variations of up to 20% due to loss of carotenoids during storage and fluctuations in the amount of diet consumed. Because our depletion and supplementation regimens changed dietary intake of zeaxanthin and -carotene from basal amounts by more than a factor of 2, small fluctuations in the dietary conditions have been ignored for simplicity of graphical presentation. In critical numerical comparisons, such as the dose-response relationship (Fig. 4), the small variations in dietary intake are accounted for.

The third monkey (S33) entered the supplementation study late, at week 75, and was immediately fed the same basal diet as the other monkeys. Prior to that time this monkey had been fed a semipurified diet with negligible carotenoid concentration (see Methods). To compare his plasma data with data from the other monkeys, we have shifted his values on the time axis. That is, his data (squares) are plotted as if he ended his low carotenoid feeding period at the same time as the other monkeys. However, S33 continued eating the basal diet for 2.5 mo (data not shown) instead of receiving a moderate supplement. This was to allow a stable eating pattern to be established after switching from the long period of consuming a very different diet. When he had fully adapted to the basal diet, he was given the high supplement at week 91 for 6 wk and then returned to basal conditions. Again, for comparability, his data are shifted on the time axis of the graph as if he had started the high supplement at the same time as the other two monkeys.

For all three monkeys, the data show that plasma responded rapidly to all changes in dietary zeaxanthin intake, increases as well as decreases. Within two weeks of a dietary change, plasma zeaxanthin concentrations reached a new, relatively stable level. In fact, for each monkey, plasma concentrations in the depletion and in the supplementation periods do not overlap. It is also interesting that, with only two exceptions, the distinctive difference between monkeys S27 and S28 was maintained at each time point, with S27 having the higher value.

Even the smaller differences among plasma concentrations in the basal periods probably reflect differences in carotenoid intake. For example, the main basal diet used until week 88 had a relatively low carotenoid concentration compared to the diet used before the dietary sequence and the diet used to end the sequence. Consequently, plasma zeaxanthin values are slightly lower in the second basal period from week 72 to week 76 than at the very beginning or at the end. These data imply a very tight relationship between the plasma zeaxanthin concentration and dietary intake of zeaxanthin. Because all three monkeys responded similarly to the dietary manipulations, their data have been averaged for subsequent figures, using the temporal adjustments for monkey S33 shown in Fig. 3.

Plasma response: -Carotene and -Carotene.

During the moderate supplement period, the seven plasma samples taken from monkeys S27 and S28 showed small increases in -carotene and -carotene concentrations when compared with the basal diet period (data not shown). For each monkey, however, one value for -carotene was more than six-fold higher than all the rest. We believe these outliers were due to contamination from the -carotene standard that was used during the HPLC analyses and we have excluded them from further consideration. The remaining data from the moderate supplement period and data from the high supplement period indicate that in spite of receiving more than twice as much -carotene as zeaxanthin in the diet, the plasma concentrations of -carotene were less than one-tenth the concentrations of zeaxanthin.

Another striking outcome is that all monkeys appeared, on the basis of retention times, to have a disproportionate amount of -carotene in the plasma compared to the diet. This was particularly apparent in the high supplement period, when there was more -carotene than -carotene in the plasma (Fig. 1A) even though there was only about 2% as much -carotene as -carotene in the diet. This conclusion must be tempered by the fact that there was too little carotene in the blood to obtain spectral confirmation of the identity of the peaks.

Plasma response: Lutein and -Cryptoxanthin.

To examine more closely the plasma responses to dietary manipulation, we calculated for each plasma sample the ratio of lutein and zeaxanthin concentrations to the mean plasma concentrations during consumption of the relevant batch of basal diet (Fig. 4). This allowed us to take into account the fact that the basal diet changed at week 88. If the plasma values were not strongly affected by supplementation, the ratio should remain close to 1. For total lutein, this prediction was verified. It is particularly striking that plasma total lutein was not noticeably different during the high supplement from the value during the final basal period. At the same time plasma total zeaxanthin changed by more than a factor of two. This strongly suggests that zeaxanthin supplementation did not inhibit uptake of lutein from the diet.

DISCUSSION

Comparison data for humans are limited, but they show that plasma zeaxanthin and lutein of males are quite responsive to dietary manipulation. For example, both zeaxanthin and lutein in plasma increased 4- to 6-fold within 2-3 wk of dietary supplementation with 10 mg/d of each pigment (Khachik et al. 1995). Furthermore, after withdrawing supplementation, the plasma concentrations of lutein and zeaxanthin dropped rapidly. However, in another study, plasma zeaxanthin + lutein was reported to decline relatively slowly after beginning a low carotenoid diet, with an estimated half-life of 33-61 d (Rock et al. 1992). This slower decline in humans than in monkeys may be due to more extensive tissue stores in humans.

We cannot exclude the possibility that -carotene absorption was inhibited by simultaneously supplementing with zeaxanthin. Such inhibitory interactions have been observed between -carotene and other carotenoids in chicks (Lim et al. 1992) and in humans (Kostic et al. 1995, Micozzi et al. 1992). However, the inhibition would require extreme structural specificity to explain poor -carotene absorption in the present case. Lutein is a structural isomer of zeaxanthin, and yet its absorption (Fig. 4), as well as the absorption of -cryptoxanthin, was unaffected.

An unexpected finding was an apparent accumulation of -carotene in the plasma during the high supplement period. Because only 2% of the nominal -carotene supplement was -carotene, this suggests that another form of specificity may be involved. We have previously shown (Snodderly et al. 1990) for both the monohydroxy and the dihydroxy xanthophylls, that molecules based on the - ring configuration (lutein and -cryptoxanthin) are accumulated by monkeys from the diet in preference to the isomers based on the - configuration (zeaxanthin and -cryptoxanthin). Because -carotene has the - ring, it may be similarly preferred over -carotene.

Given the evidence (Snodderly 1995) for protection from a blinding disease by lutein and zeaxanthin, the retinal carotenoids, our motivation for the present study was to examine the prospect of making these carotenoids more readily available to the retina by controlled dietary supplementation. Our results suggest that this is quite feasible, and that one may be able to manipulate plasma concentrations of each of the retinal carotenoids with little impact on the other. This will facilitate exploration of the rates of accumulation of lutein and of zeaxanthin in the retina, as well as exploration of the possibility of bioconversion of lutein to meso-zeaxanthin (Bone et al. 1993). These experiments with monkeys indicate that it should be possible to increase plasma and retinal concentrations of the macular xanthophylls, lutein and zeaxanthin, by dietary control in humans at risk for retinal disease.