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Nutrient Metabolism

Novel Lycopene Metabolites Are Detectable in Plasma of Preruminant Calves after Lycopene Supplementation^1,2

Tina Sicilia, Achim Bub³, Gerhard Rechkemmer^*, Klaus Kraemer, Peter P. Hoppe and Sabine E. Kulling^**

Institute of Nutritional Physiology, Federal Research Centre for Nutrition and Food, Karlsruhe, Germany; ^* Department of Food and Nutrition, Chair of Biofunctionality of Food, Technische Universitaet Muenchen, Germany; BASF AG, Ludwigshafen, Germany; and ^** Department of Food Chemistry, Institute of Nutritional Science, University of Potsdam, Potsdam-Rehbrücke, Germany

³To whom correspondence should be addressed. E-mail: achim.bub{at}bfe.uni-karlsruhe.de.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Appropriate animal models such as preruminant calves are necessary to study the complex physiological functions of carotenoids and to relate them to possible health effects in humans. In this study, the bioavailability and metabolism of lycopene from 2 dietary supplements were compared. LycoVit® containing synthetic lycopene and Lyc-O-Mato® containing natural tomato oleoresin were administered to 2 groups of preruminant calves (each n = 8) for 14 d in daily doses of 15 mg of lycopene. Plasma was analyzed for carotenoids before the intervention period, directly after, and each day for 5 d after the end of the intervention. All-trans and 5-cis lycopene, and 3 lycopene metabolites not previously found in calf plasma were detected. These metabolites contributed 52% of the total lycopene content measured at the end of the intervention period. Based on spectroscopic data, they might be hydrogenation products, which are formed from all-trans and/or 5-cis lycopene. In the LycoVit group, total lycopene concentrations were 300% higher (286 ± 89 nmol/L) than in the Lyc-O-Mato group (72 ± 33 nmol/L) (P < 0.001). This indicates that, unlike in humans, lycopene from LycoVit and Lyc-O-Mato does not have equal bioavailabilities in preruminant calves. Therefore, the preruminant calf may not be a suitable animal model with which to study the biological and physiological effects of lycopene.

KEY WORDS: • lycopene • tomato oleoresin • bioavailability • metabolism • preruminant calf

The carotenoid lycopene, which occurs mainly in tomatoes, watermelon, and guava, is increasingly gaining scientific attention because of its potential health effects. Various epidemiologic studies showed an association between a high intake of tomatoes or tomato-based products and/or high lycopene plasma levels and a decreased risk of certain chronic diseases such as specific types of cancer and cardiovascular diseases (1,2). Anticarcinogenic effects of lycopene were shown in numerous cell culture and animal studies (3) and are also assumed in human intervention studies with prostate cancer patients (4–6). Knowledge of the bioavailability and metabolism of lycopene is a prerequisite for a causal mechanistic link between lycopene consumption, plasma concentrations, and proposed health effects. To study the bioavailability, metabolism, and mechanism of action of carotenoids in vivo, several requirements should ideally be fulfilled. First, the plasma should be free from carotenoids to exclude any carotenoid interactions. Second, the intervention should be carried out under dietary control so that different eating/feeding habits cannot affect the results. Third, invasive examinations of tissues should be possible to study the distribution, metabolism, and organ-specific storage. For ethical reasons, practically none of these requirements can be met in human dietary intervention studies. Thus, it is important to use adequate animal models that are similar to humans and do not differ in carotenoid absorption, transport, metabolism, distribution, and excretion. In addition to Mongolian gerbils, nonhuman primates, and ferrets, preruminant calves can be used an animal model especially for carotenoid absorption (7). We used preruminant calves in this study for 2 reasons: 1) because they were fed a milk-replacer, their plasma was virtually free of lycopene; and 2) most studies with preruminant calves were conducted with ß-carotene, whereas other carotenoids such as lycopene, canthaxanthin, lutein, and -carotene have been less well investigated (8).

The objective of our research was to compare the bioavailability and metabolism of synthetic and natural lycopene in an intervention study with preruminant calves, using 2 commercially available dietary supplements, LycoVit® and Lyc-O-Mato®. Human plasma and tissues contain mainly cis isomers of lycopene, whereas in tomatoes and tomato-based foods, which are the principal sources of lycopene, 67–96% of total lycopene is present in the all-trans form. Among the cis isomers, 5-cis lycopene predominates followed by 13-cis, 15-cis, and 9-cis (9). Another aim of this study was to investigate whether the isomer distribution in calf plasma is similar to that in human plasma and whether the different isomer distributions of Lyc-O-Mato and LycoVit are reflected in the plasma of the treated groups after the intervention.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Materials. LycoVit® 10% beadlets (designated as LycoVit) and synthetic lycopene were provided by BASF. Lyc-O-Mato® Beads 5% (designated as Lyc-O-Mato) were provided by LycoRed Natural Products Industries. According to our analysis, LycoVit contained 73% all-trans lycopene, 19% 5-cis lycopene, and 8% other cis isomers. Lyc-O-Mato contained 95% all-trans lycopene, no 5-cis lycopene, and 5% other cis isomers. Neurosporene was a gift from Prof. Sandmann, University of Frankfurt, Germany. The DC Biorad protein assay was purchased from Bio-Rad Laboratories. Tert-butyl methyl ether (HPLC grade) was from Riedel-de Haën. All other chemicals were purchased from Carl Roth, from VWR, or from Sigma.

    Study design and sample collection. Male preruminant calves (n = 16) were leased from one herd of Holstein dairy cattle at the age of 3 wk and housed in single pens with straw bedding. After an acclimation period of 3 d, they were randomly allotted to 2 treatment groups of 8 calves each. They were fed a milk-replacer formulated to meet or exceed all nutrients according to requirement (10) in 2 feedings/d at 0800 and 1700 h. The milk-replacer was dissolved in water (43°C) at a ratio of 1:8 (wt:v) and fed from buckets. The dry matter intake was 11 g/(kg body wt · d) to allow modest growth. The composition of the milk-replacer was published previously (11). The treatment consisted of 15 mg lycopene/d for 14 d either as LycoVit or as Lyc-O-Mato. The daily dose was freshly added to the milk-replacer for the morning feeding, and care was taken to ensure quantitative intake by rinsing the buckets with a small amount of milk-replacer. After the last dose (d 14) a washout period of 10 d ensued during which no more lycopene was given. At the end of the study, the calves were returned to the dairy herd of origin. The feeding trial was supervised and blood was drawn by a veterinarian who was an employee of BASF AG Ludwigshafen, Germany. Notice was given to the veterinary authority (Department of Veterinary Affairs and Agriculture, Landau, Germany) according to statutory provisions.

Blood samples were collected on d 0 (before the intervention), on d 14 (the last day of the intervention) and on d 15, 16, 18, 21, and 24. Plasma was separated by centrifugation (1854 x g, 4°C, 10 min) and stored at –80°C after adding 10 µL of BHT in methanol (1 g/L) to 1 mL of plasma to prevent oxidation and isomerization of lycopene.

    Carotenoid extraction from plasma. Carotenoids were extracted from 1 mL of plasma as described by Briviba et al. (12). Briefly, after protein precipitation with ice-cold ethanol, the carotenoids were extracted 3 times with 2 mL of hexane:dichloromethane (5:1, v:v, with 0.025% of BHT) in an ultrasonic bath for 5 min. The combined organic phases were evaporated to dryness under a gentle stream of nitrogen. For HPLC analysis, the residue was dissolved in 200 µL of acetone:dichloromethane (10:1, v:v, with 0.01% of BHT) and 50 µL was injected onto the HPLC column. HPLC analyses were performed immediately after the extraction of the carotenoids.

    Preparations and incubations of calf liver cytosol. A fresh calf liver was obtained from the local slaughterhouse directly after the slaughter of the calf. The liver was stored on ice for 30 min until the cytosol was prepared according to the method of Lake (13) and the protein concentration of the cytosol was determined using the BIO-RAD DC protein assay.

Cytosolic incubations were carried out in a final volume of 2 mL of phosphate buffer (0.1 mol/L, pH 7.4) containing 4 mg cytosolic protein, 10 pmol of lycopene dissolved in acetone with 0.01% of BHT, and 20 µmol of NADH. After an incubation period of 70 min, preparations were extracted and analyzed as described above. Curcumin dissolved in methanol served as a positive control (14).

    Incubations with rumen fluid. Rumen fluid was obtained from a fistulated cow and filtered through cheesecloth into a prewarmed thermos flask. Immediately after sampling, the rumen fluid was gassed with carbon dioxide to ensure anaerobic conditions. Incubations were carried out in a final volume of 150 mL. The incubation medium was prepared according to Steingass and Menke (15). Briefly, 48 mL H₂O, 24 mL buffer (3.5% NaHCO₃, 0.4% NH₄HCO₃), 24 mL macromineral solution (0.57% Na₂HPO₄, 0.62% KH₂PO₄, 0.06% MgSO₄ · 7 H₂O), and 12 µL trace element solution (0.132% CaCl₂ · 2 H₂O, 0.1% MnCl₂ · 4 H₂O, 0.01% CoCl₂ · 6 H₂O, 0.08% FeCl₃ · 6 H₂O) were mixed, gassed with CO₂, and preheated to 39°C. Subsequently, 5 mL of a reducing solution (freshly prepared by dissolving 240 mg Na₂S · 7 H₂O in 40 mL water and adding 1.7 mL of 1 mol/L NaOH), 2 mg of lycopene dissolved in 1 mL THF, and 50 mL of rumen fluid were added and incubated in a shaking water bath at 39°C overnight. After the incubation period, the reaction was stopped by adding 5 mL of phosphoric acid (1 mmol/L). Preparations were extracted 3 times with 7 mL hexane:dichloromethane (5:1, v:v, with 0.025% of BHT) each and proceeded as described above. For the HPLC analysis, the residue was dissolved in 1 mL of acetone:dichloromethane (10:1, v:v, with 0.01% of BHT), and 50 µL was used for HPLC analysis. Preparations without rumen fluid served as negative controls. As a positive control, the isoflavone daidzein was used instead of lycopene for the incubation (16). Extraction and HPLC analysis of daidzein were performed as described by Kulling et al. (17).

    HPLC analysis. Analyses were performed on a low-pressure gradient system from Shimadzu equipped with an auto injector, column oven, and photodiode array detector. The auto injector was set to 10°C and the column oven to 27°C. Separation was carried out on a 250 x 4.6 mm i.d., 5 µm, YMC "Carotenoid" S5 reversed-phase C30 column with the corresponding 10 x 4.0 mm i.d. guard column (YMC Europe GmbH). Solvent A consisted of tert-butyl methyl ether, solvent B of methanol, and solvent C of water. A linear gradient was used starting with 81% B and 4% C going to 21.1% B and 4% C within 70 min. The flow rate was 1 mL/min, and the detection wavelength was set to 472 nm for all-trans and 5-cis lycopene, 450 nm for ß-carotene, 438 nm for metabolite 1, and 455 nm for metabolites 2 and 3. Quantification was performed by external calibration with all-trans lycopene and ß-carotene standards. The concentration of a stock solution of the standard in n-hexane was measured spectrophotometrically using the following molar absorbance (m² · mol^–1) coefficients: 18490 for lycopene and 13890 for ß-carotene (18). 5-cis Lycopene and the lycopene metabolites were detected at the UV-maximum that corresponded to the detection wavelength and quantified by the all-trans lycopene calibration.

To prevent isomerization and oxidation of lycopene during the analysis, the following precautions were taken. HPLC analysis was performed immediately after the dry extraction residue was redissolved; no >8 samples were analyzed in a row because of the restricted sample stability; the autoinjector was cooled to 10°C; and the HPLC column was preconditioned with deferoxamine mesylate dissolved in methanol:water to chelate free metal ions.

    LC/MS analysis. Analyses were performed on an HP1100 system equipped with a photodiode array detector coupled to a G1946A quadrupole mass spectrometer (Agilent Technologies). Separation was carried out as described above with minor modifications. The mobile phase consisted of solvent A (tert-butyl methyl ether) and solvent B (methanol) at a flow rate of 0.9 mL/min with the following gradient: starting with 80% B, going to 4% B within 30 min, ending at 0% B at 40 min.

MS was carried out by atmospheric pressure chemical ionization (APCI) in the positive mode. The drying gas flow was set to 6 L/min, drying gas temperature to 250°C, nebulizer pressure to 206.8 kPa, vaporizer temperature to 400°C, corona current to 10 µA, capillary voltage to 2500 V, and fragmentor voltage to 70 V. Mass spectra were recorded between 200 and 1000 m/z.

    Statistics. All statistical calculations were performed using SAS 9.1 (SAS Institute). Treatment effects were determined by ANOVA with the GLM (general linear model) procedure. Effects of group, the time postintervention (d 14–18), and the group x time interaction were tested. Results are presented as means ± SD.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Plasma carotenoid concentrations. Both treatments significantly increased plasma lycopene concentrations from their undetectable levels at d 0 (Table 1). ß-Carotene was detectable in plasma at d 0, ranging from 76 to 107 nmol/L (data not shown), and did not change significantly throughout the study. These plasma ß-carotene concentrations are comparable to those in a previous study with preruminant calves fed a milk-replacer diet (11). After the intervention, all-trans lycopene, 5-cis lycopene, and the 3 lycopene metabolites, which have not been described previously in the literature, were detected (Fig. 1) and differed between the groups (ANOVA GLM group effect, P < 0.0001; Table 1). In the LycoVit group, the highest concentrations of all-trans and 5-cis lycopene and metabolites were detected on d 15. In the Lyc-O-Mato group, concentrations did not differ at d 14 and 15. During the washout period, plasma all-trans and 5-cis lycopene declined (P < 0.05) from d 14 to 18 in the LycoVit group. At d 21, lycopene could not be detected in the plasma of either group. Due to the small number of blood samples taken after the last application of lycopene, the elimination half-life was not determined. At d 15, the relative distribution of lycopene isomers revealed 31% all-trans lycopene, 17% 5-cis lycopene, with the lycopene metabolites accounting for 52% of total lycopene and metabolite 1 and metabolites 2 plus 3 occurring in comparable concentrations. Although plasma concentrations of total lycopene were 300% higher in the LycoVit group than in the Lyc-O-Mato group (P < 0.001), the relative distributions of lycopene isomers and metabolites did not differ between the groups.

View larger version (17K): [in this window] [in a new window]   FIGURE 1 HPLC-chromatogram of a concentrated calf plasma extract: smaller peaks from 16 to 21 min and the peak in front of peak #2 were not identified because they were below the limit of detection in the extract of 1 mL of plasma analyzed as described.

Metabolites 2 and 3 had UV/VIS spectra that were hypsochromically shifted by 17 nm compared with all-trans lycopene, indicating the loss of 1 conjugated double bond in the lycopene molecule. The UV/VIS spectrum of metabolite 1 was hypsochromically shifted by 34 nm compared with all-trans lycopene, indicating the loss of 2 conjugated double bonds (Fig. 2). This was supported by the molecular masses determined by LC/APCI-MS. We detected quasi molecular ions [M+H]⁺ of m/z 539.8 for metabolites 2 and 3, and m/z 541.8 for metabolite 1. Thus, the corresponding molecular masses of metabolites 2 and 3 (M = 538.8) and of metabolite 1 (M = 540.8) were 4 and 2 mass units higher than that of lycopene (M = 536.8), respectively. To obtain more information about the chemical structure of metabolite 1, we compared its spectroscopic data and retention time with those of neurosporene, a carotenoid that has 9 conjugated double bonds instead of the 11 in the lycopene molecule (Fig. 3). The UV/VIS spectrum of neurosporene showed exactly the same vibrational fine structure as metabolite 1 but was shifted bathochromically by 2 nm. Furthermore, under the separation conditions used, neurosporene eluted only slightly earlier than metabolite 1 (0.2 min). The chemical structure of metabolite 1 must therefore be very similar to that of neurosporene.

View larger version (19K): [in this window] [in a new window]   FIGURE 2 UV/VIS spectra of lycopene metabolites and all-trans lycopene from calf plasma extract.

View larger version (8K): [in this window] [in a new window]   FIGURE 3 Chemical structures of all-trans lycopene and neurosporene.

    Formation of the metabolites in vitro. The loss of conjugated double bonds of lycopene, as indicated by the hypsochromic shift of the UV/VIS spectra of the metabolites, may be the result of a hydrogenation reaction. Hydrogenation may occur in the liver by reductases or in the rumen fluid by microbial activity (16,17,19).

To identify the site of the metabolite formation, we incubated lycopene in vitro with calf liver cytosol and rumen fluid. However, no conversion of lycopene to the metabolites was detected in any of these test systems. Only the compounds that were used in each test system as positive controls showed the expected transformation to the reduced metabolites, indicating that the incubation conditions used were appropriate (Table 2).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

    Course of lycopene plasma concentrations. Lycopene plasma concentrations were highest on d 14 and 15 in the Lyc-O-Mato group and on d 15 in the LycoVit group. Within 4 d after the last lycopene dose, concentrations of all-trans and 5-cis lycopene decreased by almost half in both groups. Concentrations of the metabolites, in contrast, remained constant. In male dogs, a similar course of lycopene plasma concentrations was reported after multiple dosing for 28 d (20). The highest plasma concentrations were detected 12 h after the last lycopene dose and then decreased rapidly from 1200 to 400 nmol/L within 4 d. The elimination half-life in these dogs was 30.5 h. In contrast, plasma elimination half-life of lycopene in humans is 5 d (21). Nevertheless, we cannot explain the rapid decline of plasma lycopene concentrations below the limit of detection from d 18 to 21. Further studies with daily blood samplings during the postintervention period may help to answer this question.

    Chemical structure of lycopene metabolites. Due to the UV/VIS spectra and molecular masses of the metabolites 2 and 3, we assume that they are hydrogenation products of all-trans and 5-cis lycopene formed by the hydrogenation of 1 conjugated double bond. All-trans lycopene is a symmetrical molecule that does not possess a stereo center (Fig. 3). Hydrogenation that leads to a metabolite with only 10 conjugated double bonds must occur at the position C-5/C-6 of all-trans lycopene. Two enantiomers are possible as hydrogenation products, 5(R),6- and 5(S),6-dihydrolycopene, because C-5 becomes a stereo center through hydrogenation. It is most likely that these 2 enantiomers could not be separated on the HPLC column used in this study. Because we detected 2 peaks that are not clearly separated in the chromatogram, we assumed that one metabolite was formed from all-trans lycopene and the other from 5-cis lycopene. Two enantiomers are also possible as hydrogenation products of 5-cis lycopene, i.e., 5'(R),6'- and 5'(S),6'-dihydro-5-cis-lycopene.

Metabolite 1 has a UV/VIS spectrum showing a hypsochromic shift by 34 nm and a molecular mass that is 4 mass units higher than that of all-trans lycopene. It is possible that this metabolite was formed by the hydrogenation of 2 conjugated double bonds of either all-trans or 5-cis lycopene. The second hydrogenation step can occur at either position C-7/C-8 or at position C-5'/C-6'. In the first case, 2 enantiomers are possible, 5(R), 6,7,8- and 5(S),6,7,8-tetrahydrolycopene. In the second case, 3 products can be formed: 2 enantiomers, 5(R),6,5'(R),6'- and 5(S),6,5'(S),6'-tetrahydrolycopene, and the corresponding meso-compound. If metabolite 1 is formed from 5-cis lycopene, only 2 enantiomers are conceivable, 5'(R),6',7',8'- and 5'(S),6',7',8'-5-cis-tetrahydrolycopene.

In short, for metabolites 2 and 3, there are 2 possible isomers, 5,6-dihydrolycopene and 5',6'-dihydro-5-cis lycopene. We assumed that these 2 compounds could be separated on the HPLC column used and might therefore correspond to the 2 detected peaks. For metabolite 1, there were 3 theoretically possible isomers, i.e., 5,6,7,8-tetrahydrolycopene, 5,6,5'6'-tetrahydrolycopene, and 5',6',7',8'-tetrahydro-5-cis-lycopene, but only 1 peak was detected in the chromatogram. Thus, the HPLC peak cannot definitely be assigned to one of these compounds. To obtain more information on the stereochemistry (R-/S-configuration) of the metabolites, the separation would have to be carried out on a chiral HPLC column, which was not used in this study. Further studies are required to identify the chemical structure of the lycopene metabolites.

    Formation site of lycopene metabolites. The discovery of the 3 lycopene metabolites, possibly hydrogenation products, raises the question of their formation site. Due to our extraction and analyzing protocols, the formation of artifacts can certainly be excluded. Because hydrogenation is a usual biotransformation reaction for unsaturated compounds, we propose that lycopene metabolites are formed within the digestive tract of the calves. Although preruminant calves lack the typical reducing anaerobic rumen microflora of adult cows, we addressed this issue and incubated lycopene with cow’s fresh rumen fluid under anaerobic conditions. According to the results of our positive controls, the experimental procedure worked sufficiently. However, no conversion of lycopene was observed during the incubation, indicating that the rumen microflora of adult cows is not likely a site of metabolite formation. It must be considered that the microflora in the rumen of preruminant calves differs from that of adult cows. Mainly aerobic bacteria are present in the rumen of preruminant calves, whereas in adult cows whose rumen fluid was used in this study, protozoa and anaerobic bacteria prevail (19). Rumen fluid from preruminant calves was not available; therefore, the rumen cannot be excluded as the site of metabolite formation.

The proposed reduction products of lycopene may also be generated in the liver by reductases. Hydrogenation of double bonds is in most cases catalyzed by the cytosolic fraction of the liver. For example, the enzymatic source of curcumin reduction products was found to be exclusively cytosolic (14). Our experiments in vitro with calf liver cytosolic fractions did not result in any conversion of lycopene to the metabolites detected in the plasma. Thus, cytosolic reductases of the liver are unlikely to be responsible for metabolite formation. However, we could not exclude the possibility that the hydrogenation of the double bond that leads to the lycopene metabolites might be catalyzed by microsomal reductases of the liver. Therefore, the liver also could not be excluded completely as the site of lycopene metabolite formation.

    Differences in lycopene absorption from Lyc-O-Mato and LycoVit. The treatment groups did not differ in their relative carotenoid distribution, but they did differ in their absolute concentrations. In the LycoVit group, plasma total lycopene was 300% higher than in the Lyc-O-Mato group. This result was surprising because plasma lycopene concentrations (LycoVit 0.91 ± 0.3 µmol/L, Lyc-O-Mato 0.95 ± 0.2 µmol/L) and the change from baseline (0.57 ± 0.26 vs. 0.58 ± 0.32 µmol/L) did not differ among the groups in a human study with identical lycopene preparations given at 15 mg/d for 28 d (22). The differences in plasma responses in the calves could be due to the difference in preparation. LycoVit contains 10% lycopene incorporated into a matrix of cornstarch, gelatin, sucrose, with dl--tocopherol and ascorbylpalmitate as antioxidants (23). Lyc-O-Mato contains only 5% lycopene incorporated into a matrix of gelatin, sugars, ascorbylpalmitate, and vitamin E. These differences in the formulation matrices and in the amounts of lycopene incorporated into these matrices may contribute to better lycopene absorption from LycoVit compared with Lyc-O-Mato in preruminant calves. It might be characteristic of preruminant calves not to be able to digest LycoVit and Lyc-O-Mato comparably. In the human study, however, these matrix differences had no effects on lycopene plasma concentrations (22). Further studies are required to investigate whether differences in lycopene tissue storage contribute to the observed differences in plasma concentrations.

    Lycopene isomer distribution in plasma. We also investigated lycopene isomers in plasma because the 2 lycopene preparations, LycoVit and Lyc-O-Mato, differed in their isomer distribution. LycoVit contained 73% all-trans lycopene, 19% 5-cis lycopene, and 8% other cis isomers, whereas Lyc-O-Mato contained no 5-cis lycopene and only 5% other cis isomers. Despite these differences, the same isomer distribution was detected in the plasma of both treatment groups after the intervention on d 15 (32% all-trans lycopene, 16% 5-cis lycopene, and 52% metabolites). Apart from the lycopene metabolites, the 5-cis isomer is the predominant cis lycopene in calf plasma as in human plasma (9,24). Although Lyc-O-Mato contains no 5-cis lycopene, 16% 5-cis lycopene was detected in plasma of the Lyc-O-Mato group. Therefore, the occurrence of the lycopene 5-cis isomer seems to be independent of the lycopene isomers ingested. We propose that there is no preference in lycopene absorption between the different lycopene isomers and that isomerization to 5-cis lycopene may take place in the digestive tract and/or in plasma. Some authors, however, suggested that cis lycopene is more bioavailable than all-trans lycopene most likely because of a better solubility in bile acid micelles, a lower tendency to aggregate, and a preferential incorporation into chylomicrons (25,26). We do not agree with this assumption because in these studies, no discrimination was made between 5-cis and other cis isomers; furthermore, isomerization in the small intestine, the mucosa, or the lymph was not excluded. Our results do not support the hypothesis that cis lycopene is more bioavailable than all-trans lycopene.

In summary, a 2-wk daily supplementation of lycopene as LycoVit to preruminant calves resulted in 300% higher plasma concentrations than after Lyc-O-Mato, which is in contrast to a human study (22). In addition to all-trans and cis lycopene, 3 lycopene metabolites were detected that have not been described to date, isomers that do not occur in human plasma after LycoVit/Lyc-O-mato supplementation (12). These metabolites might be formed by hydrogenation from all-trans or 5-cis lycopene. The site of metabolic transformation, however, was not found in our studies in vitro. It is important to investigate whether these lycopene metabolites have a relevant physiological function. Finally, the differences observed between humans and calves indicate some limitation to the suitability of preruminant calves as an animal model for studying human lycopene absorption and metabolism.

	ACKNOWLEDGMENTS

 
The authors thank the research group of Prof. K. Albert, University of Tübingen, for the LC/NMR analyses and Prof. Steingass, University of Hohenheim, for providing the rumen fluid. We thank L. Korn for his statistical consultation.

	FOOTNOTES

 
¹ Presented in part at the 32nd Deutscher Lebensmittelchemikertag 2003, October 8–9, 2003, Munich, Germany [Scilia T, Kulling SE, Hoppe PP, Krämer K, Bub A, Rechkemmer G. Bioavailability and metabolism of lycopene in the preruminant calf (abstract). Lebensmittelchemie. 2004;58:28] and at the 42nd DGE congress, March 11–12, 2004, Freising-Weihenstephan, Germany [Scilia T, Kulling SE, Hoppe PP, Krämer K, Bub A, Rechkemmer G. Comparing the bioavailability of two lycopene preparations in the preruminant calf (abstract). Proc Ger Nutr Soc. 2004;6:68].

² Supported by the Federal Ministry of Consumer Protection, Food and Agriculture (BMVEL) and a grant of the Federal Ministry of Education and Research (BMBF-BEI032/0312248H/6).

Manuscript received 30 June 2005. Initial review completed 12 July 2005. Revision accepted 15 August 2005.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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