Human and Clinical Nutrition
uptake of Lycopene and Its Geometrical Isomers Is Greater from Heat-Processed than from unprocessed Tomato Juice in Humans1 WILHELM STAHL AND HELMUT Institut fürPhysiologische
SÕES2
Chemie I, Universität Düsseldorf, W-4000 Düsseldorf, Germany same extent or even more than ß-carotene to total carotenoids in human tissues (Parker 1989, Schmitz et al. 1991). Little information is available on the absorption, distribution and metabolism (biokinetics) of this compound (Mathews-Roth et al. 1990, Snodderly et al. 1990). In recent studies, differences were found in the organ distribution of lycopene and ßcarotene. Lycopene was present in relatively high concentrations in testes compared with other tissues such as liver or adrenal glands (Kaplan et al. 1990, Stahl et al. 1992), and it occurs in the form of several geometrical isomers (Krinsky et al. 1990b, Stahl et al. 1992), which might also possess different biological properties. Here we studied the uptake of lycopene from a dietary source.
ABSTRACT Lycopene and ß-caroteneare the most abundant carotenoids ¡nhuman blood and tissues. Although lacking provitamin A activity, lycopene may be biologically active by contributing to the antioxidative defense system of the organism. We studied the uptake of lycopene from processed (boiled with 1% corn oil for 1 h) and unprocessed tomato juice in humans. Lycopene concentrations in human serum increased only when processed tomato juice was consumed. Lycopene uptake varied with individuals, but peak serum concen trations were always reached between 24 and 48 h. The carotenoid was eliminated from serum with a half-life of 2-3 d. The increase in peak serum concentrations was dose-dependent but not linear with the dose. Repeated doses led to a continual rise of lycopene in human serum. Of the different geometrical isomers (all-trans, 9-c/s and 13-cis), the c/s isomers seemed to be somewhat better absorbed than the all-trans form. J. Mutr. 122: 2161-2166, 1992.
MATERIALS AND METHODS INDEXING KEY WORDS:
•lycopene •carotenoids •biokinetics •cis-trans isomers •humans
Since carotenoids have been shown to be effective in the prevention of several types of cancer, interest has grown in their biological activity beyond their importance as provitamin A (Black and MathewsRoth 1991, Peto et al. 1981, Pung et al. 1989, Ziegler 1989). Although the biochemical mechanisms under lying these cancer-preventing properties are still unknown, it has been suggested that quenching of harmful reactive oxygen species might be involved in such processes (Burton and Ingold 1984, Foote and Denny 1968, Krinsky 1989). In vitro studies proved that some naturally occurring (Di Mascio et al. 1989) and synthetic carotenoids (Devasagayam et al. 1992) without provitamin A activity are superior to ßcarotene in quenching singlet oxygen. Lycopene was found to be especially active (Conn et al. 1991, Di Mascio et al. 1989). This carotenoid contributes to the 0022-3166/92 $3.00
Reagents. All-trans ß-carotene, a-carotene, lycopene, cryptoxanthin and canthaxanthin were kind gifts from J. Bausch and H. E. Keller (Hoffmann-La Roche Co. Ltd., Basel, Switzerland). Canthaxanthin2,4-dinitrophenylhydrazone was synthesized as described earlier (Stahl et al. 1992). All other chem icals were at least analytical grade and obtained from E. Merck (Darmstadt, Germany). Tomato juice and corn oil were purchased at local distributors. Tomato juice with 1% corn oil (by volume) was heated under stirring at 100°Cfor 1 h; the mixture was allowed to cool in the dark at 5°C,and 5 mL was withdrawn for carotenoid analysis. Unprocessed tomato juice was prepared by stirring it with 1% corn oil for 1 h at room temperature. Study design. Six healthy adults (22 to 36 y old, one female and five male) took part in the study.
1Supported by the Ministerium für Wissenschaft und Forschung Nordrhein-Westfalen and by the Bundesministerium für Forschung und Technologie, Bonn. 2To whom correspondence should be addressed.
1992 American Institute of Nutrition. Received 17 March 1992. Accepted 2 July 1992. 2161
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Written consent was obtained from each participant. Each consumed defined amounts of tomato juice within a 2-h period beginning after the first blood sample (basal value) was collected. Depending on the desired dose and the concentration of lycopene in the tomato juice, the participants consumed between 200 and 700 mL. For absorption studies three individuals consumed heated tomato juice equivalent to a single lycopene dose of 2.5 umol/kg body wt. Two subjects consumed unheated tomato juice and two subjects (controls) did not consume tomato juice. To determine dose dependence of lycopene ab sorption, one person consumed various amounts of heated tomato juice equivalent to single lycopene doses of 0.35, 1.25 and 2.5 umol/kg body wt in three different experiments. To determine accumulation of lycopene, a single person repeatedly consumed heated tomato juice equivalent to repeated lycopene doses of 0.35 umol/kg body wt. Tomato juice was consumed once a day for a 4-d period. During the studies the participants consumed a diet low in carotenoids. All participants were advised to avoid tomatoes and tomato products in particular, and other colored vegetables and fruits. Compliance was monitored by daily interview. Such a diet lo wered serum lycopene concentrations in the two sub jects who did not consume tomato juice (controls). Sample preparation. Blood samples were collected in syringes (without anticoagulants) daily at 1400 h and centrifuged for 15 min at 2800 x g to obtain serum. The serum was stored at -70°C until analysis. Serum (500 uL) was diluted 1:1 with buffer and the carotenoids extracted into hexane-dichloromethane as previously described in detail (Stahl et al. 1992). The organic phase was evaporated under nitrogen in the dark. Chromatography. The residues were dissolved in 100-200 uL of HPLC eluent directly before analysis. If necessary, 20 uL of dichloromethane was added to obtain a clear solution. The HPLC analysis was per formed on a 5-um RP 18 endcapped column (4 x 250 mm) (E. Merck) with methanol-acetonitrile-dichloromethane-water (7:7:2:0.16), flow rate of 1 mL/min and detection at 460 nm. The solvent was degassed by sonification. A diode array detector (model 168, Beckman, Munich, Germany) was used for spectrophotometric peak identification. Calculations. Carotenoid concentrations in samples were calculated from a calibration curve generated from peak height ratios of the carotenoids to an internal standard or peak heights of carotenoids in calibration samples. The intra-assay precision varied between serum samples, with the coefficient of variation ranging from 5 to 15%. The recovery of internal standard and carotenoids was -90%, and the detection limit was -10 nmol/L serum. Downloaded from https://academic.oup.com/jn/article-abstract/122/11/2161/4754717 by guest on 22 January 2018
m
8
CD "En
B
I
O CO
a o o
O
—I— 10 Time(min)
—I— 20
OS O
o
tÃo
ID
1
10 Time (min)
20
FIGURE 1 HPLC-chromatograms of unheated (A) or heated (B) tomato juice and of a human serum sample 24 h after consumption of heated tomato juice (C). Standard:
ABSORPTION
I
01
2163
OF LYCOPENE IN HUMANS
234567
2
3 /, Time (days)
5
6
7
FIGURE 3 Increase in lycopene concentrations over the basal value after the consumption of different amounts of heated tomato juice equivalent to lycopene doses 0.35 (A), 1.25 («)and 2.5 (A) umol/kg body wt by a single subject.
Ti me (days) FIGURE 2 Serum concentrations of total lycopene after the consumption of heated (•) or unheated (o) tomato juice by five subjects. The lycopene dose was 2.5 (¿mol/kgbody wt.
For calculation of the area under the curve (AUC), the lycopene concentrations at the start of the experi ments (i.e., basal levels) were subtracted from the lycopene concentrations determined at later time points ([Ly]t- [Ly]o).The AUC values were calculated according to the trapezoidal rule. The half-life of lycopene in serum was calculated as the time in which the maximum serum concentration of lycopene decreased to half of the starting value.
RESULTS Influence of food processing on lycopene serum concentrations. To study the uptake of lycopene and its geometrical isomers from the diet, tomato juice was selected as a source. Tomato juice contains 50-120 mg/L of lycopene, which occurs mainly in the all-trans configuration (Fig. IA). Heating the juice induces trans-to-cis isomerization. After l h at 100°C the isomerie composition of the carotenoid had changed, and 20-30% of total lycopene consisted of cis-isomers (Fig. Iß).As previously described (Stahl et al. 1992), two isomers have been tentatively assigned by retention times and diode array data as 9- and 13-cis lycopene. However, other geometrical isomers likely are present and co-elute with these peaks. A typical chromatogram of carotenoids in human serum samples is presented in Figure 1C. Serum con centrations of total lycopene obtained from different individuals after the consumption of heated or unDownloaded from https://academic.oup.com/jn/article-abstract/122/11/2161/4754717 by guest on 22 January 2018
heated tomato juice are shown in Figure 2. The intake of unheated tomato juice did not increase serum lycopene concentrations; similar to results in the con trols (data not shown), the serum concentrations decreased with the time. The consumption of heated tomato juice, however, was followed by an increase of total lycopene concentrations in the serum (Fig. 2). To ensure an increase in serum lycopene, all other ex periments were performed with heated tomato juice. Absorption of total lycopene and individual differ ences. For individuals consuming heated tomato juice (see Fig. 2), peak serum concentrations of total lycopene were observed at 24-48 h after intake. The half-life (ti/2)of lycopene in human serum was be tween 2 and 3 d. The data showed that there are interindividual differences in lycopene uptake from a dietary source. After consumption of the same amount of lycopene, increases in serum lycopene con centrations ranged from 80 to 350 nmol/L over the basal concentration. Dose-dependence and accumulation. Data on dosedependence of lycopene uptake from a dietary source are presented in Figure 3. After a dose of 0.35 umol/kg body wt, total lycopene in serum was elevated 85 nmol/L above the basal level. Higher doses of lycopene raised the serum concentrations by -170 nmol/L (1.25 umol/kg body wt) and 260 nmol/L (2.5 umol/kg body wt) over the basal level. The AUC values also increased with the dose from 6.2 umol L"1 h at 0.35 umol/kg body wt and 11.5 umol L"1 h at 1.25 umol/kg body wt to 18.5 umol L"1 h at 2.5 umol/ kg body wt. The uptake of lycopene was dose de pendent but not strictly linear with dose. Relatively more lycopene was absorbed when lower amounts were consumed. Serum concentrations and AUC values increased only twofold when the dose was raised 3.5-fold from 0.35 to 1.25 umol/kg body wt. A
STAHL AND SÕES
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13-cis
O 1D 1
2
1
1
2i Ti me (days)
3i
U
FIGURE 4 Increase in total lycopene and its isomers after repeated consumption of heated tomato juice (0.35 umol lycopene/kg body wt) by one subject. Arrows indicate time of consumption.
further twofold increase of the dose from 1.25 to 2.5 umol/kg body wt led to only a 1.5-fold rise in both lycopene serum concentrations and AUC values. Figure 4 shows the elevation of serum total lycopene and three lycopene isomers (all-trans, 9-cis and 13-cis) after repeated consumption of heated tomato juice providing 0.35 umol lycopene/kg body wt each time. The accumulation of total lycopene and lycopene isomers in serum was nearly linear. The total dose was 1.4 umol/kg body wt and led to an increase of total lycopene by 320 nmol/L over the base level. This is a similar increase in total lycopene found with the same individual after the intake of a single dose of 2.5 umol/kg body wt (350 nmol/L), emphasizing that lower doses are relatively absorbed better than higher ones. These results were confirmed in a second experiment with another subject (data not shown). Absorption of different lycopene isomers. Figure 5 shows an example of the serum concentrations of total lycopene and its isomers ali-trans, 9-cis and 13-cis after intake of heated tomato juice. The amount of tomato juice was equivalent to a dose of 2.5 umol total lycopene/kg body wt. The carotenoid was composed of 80% all-trans, 5% 9-cis and 15% 13-cis lycopene, corresponding to doses of 2 umol alltrans, 0.13 umol 9-cis and 0.38 umol 13-cis/kg body wt. All isomers reached the highest concentrations in serum after 24-48 h. Total lycopene increased by 260 nmol/L, all-trans by 170 nmol/L, 9-cis by 60 nmol/L and 13-cis by 30 nmol/L over the basal level. It seemed that, given their concentrations in the tomato Downloaded from https://academic.oup.com/jn/article-abstract/122/11/2161/4754717 by guest on 22 January 2018
~r~ —i— —r 3 4 5 6 Time (days)
7
FIGURE 5 Serum concentrations of total lycopene and its isomers after consumption of a single dose of 2.5 umol/ kg body wt of lycopene in heated tomato juice by one subject.
juice, the cis isomers were absorbed slightly better, or were metabolized to a lesser extent, than all-trans lycopene (see Fig. 5). Other carotenoids. The serum concentrations of cccarotene, ß-carotene and canthaxanthin were also de termined in the present study. In all experiments a continuous loss of all three compounds was observed. This likely was due to the consumption of a carotenoid-poor diet and may also be an indicator of participants' compliance.
DISCUSSION Several studies have been reported on the ab sorption, distribution and metabolism of ß-carotene and canthaxanthin in different animal species and in humans (Brown et al. 1989; Krinsky et al. 1990a, Mathews-Roth 1990, Prince et al. 1991, RibayaMercado et al. 1989, Shapiro et al. 1984). Pure carote noids in various supplements as well as fruits and vegetables as dietary sources have been used to study carotenoid biokinetics. 14C-Labeled lycopene has been applied to monkeys for investigations on the biokinetics (Mathews-Roth et al. 1990). Unfor tunately, no pure lycopene is available that can be used in human studies according to the Helsinki Con ference Conventions. We therefore took tomato juice as a lycopene source, because tomatoes are among the vegetables with the highest lycopene content. No increase in lycopene serum concentrations, however, was found after the intake of 700 mL of juice (130 umol of lycopene), which is in agreement with a previous study in which the consumption of tomato
ABSORPTION
OF LYCOPENE IN HUMANS
juice (containing 22 (¿molof lycopene) did not in crease plasma concentrations of that compound (Brown et al. 1989). It has been reported that processing of food in fluences the availability of carotenoids from the diet (Erdman 1988); especially, mild cooking is thought to contribute to availability. We found this to be true for lycopene from tomato juice. Heating a mixture of juice and 1% corn oil for l h before consumption led to an increase in lycopene serum concentrations. Many different factors can influence the absorption of carotenoids from the diet, so that it remains to be studied which particular process related to heating improves the availability of lycopene. Possible mechanisms might be thermally induced rupture of cell walls accompanied by release of lycopene from the cells, heat-improved extraction of lycopene into the lipophilic corn oil as vehicle, or a combination of several other factors. Regardless of the mechanism, heating with small amounts of lipophilic oil improves absorption of lycopene from the diet, as is known for ß-carotene(Erdman 1988). This should be kept in mind when interpreting epidemiological studies on nutritional habits. Absorption of lycopene from the diet is influenced additionally by other factors. As described for ß-carotene(Brown et al. 1989), the in crease in lycopene serum concentrations differed be tween individuals consuming amounts of tomato juice equivalent to equal doses of lycopene based on body weight. These differences in absorption presumably con tribute to the inter-individual differences in the plasma concentrations that have been described in several studies (Krinsky et al. 1990b, Parker 1989, Stahl et al. 1992). Differences in LDL assembly could also be of importance in this context. At low doses the efficiency of lycopene absorption from the diet is higher than with higher doses, as described for ß-carotene(Brown et al. 1989, Erdman 1988). Two biokinetic parameters of lycopene determined in this study are maximum concentration of lycopene at 24-48 h and elimination of the compound from serum with a half-life of 2-3 d. These cmaxvalues in humans are within the range found in a study with monkeys (8-48 h) in which, however, only peak radi oactivity in plasma after application of labeled lycopene was measured. In rats, peak plasma radioac tivity was determined between 4 and 8 h, which is possibly due to substantially different biokinetics of carotenoids compared with primates (Mathews-Roth et al. 1990). In a study with ß-carotenefrom dietary (carrots) or synthetic sources, cmaxvalues were found between 24 and 48 h after consumption (Brown et al. 1989). The elimination from plasma was somewhat slower (4-8 d) than the time reported here for lycopene (2-3 d). Although these studies were different in design, one Downloaded from https://academic.oup.com/jn/article-abstract/122/11/2161/4754717 by guest on 22 January 2018
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might conclude that lycopene and ß-caroteneare similarly absorbed from the diet but differently dis tributed, accumulated, metabolized and/or excreted. Several reports exist on differences in the availa bility and the accumulation of geometrical isomers of ß-carotene.In rat and chicken livers a higher accumu lation of 9-cis ß-carotenewas observed compared with the all-trans isomer (Ben-Amotz et al. 1989, Mokady et al. 1990). In humans, however, a predominant ab sorption of all-trans ß-carotenewas described (Jensen et al. 1987). With lycopene we found better availa bility of the cis isomers compared with the all-irans compound. This difference might be a result of different amounts of isomers used in our study, whereas Jensen et al. (1987) applied approximately equal doses of the ß-caroteneisomers. We are aware, however, that other factors difficult to control in studies with humans and food as a source of carote noids might influence the absorption of lycopene and its isomers. Although the processed tomato juice contained only minor amounts of 9-CJ'slycopene, the concen tration of this isomer also increased in serum. From that point of view one might speculate that there are in-vivo isomerization mechanisms, possibly enzyme mediated.
ACKNOWLEDGMENT The technical assistance by Elisabeth Bloch is gratefully acknowledged.
LITERATURECITED Ben-Amotz, A., Mokady, S., Edelstein, S. & Avron, M. (1989) Bioavailability of a natural isomer mixture as compared with synthetic all-trans-ß-carotene in rats and chicks. J. Nutr. 119: 1013-1019. Black, H. S. & Mathews-Roth, M. M. (1991) Protective role of butylated hydroxytoluene and certain carotenoids in photocarcinogenesis. Photochem. Photobiol. 53: 707-716. Brown, E. D., Micozzi, M. S., Craft, N. E., Bieri, J. G., Beecher, G., Edwards, B. K., Rose, A., Taylor, P. R. & Smith, }. C. (1989) Plasma carotenoids in normal men after a single ingestion of vegetables or purified ß-carotene. Am. J. Clin. Nutr. 49: 1258-1265. Burton, G. W. & Ingold, K. U. (1984) ß-Carotene:an unusual type of lipid antioxidant. Science (Washington, DC) 224: 569-573. Conn, P. F., Schalen, W. & Truscott, T. G. (1991) The singlet oxygen and carotenoid interaction. J. Photochem. Photobiol. B. 11: 41-47. Devasagayam, T.P.A., Werner, T., Ippendorf, H., Martin, H. D. & Sies, H. (1992) Synthetic carotenoids, novel polyene polyketones and new capsorubin isomers as efficient quenchers of singlet molecular oxygen. Photochem. Photobiol. 55: 511-514. Di Mascio, P., Kaiser, S. & Sies, H. (1989) Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch. Biochem. Biophys. 274: 532-538. Erdman, f. (1988) The physiologic chemistry of carotenes in man. Clin. Nutr. 7: 101-106.
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Foote, C. S. & Denny, R. W. (1968) Chemistry of singlet oxygen. VII. Quenching by ß-carotene. I. Am. Chem. Soc. 90: 6233-6235. Jensen, C. D., Howes, T. W., Spiller, G. A., Pattison, T. S., Whittam, ]. H. & Scala, J. (1987) Observations on the effects of ingesting cj's- and frans-beta-carotene isomers on human serum concentrations. Nutr. Rep. Int. 35: 413-422. Kaplan, L. A., Lau, J. M. & Stein, E. A. (1990) Carotenoid compo sition, concentrations, and relationships in various human organs. Clin. Physiol. Biochem. 8: 1-10. Krinsky, N. I. (1989) Antioxidant functions of carotenoids. Free Radical Biol. & Med. 7: 617-635. Krinsky, N. I., Mathews-Roth, M. M., Welankiwar, S., Sehgal, P. K., Lausen, N.C.G. & Russen, M. (1990a| The metabolism of [14C]ß-carotene and the presence of other carotenoids in rats and monkeys. J. Nutr. 120: 81-87. Krinsky, N. L, Russe«,M. D., Handelman, G. J. & Snodderly, D. M. 11wob! Structural and geometrical isomers of carotenoids in human plasma. J. Nutr. 120: 1654-1662. Mathews-Roth, M. M. (1990) Plasma concentrations of carotenoids after large doses of ß-carotene.Am. J. Clin. Nutr. 52: 500-501. Mathews-Roth, M. M., Welankiwar, S., Sehgal, P. K., Lausen, N.C.G., Russe«,M. &. Krinsky, N. I. (1990) Distribution of [14C]canthaxanthin and |14C]lycopene in rats and monkeys. J. Nutr. 120: 1205-1213. Mokady, S., Avron, M. & Ben-Amotz, A. (1990) Accumulation in chick livers of 9-cj's versus M-trans ß-carotene.J. Nutr. 120: 889-892. Parker, R. S. (1989) Carotenoids in human blood and tissues. ). Nutr. 119: 101-104.
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Peto, R., Doll, R., Buckley, J. D. & Sporn, M. B. (1981) Can dietary beta-carotene materially reduce human cancer rates? Nature (Lond.) 290: 201-208. Prince, M. R., Frisoli, J. K., Goetschkes, M. M., Stringham, J. M. & LaMuraglia, G. M. (1991) Rapid serum carotene loading with high-dose ß-carotene:clinical implications. J. Cardiovasc. Pharmacol. 17: 343-347. Pung, A., Rundhaug, J. E., Yoshizawa, C. N. & Bertram, J. S. (1988) ß-Carotene and canthaxanthin inhibit chemically and physically induced neoplastic transformations in 10T1/2 cells. Carcinogenesis 9: 1533-1539. Ribaya-Mercado, J. D., Holmgren, S. C., Fox, J. G. & Russell, R. M. (1989) Dietary ß-caroteneabsorption and metabolism in ferrets and rats. J. Nutr. 119: 665-668. Schmilz, H. H., Poor, C. L., Wellman, R. B. & Erdman, ]. Vf., Jr. (1991) Concentrations of selected carotenoids and vitamin A in human liver, kidney and lung tissue. J. Nutr. 121: 1613-1621. Shapiro, S. S., Mo«,D. J. & Machlin, L. J. (1984) Kinetic charac teristics of ß-carotene uptake and depletion in rat tissue. J. Nutr. 114: 1924-1933. Snodderly, D. M., Russett, M. D., Land, R. I. & Krinsky, N. I. (1990) Plasma carotenoids of monkeys (Macaca fascicularis and SaimirÃ-sciureus] fed a nonpurified diet. J. Nutr. 120: 1663-1671. Stahl, W., Schwarz, W., Sundquist, A. R. & Sies, H. (1992) cis-trans Isomers of lycopene and ß-carotene in human serum and tissues. Arch. Biochem. Biophys. 294: 173-177. Ziegler, R. G. (1989) A review of epidemiologie evidence that carotenoids reduce the risk of cancer. J. Nutr. 119: 116-122.
uptake of Lycopene and Its Geometrical Isomers Is Greater from Heat-Processed than from unprocessed Tomato Juice in Humans1 WILHELM STAHL AND HELMUT Institut fürPhysiologische
SÕES2
Chemie I, Universität Düsseldorf, W-4000 Düsseldorf, Germany same extent or even more than ß-carotene to total carotenoids in human tissues (Parker 1989, Schmitz et al. 1991). Little information is available on the absorption, distribution and metabolism (biokinetics) of this compound (Mathews-Roth et al. 1990, Snodderly et al. 1990). In recent studies, differences were found in the organ distribution of lycopene and ßcarotene. Lycopene was present in relatively high concentrations in testes compared with other tissues such as liver or adrenal glands (Kaplan et al. 1990, Stahl et al. 1992), and it occurs in the form of several geometrical isomers (Krinsky et al. 1990b, Stahl et al. 1992), which might also possess different biological properties. Here we studied the uptake of lycopene from a dietary source.
ABSTRACT Lycopene and ß-caroteneare the most abundant carotenoids ¡nhuman blood and tissues. Although lacking provitamin A activity, lycopene may be biologically active by contributing to the antioxidative defense system of the organism. We studied the uptake of lycopene from processed (boiled with 1% corn oil for 1 h) and unprocessed tomato juice in humans. Lycopene concentrations in human serum increased only when processed tomato juice was consumed. Lycopene uptake varied with individuals, but peak serum concen trations were always reached between 24 and 48 h. The carotenoid was eliminated from serum with a half-life of 2-3 d. The increase in peak serum concentrations was dose-dependent but not linear with the dose. Repeated doses led to a continual rise of lycopene in human serum. Of the different geometrical isomers (all-trans, 9-c/s and 13-cis), the c/s isomers seemed to be somewhat better absorbed than the all-trans form. J. Mutr. 122: 2161-2166, 1992.
MATERIALS AND METHODS INDEXING KEY WORDS:
•lycopene •carotenoids •biokinetics •cis-trans isomers •humans
Since carotenoids have been shown to be effective in the prevention of several types of cancer, interest has grown in their biological activity beyond their importance as provitamin A (Black and MathewsRoth 1991, Peto et al. 1981, Pung et al. 1989, Ziegler 1989). Although the biochemical mechanisms under lying these cancer-preventing properties are still unknown, it has been suggested that quenching of harmful reactive oxygen species might be involved in such processes (Burton and Ingold 1984, Foote and Denny 1968, Krinsky 1989). In vitro studies proved that some naturally occurring (Di Mascio et al. 1989) and synthetic carotenoids (Devasagayam et al. 1992) without provitamin A activity are superior to ßcarotene in quenching singlet oxygen. Lycopene was found to be especially active (Conn et al. 1991, Di Mascio et al. 1989). This carotenoid contributes to the 0022-3166/92 $3.00
Reagents. All-trans ß-carotene, a-carotene, lycopene, cryptoxanthin and canthaxanthin were kind gifts from J. Bausch and H. E. Keller (Hoffmann-La Roche Co. Ltd., Basel, Switzerland). Canthaxanthin2,4-dinitrophenylhydrazone was synthesized as described earlier (Stahl et al. 1992). All other chem icals were at least analytical grade and obtained from E. Merck (Darmstadt, Germany). Tomato juice and corn oil were purchased at local distributors. Tomato juice with 1% corn oil (by volume) was heated under stirring at 100°Cfor 1 h; the mixture was allowed to cool in the dark at 5°C,and 5 mL was withdrawn for carotenoid analysis. Unprocessed tomato juice was prepared by stirring it with 1% corn oil for 1 h at room temperature. Study design. Six healthy adults (22 to 36 y old, one female and five male) took part in the study.
1Supported by the Ministerium für Wissenschaft und Forschung Nordrhein-Westfalen and by the Bundesministerium für Forschung und Technologie, Bonn. 2To whom correspondence should be addressed.
1992 American Institute of Nutrition. Received 17 March 1992. Accepted 2 July 1992. 2161
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Written consent was obtained from each participant. Each consumed defined amounts of tomato juice within a 2-h period beginning after the first blood sample (basal value) was collected. Depending on the desired dose and the concentration of lycopene in the tomato juice, the participants consumed between 200 and 700 mL. For absorption studies three individuals consumed heated tomato juice equivalent to a single lycopene dose of 2.5 umol/kg body wt. Two subjects consumed unheated tomato juice and two subjects (controls) did not consume tomato juice. To determine dose dependence of lycopene ab sorption, one person consumed various amounts of heated tomato juice equivalent to single lycopene doses of 0.35, 1.25 and 2.5 umol/kg body wt in three different experiments. To determine accumulation of lycopene, a single person repeatedly consumed heated tomato juice equivalent to repeated lycopene doses of 0.35 umol/kg body wt. Tomato juice was consumed once a day for a 4-d period. During the studies the participants consumed a diet low in carotenoids. All participants were advised to avoid tomatoes and tomato products in particular, and other colored vegetables and fruits. Compliance was monitored by daily interview. Such a diet lo wered serum lycopene concentrations in the two sub jects who did not consume tomato juice (controls). Sample preparation. Blood samples were collected in syringes (without anticoagulants) daily at 1400 h and centrifuged for 15 min at 2800 x g to obtain serum. The serum was stored at -70°C until analysis. Serum (500 uL) was diluted 1:1 with buffer and the carotenoids extracted into hexane-dichloromethane as previously described in detail (Stahl et al. 1992). The organic phase was evaporated under nitrogen in the dark. Chromatography. The residues were dissolved in 100-200 uL of HPLC eluent directly before analysis. If necessary, 20 uL of dichloromethane was added to obtain a clear solution. The HPLC analysis was per formed on a 5-um RP 18 endcapped column (4 x 250 mm) (E. Merck) with methanol-acetonitrile-dichloromethane-water (7:7:2:0.16), flow rate of 1 mL/min and detection at 460 nm. The solvent was degassed by sonification. A diode array detector (model 168, Beckman, Munich, Germany) was used for spectrophotometric peak identification. Calculations. Carotenoid concentrations in samples were calculated from a calibration curve generated from peak height ratios of the carotenoids to an internal standard or peak heights of carotenoids in calibration samples. The intra-assay precision varied between serum samples, with the coefficient of variation ranging from 5 to 15%. The recovery of internal standard and carotenoids was -90%, and the detection limit was -10 nmol/L serum. Downloaded from https://academic.oup.com/jn/article-abstract/122/11/2161/4754717 by guest on 22 January 2018
m
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CD "En
B
I
O CO
a o o
O
—I— 10 Time(min)
—I— 20
OS O
o
tÃo
ID
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FIGURE 1 HPLC-chromatograms of unheated (A) or heated (B) tomato juice and of a human serum sample 24 h after consumption of heated tomato juice (C). Standard:
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2
3 /, Time (days)
5
6
7
FIGURE 3 Increase in lycopene concentrations over the basal value after the consumption of different amounts of heated tomato juice equivalent to lycopene doses 0.35 (A), 1.25 («)and 2.5 (A) umol/kg body wt by a single subject.
Ti me (days) FIGURE 2 Serum concentrations of total lycopene after the consumption of heated (•) or unheated (o) tomato juice by five subjects. The lycopene dose was 2.5 (¿mol/kgbody wt.
For calculation of the area under the curve (AUC), the lycopene concentrations at the start of the experi ments (i.e., basal levels) were subtracted from the lycopene concentrations determined at later time points ([Ly]t- [Ly]o).The AUC values were calculated according to the trapezoidal rule. The half-life of lycopene in serum was calculated as the time in which the maximum serum concentration of lycopene decreased to half of the starting value.
RESULTS Influence of food processing on lycopene serum concentrations. To study the uptake of lycopene and its geometrical isomers from the diet, tomato juice was selected as a source. Tomato juice contains 50-120 mg/L of lycopene, which occurs mainly in the all-trans configuration (Fig. IA). Heating the juice induces trans-to-cis isomerization. After l h at 100°C the isomerie composition of the carotenoid had changed, and 20-30% of total lycopene consisted of cis-isomers (Fig. Iß).As previously described (Stahl et al. 1992), two isomers have been tentatively assigned by retention times and diode array data as 9- and 13-cis lycopene. However, other geometrical isomers likely are present and co-elute with these peaks. A typical chromatogram of carotenoids in human serum samples is presented in Figure 1C. Serum con centrations of total lycopene obtained from different individuals after the consumption of heated or unDownloaded from https://academic.oup.com/jn/article-abstract/122/11/2161/4754717 by guest on 22 January 2018
heated tomato juice are shown in Figure 2. The intake of unheated tomato juice did not increase serum lycopene concentrations; similar to results in the con trols (data not shown), the serum concentrations decreased with the time. The consumption of heated tomato juice, however, was followed by an increase of total lycopene concentrations in the serum (Fig. 2). To ensure an increase in serum lycopene, all other ex periments were performed with heated tomato juice. Absorption of total lycopene and individual differ ences. For individuals consuming heated tomato juice (see Fig. 2), peak serum concentrations of total lycopene were observed at 24-48 h after intake. The half-life (ti/2)of lycopene in human serum was be tween 2 and 3 d. The data showed that there are interindividual differences in lycopene uptake from a dietary source. After consumption of the same amount of lycopene, increases in serum lycopene con centrations ranged from 80 to 350 nmol/L over the basal concentration. Dose-dependence and accumulation. Data on dosedependence of lycopene uptake from a dietary source are presented in Figure 3. After a dose of 0.35 umol/kg body wt, total lycopene in serum was elevated 85 nmol/L above the basal level. Higher doses of lycopene raised the serum concentrations by -170 nmol/L (1.25 umol/kg body wt) and 260 nmol/L (2.5 umol/kg body wt) over the basal level. The AUC values also increased with the dose from 6.2 umol L"1 h at 0.35 umol/kg body wt and 11.5 umol L"1 h at 1.25 umol/kg body wt to 18.5 umol L"1 h at 2.5 umol/ kg body wt. The uptake of lycopene was dose de pendent but not strictly linear with dose. Relatively more lycopene was absorbed when lower amounts were consumed. Serum concentrations and AUC values increased only twofold when the dose was raised 3.5-fold from 0.35 to 1.25 umol/kg body wt. A
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13-cis
O 1D 1
2
1
1
2i Ti me (days)
3i
U
FIGURE 4 Increase in total lycopene and its isomers after repeated consumption of heated tomato juice (0.35 umol lycopene/kg body wt) by one subject. Arrows indicate time of consumption.
further twofold increase of the dose from 1.25 to 2.5 umol/kg body wt led to only a 1.5-fold rise in both lycopene serum concentrations and AUC values. Figure 4 shows the elevation of serum total lycopene and three lycopene isomers (all-trans, 9-cis and 13-cis) after repeated consumption of heated tomato juice providing 0.35 umol lycopene/kg body wt each time. The accumulation of total lycopene and lycopene isomers in serum was nearly linear. The total dose was 1.4 umol/kg body wt and led to an increase of total lycopene by 320 nmol/L over the base level. This is a similar increase in total lycopene found with the same individual after the intake of a single dose of 2.5 umol/kg body wt (350 nmol/L), emphasizing that lower doses are relatively absorbed better than higher ones. These results were confirmed in a second experiment with another subject (data not shown). Absorption of different lycopene isomers. Figure 5 shows an example of the serum concentrations of total lycopene and its isomers ali-trans, 9-cis and 13-cis after intake of heated tomato juice. The amount of tomato juice was equivalent to a dose of 2.5 umol total lycopene/kg body wt. The carotenoid was composed of 80% all-trans, 5% 9-cis and 15% 13-cis lycopene, corresponding to doses of 2 umol alltrans, 0.13 umol 9-cis and 0.38 umol 13-cis/kg body wt. All isomers reached the highest concentrations in serum after 24-48 h. Total lycopene increased by 260 nmol/L, all-trans by 170 nmol/L, 9-cis by 60 nmol/L and 13-cis by 30 nmol/L over the basal level. It seemed that, given their concentrations in the tomato Downloaded from https://academic.oup.com/jn/article-abstract/122/11/2161/4754717 by guest on 22 January 2018
~r~ —i— —r 3 4 5 6 Time (days)
7
FIGURE 5 Serum concentrations of total lycopene and its isomers after consumption of a single dose of 2.5 umol/ kg body wt of lycopene in heated tomato juice by one subject.
juice, the cis isomers were absorbed slightly better, or were metabolized to a lesser extent, than all-trans lycopene (see Fig. 5). Other carotenoids. The serum concentrations of cccarotene, ß-carotene and canthaxanthin were also de termined in the present study. In all experiments a continuous loss of all three compounds was observed. This likely was due to the consumption of a carotenoid-poor diet and may also be an indicator of participants' compliance.
DISCUSSION Several studies have been reported on the ab sorption, distribution and metabolism of ß-carotene and canthaxanthin in different animal species and in humans (Brown et al. 1989; Krinsky et al. 1990a, Mathews-Roth 1990, Prince et al. 1991, RibayaMercado et al. 1989, Shapiro et al. 1984). Pure carote noids in various supplements as well as fruits and vegetables as dietary sources have been used to study carotenoid biokinetics. 14C-Labeled lycopene has been applied to monkeys for investigations on the biokinetics (Mathews-Roth et al. 1990). Unfor tunately, no pure lycopene is available that can be used in human studies according to the Helsinki Con ference Conventions. We therefore took tomato juice as a lycopene source, because tomatoes are among the vegetables with the highest lycopene content. No increase in lycopene serum concentrations, however, was found after the intake of 700 mL of juice (130 umol of lycopene), which is in agreement with a previous study in which the consumption of tomato
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OF LYCOPENE IN HUMANS
juice (containing 22 (¿molof lycopene) did not in crease plasma concentrations of that compound (Brown et al. 1989). It has been reported that processing of food in fluences the availability of carotenoids from the diet (Erdman 1988); especially, mild cooking is thought to contribute to availability. We found this to be true for lycopene from tomato juice. Heating a mixture of juice and 1% corn oil for l h before consumption led to an increase in lycopene serum concentrations. Many different factors can influence the absorption of carotenoids from the diet, so that it remains to be studied which particular process related to heating improves the availability of lycopene. Possible mechanisms might be thermally induced rupture of cell walls accompanied by release of lycopene from the cells, heat-improved extraction of lycopene into the lipophilic corn oil as vehicle, or a combination of several other factors. Regardless of the mechanism, heating with small amounts of lipophilic oil improves absorption of lycopene from the diet, as is known for ß-carotene(Erdman 1988). This should be kept in mind when interpreting epidemiological studies on nutritional habits. Absorption of lycopene from the diet is influenced additionally by other factors. As described for ß-carotene(Brown et al. 1989), the in crease in lycopene serum concentrations differed be tween individuals consuming amounts of tomato juice equivalent to equal doses of lycopene based on body weight. These differences in absorption presumably con tribute to the inter-individual differences in the plasma concentrations that have been described in several studies (Krinsky et al. 1990b, Parker 1989, Stahl et al. 1992). Differences in LDL assembly could also be of importance in this context. At low doses the efficiency of lycopene absorption from the diet is higher than with higher doses, as described for ß-carotene(Brown et al. 1989, Erdman 1988). Two biokinetic parameters of lycopene determined in this study are maximum concentration of lycopene at 24-48 h and elimination of the compound from serum with a half-life of 2-3 d. These cmaxvalues in humans are within the range found in a study with monkeys (8-48 h) in which, however, only peak radi oactivity in plasma after application of labeled lycopene was measured. In rats, peak plasma radioac tivity was determined between 4 and 8 h, which is possibly due to substantially different biokinetics of carotenoids compared with primates (Mathews-Roth et al. 1990). In a study with ß-carotenefrom dietary (carrots) or synthetic sources, cmaxvalues were found between 24 and 48 h after consumption (Brown et al. 1989). The elimination from plasma was somewhat slower (4-8 d) than the time reported here for lycopene (2-3 d). Although these studies were different in design, one Downloaded from https://academic.oup.com/jn/article-abstract/122/11/2161/4754717 by guest on 22 January 2018
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might conclude that lycopene and ß-caroteneare similarly absorbed from the diet but differently dis tributed, accumulated, metabolized and/or excreted. Several reports exist on differences in the availa bility and the accumulation of geometrical isomers of ß-carotene.In rat and chicken livers a higher accumu lation of 9-cis ß-carotenewas observed compared with the all-trans isomer (Ben-Amotz et al. 1989, Mokady et al. 1990). In humans, however, a predominant ab sorption of all-trans ß-carotenewas described (Jensen et al. 1987). With lycopene we found better availa bility of the cis isomers compared with the all-irans compound. This difference might be a result of different amounts of isomers used in our study, whereas Jensen et al. (1987) applied approximately equal doses of the ß-caroteneisomers. We are aware, however, that other factors difficult to control in studies with humans and food as a source of carote noids might influence the absorption of lycopene and its isomers. Although the processed tomato juice contained only minor amounts of 9-CJ'slycopene, the concen tration of this isomer also increased in serum. From that point of view one might speculate that there are in-vivo isomerization mechanisms, possibly enzyme mediated.
ACKNOWLEDGMENT The technical assistance by Elisabeth Bloch is gratefully acknowledged.
LITERATURECITED Ben-Amotz, A., Mokady, S., Edelstein, S. & Avron, M. (1989) Bioavailability of a natural isomer mixture as compared with synthetic all-trans-ß-carotene in rats and chicks. J. Nutr. 119: 1013-1019. Black, H. S. & Mathews-Roth, M. M. (1991) Protective role of butylated hydroxytoluene and certain carotenoids in photocarcinogenesis. Photochem. Photobiol. 53: 707-716. Brown, E. D., Micozzi, M. S., Craft, N. E., Bieri, J. G., Beecher, G., Edwards, B. K., Rose, A., Taylor, P. R. & Smith, }. C. (1989) Plasma carotenoids in normal men after a single ingestion of vegetables or purified ß-carotene. Am. J. Clin. Nutr. 49: 1258-1265. Burton, G. W. & Ingold, K. U. (1984) ß-Carotene:an unusual type of lipid antioxidant. Science (Washington, DC) 224: 569-573. Conn, P. F., Schalen, W. & Truscott, T. G. (1991) The singlet oxygen and carotenoid interaction. J. Photochem. Photobiol. B. 11: 41-47. Devasagayam, T.P.A., Werner, T., Ippendorf, H., Martin, H. D. & Sies, H. (1992) Synthetic carotenoids, novel polyene polyketones and new capsorubin isomers as efficient quenchers of singlet molecular oxygen. Photochem. Photobiol. 55: 511-514. Di Mascio, P., Kaiser, S. & Sies, H. (1989) Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch. Biochem. Biophys. 274: 532-538. Erdman, f. (1988) The physiologic chemistry of carotenes in man. Clin. Nutr. 7: 101-106.
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Foote, C. S. & Denny, R. W. (1968) Chemistry of singlet oxygen. VII. Quenching by ß-carotene. I. Am. Chem. Soc. 90: 6233-6235. Jensen, C. D., Howes, T. W., Spiller, G. A., Pattison, T. S., Whittam, ]. H. & Scala, J. (1987) Observations on the effects of ingesting cj's- and frans-beta-carotene isomers on human serum concentrations. Nutr. Rep. Int. 35: 413-422. Kaplan, L. A., Lau, J. M. & Stein, E. A. (1990) Carotenoid compo sition, concentrations, and relationships in various human organs. Clin. Physiol. Biochem. 8: 1-10. Krinsky, N. I. (1989) Antioxidant functions of carotenoids. Free Radical Biol. & Med. 7: 617-635. Krinsky, N. I., Mathews-Roth, M. M., Welankiwar, S., Sehgal, P. K., Lausen, N.C.G. & Russen, M. (1990a| The metabolism of [14C]ß-carotene and the presence of other carotenoids in rats and monkeys. J. Nutr. 120: 81-87. Krinsky, N. L, Russe«,M. D., Handelman, G. J. & Snodderly, D. M. 11wob! Structural and geometrical isomers of carotenoids in human plasma. J. Nutr. 120: 1654-1662. Mathews-Roth, M. M. (1990) Plasma concentrations of carotenoids after large doses of ß-carotene.Am. J. Clin. Nutr. 52: 500-501. Mathews-Roth, M. M., Welankiwar, S., Sehgal, P. K., Lausen, N.C.G., Russe«,M. &. Krinsky, N. I. (1990) Distribution of [14C]canthaxanthin and |14C]lycopene in rats and monkeys. J. Nutr. 120: 1205-1213. Mokady, S., Avron, M. & Ben-Amotz, A. (1990) Accumulation in chick livers of 9-cj's versus M-trans ß-carotene.J. Nutr. 120: 889-892. Parker, R. S. (1989) Carotenoids in human blood and tissues. ). Nutr. 119: 101-104.
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Peto, R., Doll, R., Buckley, J. D. & Sporn, M. B. (1981) Can dietary beta-carotene materially reduce human cancer rates? Nature (Lond.) 290: 201-208. Prince, M. R., Frisoli, J. K., Goetschkes, M. M., Stringham, J. M. & LaMuraglia, G. M. (1991) Rapid serum carotene loading with high-dose ß-carotene:clinical implications. J. Cardiovasc. Pharmacol. 17: 343-347. Pung, A., Rundhaug, J. E., Yoshizawa, C. N. & Bertram, J. S. (1988) ß-Carotene and canthaxanthin inhibit chemically and physically induced neoplastic transformations in 10T1/2 cells. Carcinogenesis 9: 1533-1539. Ribaya-Mercado, J. D., Holmgren, S. C., Fox, J. G. & Russell, R. M. (1989) Dietary ß-caroteneabsorption and metabolism in ferrets and rats. J. Nutr. 119: 665-668. Schmilz, H. H., Poor, C. L., Wellman, R. B. & Erdman, ]. Vf., Jr. (1991) Concentrations of selected carotenoids and vitamin A in human liver, kidney and lung tissue. J. Nutr. 121: 1613-1621. Shapiro, S. S., Mo«,D. J. & Machlin, L. J. (1984) Kinetic charac teristics of ß-carotene uptake and depletion in rat tissue. J. Nutr. 114: 1924-1933. Snodderly, D. M., Russett, M. D., Land, R. I. & Krinsky, N. I. (1990) Plasma carotenoids of monkeys (Macaca fascicularis and SaimirÃ-sciureus] fed a nonpurified diet. J. Nutr. 120: 1663-1671. Stahl, W., Schwarz, W., Sundquist, A. R. & Sies, H. (1992) cis-trans Isomers of lycopene and ß-carotene in human serum and tissues. Arch. Biochem. Biophys. 294: 173-177. Ziegler, R. G. (1989) A review of epidemiologie evidence that carotenoids reduce the risk of cancer. J. Nutr. 119: 116-122.
