Identification, Quantification, and Relative Concentrations of

Thirty-four carotenoids, including 13 geometrical isomers and eight metabolites, in breast milk and serum of three lactating mothers have been separat...
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Anal. Chem. 1997, 69, 1873-1881

Identification, Quantification, and Relative Concentrations of Carotenoids and Their Metabolites in Human Milk and Serum Frederick Khachik,*,† Christopher J. Spangler, and J. Cecil Smith, Jr.

Carotenoid Research Unit, Beltsville Human Nutrition Research Center, Building 161 BARC-East, U.S. Department of Agriculture, ARS, Beltsville, Maryland 20705 Louise M. Canfield

Department of Biochemistry, University of Arizona, Tucson, Arizona 85721 Andrea Steck and Hanspeter Pfander

Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland

Thirty-four carotenoids, including 13 geometrical isomers and eight metabolites, in breast milk and serum of three lactating mothers have been separated, identified, quantified, and compared by high-performance liquid chromatography (HPLC)-photodiode array (PDA) detectionmass spectrometry (MS). Among the metabolites were two oxidation products of lycopene and four of lutein/ zeaxanthin. In addition, two metabolites of lutein, formed as a result of dehydration of this dihydroxycarotenoid under acidic conditions similar to those of the stomach, have also been identified in plasma and breast milk. The oxidative metabolites of lycopene with a novel fivemembered-ring end group have been identified as epimeric 2,6-cyclolycopene-1,5-diols by comparison of their HPLC-UV/visible-MS profiles with those of fully characterized (1H- and 13C-NMR spectroscopy) synthetic compounds. The HPLC procedures employed also detected vitamin A, two forms of vitamin E (γ- and r-tocopherol), and two non-carotenoid food components, i.e., piperine and caffeine, in serum and breast milk.

Breast milk carotenoids provide an important source of vitamin A for the infant1,2 and may contribute significantly to protection of the nursing infant from respiratory and gastrointestinal infection.3-5 Thus, the carotenoids of human milk are important for developing and maintaining good health of infants and are particularly relevant in developing countries, where a reliable † Also a member of the Department of Chemistry, Catholic University of America, Washington, DC 20064. (1) Giuliano, A. R.; Neilson, E. M.; Yap, H. H.; Baier, M.; Canfield, L. M. J. Nutr. Biochem. 1994, 5, 551-556. (2) Wallingford, J. C.; Underwood, B. A. Vitamin A deficiency in pregnancy, lactation and the nursing Child. In Vitamin A deficiency and its control; Bauernfeind, J. C., Ed.; Academic Press: New York, 1986; pp 101-152. (3) Krinsky, N. I. Annu. Rev. Nutr. 1993, 13, 561-587. (4) West, K. P.; Howard, G. R.; Sommer, A. Annu. Rev. Nutr. 1989, 9, 63-68. (5) Stoltzfus, R. J.; Hakimi, M.; Miller, K. W.; Rasmussen, K. M.; Dawiesah, S.; Habicht, J. P.; Dilbey, M. J. J. Nutr. 1993, 123, 666-673.

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source of preformed vitamin A (retinol) may not be consistently available in the mother’s diet.6 β-Carotene of human milk has been the focus of attention because of its provitamin A activity. In addition to β-carotene, several other dietary carotenoids, such as R-carotene, β-cryptoxanthin, and γ-carotene, serve as provitamin A sources. During the past 14 years, we have identified and quantified carotenoids from fruits and vegetables commonly consumed in the United States and have demonstrated that up to 50 dietary carotenoids may be absorbed and metabolized by humans.7-11 In 1992, in addition to seven carotenoids previously known in human blood, we reported the isolation and characterization of 11 new carotenoids, including several of their metabolites.9,12 In recent years, the protective role of carotenoid-rich fruits and vegetables in prevention of cancer,13,14 heart disease,15,16 and advanced age-related macular degeneration17 has become more evident from several epidemiological studies. Unfortunately, (6) Newman, V. Food Nutr. Bull. 1994, 15, 161-176. (7) Khachik, F.; Beecher, G. R.; Goli, M. B.; Lusby, W. R. Pure Appl. Chem. 1991, 63 (1), 71-80. (8) Khachik, F.; Beecher, G. R.; Goli, M. B.; Lusby, W. R. Separation and quantification of carotenoids in foods. In Methods in Enzymology; Packer, L., Ed.; Academic Press: New York, 1992; Vol. 213A, pp 347-359. (9) Khachik, F.; Beecher, G. R.; Goli, M. B.; Lusby, W. R.; Smith, J. C., Jr. Anal. Chem. 1992, 64, 2111-2122. (10) Khachik, F.; Beecher, G. R.; Goli, M. B.; Lusby, W. R.; Daitch, C. E. Separation and quantification of carotenoids in human plasma. In Methods in Enzymology; Packer, L., Ed.; Academic Press: New York, 1992; Vol. 213A, pp 205-219. (11) Khachik, F.; Englert, G.; Daitch, C. E.; Beecher, G. R.; Lusby, W. R.; Tonucci, L. H. J. Chromatogr. Biomed. Appl. 1992, 582, 153-166. (12) Khachik, F.; Englert, G.; Beecher, G. R. J. Chromatogr. Biomed. Appl. 1995, 670, 219-233. (13) Micozzi, M. S. In Nutrition and cancer prevention; Moon, T., Micozzi, M. S., Eds.; Marcel Dekker: New York, 1989; pp 213-241. (14) van Poppel, G. Eur. J. Cancer 1993, 29A, 1335-1344. (15) Morris, D. L.; Kritchevsky, S. B.; Davis, C. E. JAMA, J. Am. Med. Assoc. 1994, 272, 1439-1441. (16) Gaziano, J. M.; Manson, J. E.; Branch, L. G.; Colditz, G. A.; Willet, W. C.; Buring, J. E. Ann. Epidemiol. 1995, 5, 255-260. (17) Seddon, J. M.; Ajani, U. A.; Sperduto, R. D.; Hiller, R.; Blair, N.; Burton, T. C.; Farber, M. D.; Gragoudas, E. S.; Haller, J.; Miller, D. T.; Yannuzzi, L. A.; Willet, W. JAMA, J. Am. Med. Assoc. 1994, 272, 1413-1420.

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except for β-carotene, the study of the metabolism, function, and biological activity of non-vitamin A active carotenoids has been largely neglected. Therefore, as other carotenoids without provitamin A function are identified, the isolation and quantitation of the full complement of carotenoids in human serum and milk is fundamental to our understanding of the role of these compounds in the health and well-being of adults as well as the nursing infant. The separation of a limited number of carotenoids in human milk has been previously reported;1 however, the detailed separation and characterization of the complete spectrum of the dietary carotenoids and their metabolites have not been investigated. We describe the separation, characterization, and quantification of 34 carotenoids, including 13 geometrical isomers and eight metabolites, vitamin A, and two forms of vitamin E (γ- and R-tocopherol) in breast milk and serum of three nursing mothers by highperformance liquid chromatography (HPLC)-UV/visible-Mass Spectrometry (MS). Two metabolites of lycopene with a novel five-membered-ring end group have been identified in serum and breast milk. The qualitative and quantitative distribution of carotenoids and their metabolites in human milk and serum from three lactating mothers have been compared. EXPERIMENTAL SECTION (A) Subjects and Methods. (1) Population Selection. Subjects were three healthy lactating females living in the Tucson, AZ, metropolitan area. The women were more than 1 month postpartum, and breast milk was the sole source of nutrients for their infants. Mothers were nonsmokers over the age of 18, had no chronic diseases, were not receiving over-the-counter or prescribed medication or steroid contraceptives, and had older children with normal growth patterns. (2) Procedures. Prior to sample collection, subjects signed informed consent forms in accordance with regulations of the University of Arizona Human Subjects Committee. Samples were collected in the residences of the mothers. The field research team collected fasting (e.g., g12 h) blood from the mothers before breakfast. (3) Collection of Milk Samples. The total milk volume of one breast was collected under subdued lighting by electric breast pump (Ameda Egnell, Cary, IL) into sterile polypropylene containers or glass bottles. To ensure that residual hind milk did not contaminate breast milk samples, 2-3 h prior to collection the baby nursed from the breast to be sampled, and the breast was then completely emptied using the breast pump. After collection, the milk samples were transported directly to the laboratory, where they were stored at -70 °C until analysis. (4) Collection of Serum. Blood samples were collected from mothers by antecubital puncture into 15-mL vacuum tubes and transported directly to the laboratory, held at 25 °C for 30 min to 1 h, and centrifuged to yield ∼6.0 mL of sera; these were immediately stored at -70 °C until analysis. (5) Extraction of Human Serum. Sera (6 mL) were treated with ethanol (6 mL) containing 0.1% butylated hydroxytoluene (BHT) to precipitate the proteins; the supernatant was extracted twice with hexane as previously described.1 The serum extracts were evaporated to dryness and redissolved in 0.15 mL of HPLC solvents (eluent B). (6) Enzymatic Extraction of Breast Milk. Volumes of 124, 128, and 176 mL of milk samples from three lactating mothers were extracted. A typical procedure for extraction of 100 mL of milk was as follows. To 100 mL of milk were added 0.6 g of bile 1874

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salts (50% sodium cholate and 50% sodium deoxycholate) and 1 g of MgCO3. The mixture was vortexed for 30 s and shaken at 37 °C for 15 min. This was followed by addition of 2 mL of 50 mg/ mL pronase E in physiologically buffered saline (PBS) solution and 4 mL of 50 mg/mL lipase in PBS solution. The mixture was incubated at 37 °C for 1 h. Ethanol (100 mL) containing BHT (0.1%) was added, and the mixture was vortexed for 30 s. The mixture was then extracted twice with hexane (2 × 50 mL) containing BHT (0.1%). The hexane layers were combined, dried over sodium sulfate, and evaporated to dryness. The residue was dissolved in ∼3-4 mL of dichloromethane and filtered through a 0.45-µm disposable filter assembly (Baxter, Scientific Product Division, McGaw Park, IL) into a 5-mL graduated microsample vial. The solvent was evaporated under nitrogen, and HPLC solvents (eluent B) were added until the total volume of the extract was 300 µL. Recovery of carotenoids was estimated using the method of exhaustive extraction as described previously.1 (7) Qualitative and Quantitative HPLC Analyses of Serum and Milk. Qualitative and quantitative HPLC-MS analyses of serum and milk carotenoids were performed on reversed-phase (eluent A, qualitative) and normal-phase (eluent B, qualitative and quantitative) columns using HPLC system 1. For quantitative HPLC analyses of carotenoids by reversed-phase chromatography, milk and serum extracts were diluted 10-fold and analyzed by HPLC system 2 (eluent C), which was equipped with a more sensitive detector. The injection volume in all the above HPLC analyses was 20 µL. (8) Source of Human Plasma and Milk Pools. A plasma pool was used to ensure the consistency and the reproducibility of the hydrolysis, extraction, and analytical procedures. A typical pool consisted of a unit (∼360 mL) of plasma (American Red Cross, Baltimore, MD), which was thoroughly mixed, and small volumes (∼1 mL) were transferred to freezer vials and stored at -70 °C. Extraction and HPLC results from these samples employing ethyl β-apo-8′-carotenoate as an internal standard were used to assess within- and between-day variations. A breast milk pool was constructed from milk of seven Tucson mothers for standardization of extraction and HPLC analysis of individual samples using ethyl β-apo-8′-carotenoate as an external standard. (9) Verification of Accuracy. The focus of this study was to identify the whole spectrum of carotenoids in human milk and serum which have not been previously identified. Therefore, because the use of an internal standard in the extraction of the samples could possibly interfere with the presence of unknown carotenoids, no internal standard in the extraction of human milk and serum was used. This is because carotenoids are carried in the milk fat globule, and the added internal standard cannot enter the fat globule and equilibrate with endogenous carotenoids to provide useful information about the efficiency of carotenoid extraction. However, ethyl β-apo-8′-carotenoate was used as an external standard to determine the recovery and reproducibility of the HPLC analysis of carotenoids in the extracts from human milk and serum. Further, to monitor the accuracy and reproducibility of the HPLC analysis (system 2), a solution containing known concentrations of ethyl β-apo-8′-carotenoate (external standard), retinol, R-tocopherol, lutein, R- and β-cryptoxanthin, and R- and β-carotene was routinely analyzed. Extraction and HPLC analysis of plasma pool samples (American Red Cross, Baltimore, MD), employing ethyl β-apo-8′-carotenoate as an internal standard,

was used to assess within- and between-day variations. Freezedried sera samples from the National Institute of Standards and Technology (NIST, Gaithersburg, MD) were also extracted using the internal standard and analyzed by HPLC system 2 as part of our laboratory’s participation in a quality assurance for quantification of carotenoids and fat-soluble vitamins. Certified value for total β-carotene was based on the mean of laboratory results, with verification based on measurement at NIST using two or more different methods. These studies revealed that the recovery and accuracy of the HPLC analyses were greater than 95%. Furthermore, the recoveries of the internal standard, ethyl β-apo-8′carotenoate, from repeated extraction of the above pool plasma samples were in the range of 90-95%. These were determined by the HPLC peak area of the internal standard before and after extraction and workup procedures. (B) Chromatographic Procedures. (1) HPLC System 1. A Beckman Model 114M ternary solvent delivery system equipped with a Beckman Model 421 controller was interfaced into a Hewlett-Packard (HP) 1040A rapid-scanning UV/visible photodiode array detector. The data were stored and processed by a HP 9000/Series 300 (Chem-Station) computing system, in combination with a HP Model 9153B disk drive, a color display monitor (Model 35741), and a Model 7470A plotter. The absorption spectra of the carotenoids were recorded between 200 and 600 nm at a rate of 12 spectra/min. (2) Reversed-Phase Separations (Eluent A). Qualitative separations with HPLC system 1 employed a Microsorb (25-cm length × 4.6-mm i.d.) C18 (5-µm spherical particles) column (Rainin Instrument Co., Woburn, MA), which was protected with a Brownlee guard cartridge (3-cm length × 4.6-mm i.d.) packed with spheri-5-C18 (5-µm particle size). Eluent A consisted of an isocratic mixture of acetonitrile (85%), methanol (10%), dichloromethane (2.5%), and hexane (2.5%) at time 0, followed by a linear gradient beginning at 10 min and completed at 40 min. The final composition of the gradient mixture was acetonitrile (45%), dichloromethane (22.5%), hexane (22.5%), and methanol (10%). The column flow rate was 0.70 mL/min. At the end of the gradient, the column was re-equilibrated for 15 min under the initial isocratic conditions. With this eluent, the HPLC separations were monitored and optimized at seven different wavelengths (470, 445, 400, 350, 325, 290, and 276 nm) simultaneously to ensure the detection of all components in serum and milk extracts. (3) Normal-Phase Separations (Eluent B). Qualitative and quantitative separations with HPLC system 1 were also carried out on a silica-based, nitrile-bonded (25-cm length × 4.6-mm i.d.; 5-µm spherical particle) column (Regis Chemical Co., Morton Grove, IL), which was protected with a Brownlee nitrile-bonded guard cartridge (3-cm length × 4.6-mm i.d.; 5-µm particle size). Eluent B consisted of an isocratic mixture of hexane (74.65%), dichloromethane (25.00%), methanol (0.25%), and N,N-diisopropylethylamine (0.10%). The column flow rate was 0.7 mL/min. For reproducible separations with this eluent, an accurate composition of each solvent, particularly that of methanol, was maintained by preparing this HPLC eluent immediately prior to use. This is because volatility of hexane and dichloromethane may result in evaporation. The wavelengths monitored with this eluent were 445, 340, 325, and 276 nm. (4) HPLC-MS Interface System. The HPLC system 1 was interfaced into a Hewlett-Packard Model 5989A particle beam mass spectrometer. Reversed-phase (eluent A) and normal-phase

(eluent B) separations were employed to separate and identify milk and serum carotenoids. Eluate from the HPLC was divided in a ratio of 1:3, with the lesser amount entering the particle beam interface, which was operated at a desolvation temperature of 45 °C. Electron capture negative ionization (ECNI) was achieved using methane at a pressure of 0.85 Torr and a source temperature of 250 °C. Spectra were collected from m/z 100 to 700 using a scan cycle time of 1.5 s. (5) HPLC System 2. A Beckman system Gold equipped with a programmable solvent Module 126, a UV/visible photodiode array detector Module 168, and an autosampler 507 (cooled to 13 °C with a Haake FX circulatory bath). The data were stored and processed on an IBM personal computing system Model 55SX with a color display monitor. Reversed-phase analytical separations were carried out on a Microsorb column protected with a guard column as described previously. The column was equipped with a column water jacket (Rainin Instrument Co.) and maintained at 25 °C with a Haake FS circulatory bath. The solvent module consisted of two pumps. Pump A pumped a mixture of acetonitrile/methanol (9/1 v/v), and pump B pumped a mixture of hexane/dichloromethane/methanol/N,N-diisopropylethylamine (4.5/4.5/0.99/0.01 v/v/v/v/v). At time zero, an isocratic mixture of acetonitrile (85.5%), methanol (9.995%), dichloromethane (2.25%), hexane (2.25%), and N,N-diisopropylethylamine (0.005%) (95% pump A, 5% pump B) was pumped for 10 min. After 10 min, a linear gradient was run for 30 min, resulting in a final composition of acetonitrile (40.5%), methanol (9.95%), dichloromethane (24.75%), hexane (24.75%), and N,N-diisopropylethylamine (0.055%) (45% pump A, 55% pump B). The column flow rate was 0.70 mL/min. At the end of the gradient, the column was equilibrated under the initial isocratic conditions for 15 min. The wavelengths of the photodiode array detector, which was only capable of monitoring two channels simultaneously, were changed at various intervals to ensure the detection of carotenoids vitamins A and E at their main absorption maxima (see Table 1). (6) Reagents and Materials. The reference samples of carotenoids were either synthesized or isolated from natural sources according to our previously published procedures.8-12 The reference samples of 2,6-cyclolycopene-1,5-diols I and II were prepared by oxidation of lycopene with m-chloroperbenzoic acid followed by acidic hydrolysis.18-21 Ethyl β-apo-8′-carotenoate (Fluka Chemical Co., New York, NY) was employed as both external and internal standards in extraction and separation of carotenoids from plasma and milk by HPLC system 2. Retinol and γ- and R-tocopherols were obtained from Eastman Kodak Co. (Rochester, NY). Piperine, caffeine, BHT, and N,N-diisopropylethylamine were purchased from Aldrich Chemical Co. (Milwaukee, WI). Bile salts, pronase E, and lipase (Type VII from Candida cylindracea) were from Sigma Chemical Co. (St. Louis, MO). HPLC-grade solvents acetonitrile, dichloromethane, hexane, and (18) Khachik, F.; Beecher, G. R.; Smith, J. C., Jr. J. Cell. Biochem. 1995, 22, 236-246. (19) Khachik, F.; Beecher, G. R.; Steck, A.; Pfander, H. Partial synthesis of the oxidative metabolites of lycopene isolated from human plasma, Presented at the 11th International Symposium on Carotenoids, Leiden, The Netherlands, Aug 18-23, 1996. (20) Khachik, F.; Steck, A.; Pfander, H. Bioavailability, metabolism, and possible mechanism of chemoprevention by lutein and lycopene in humans. Proceedings of the Second International Conference on Food Factors: Chemistry and Cancer Prevention; Ohigashi, H., Ed.; Springer-Verlag: Tokyo, 1997 (in press). (21) Tonucci, L. H.; Holden, J. M.; Beecher, G. R.; Khachik, F.; Davis, C. S.; Mulokozi, G. J. Agric. Food Chem. 1995, 43, 579-586.

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Table 1. HPLC Peak Identification of Carotenoids in Human Serum and Milk Extracts from Their Wavelengths of Absorption Maxima and Mass Spectral Data Determined by HPLC Photodiode Array Detection Mass Spectrometry in the Order of Elution with Eluents B and A peak no.

serum/milk carotenoidsb

absorption maxima (nm)c

molecular mass (m/z)d

1a 2a 3a 4a 5a 6 7 8 9a 10 11a 12 13 14 15 16 17

Eluent B ,-carotene-3,3′-dione 3′-hydroxy-,-caroten-3-one 2,6-cyclolycopene-1,5-diol I 3-hydroxy-β,-caroten-3′-one (Z)-3-hydroxy-β,-caroten-3′-one (3S,6S,3′S,6′S)-,-carotene-3,3′-diol (lactucaxanthin) (13Z,13′Z,3R,3′R,6′R)-β,-carotene-3,3′-diol [(13Z,13′Z,3R,3′R,6′R)-lutein] (all-E,3R,3′R,6′R)-β,-carotene-3,3′-diol [(all-E,3R,3′R,6′R)-lutein] 2,6-cyclolycopene-1,5-diol II (all-E,3R,3′R)-β,β-carotene- 3,3′-diol [(all-E,3R,3′R)-zeaxanthin] (all-E,3R,3′S,6′R)-β,- carotene-3,3′-diol [(all-E)-3′-epilutein] (9Z,3R,3′R,6′R)-lutein (9′Z,3R,3′R,6′R)-lutein (13Z)-lutein + (13′Z)-lutein (9Z)-zeaxanthin (13Z)-zeaxanthin (15Z)-zeaxanthin

420, 442, 472 422, 442, 472 434, 458, 492 (424), 448, 476 (420), 442, 470 416, 442, 470 274, 336, 410, 432, 460 (424), 448, 476 432, 458, 490 (428), 454, 482 (424), 448, 476 334, (420), 442, 470 332, (420), 444, 472 334, (418), 440, 468 340, (424), 450, 474 338, (419), 446, 472 338, (426), 450, 478

564 566, 548 (M - H2O) 570 566, 548 (M - H2O) 566, 548 (M - H2O) 568, 550 (M - H2O) 568, 550 (M - H2O) 568, 550 (M - H2O) 570 568 568, 550 (M - H2O) 568, 550 (M - H2O) 568, 550 (M - H2O) 568, 550 (M - H2O) 568 568 568

18a 19a 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Eluent A (3R,6′R)-3-hydroxy-3′,4′-didehydro-β,γ-carotene (3R,6′R)-3-hydroxy-2′,3′-didehydro-β,-carotene (2′,3′-anhydrolutein) β,-caroten-3-ol (R-cryptoxanthin) 3-hydroxy-β-carotene (β-cryptoxanthin) (Z)-3-hydroxy-β-carotene [(Z)-β-cryptoxanthin] ψ,ψ-carotene (lycopene) (Z)-ψ,ψ-carotene [(Z)-lycopene] 7,8-dihydro-ψ,ψ-carotene (neurosporene) β,ψ-carotene (γ-carotene) 7,8,7′,8′-tetrahydro-ψ,ψ-carotene (ζ-carotene) β,-carotene (R-carotene) (all-E)-β,β-carotene [(all-E)-β-carotene] (9Z)-β,β-carotene [(9Z)-β-carotene] (13Z)-β,β-carotene [(13Z)-β-carotene] (all-E)- or (Z)-7,8,11,12,7′,8′-hexahydro-ψ,ψ-carotene [(all-E)- or (Z)-phytofluene] [(Z)- or (all-E)-phytofluene] 7,8,11,12,7′,8′,11′,12′-octahydro-ψ,ψ-carotene (phytoene)

334, (424), 446, 476 336, (424), 448, 476 (424), 446, 476 (428), 454, 480 (424), 450, 476 446, 474, 502 348, 362, 438, 466, 494 418, 442, 470 (440), 462, 492 378, 400-402, 426 (428), 446-448, 474 (430), 454, 478 340, (426), 450, 474 340, (424), 448, 472 334, 350, 368 334, 350, 368 (276), 286, (295)

550 550 552 552 552 536 536 538 536 540 536 536 536 536 e e e

a Refers to carotenoid metabolites. b Common names for certain carotenoids are shown in parentheses. c Values in parentheses represent points of inflection. d The molecular ions appeared as the base peak (100% intensity). In some cases, the ion due to the loss of H2O from the molecular parent ion (M) could also be observed. e Due to the coelution of cholestryl esters with this compound, its molecular parent ion was not observed by HPLC-MS.

methanol (Baxter Scientific Division, McGaw Park, IL) were used without further purification. RESULTS AND DISCUSSION In a 1992 publication, we reported the separation and identification of 18 carotenoids from extracts of human plasma.9,10 These included several oxidative metabolites of lutein, zeaxanthin, and lycopene. In the course of the past several years, we have confirmed the identity and the chemical structure of several of these plasma carotenoids by detailed nuclear magnetic resonance (NMR) spectroscopy.11,12 In the present report, we have established the identity of 34 carotenoids, including 13 geometrical isomers and eight metabolites, in human serum and have detected the same carotenoids in the extracts of human breast milk. In contrast to our 1992 paper, we have combined the 13 geometrical isomers of carotenoids with 21 of their all-E (all-trans) isomers to report a total of 34 carotenoids in human milk and serum. A detailed description of the qualitative and quantitative distribution of serum and breast milk carotenoids follows. (A) Qualitative Distribution of Serum and Breast Milk Carotenoids. The carotenoids detected in human serum of the three lactating mothers were also present in their breast milk, 1876 Analytical Chemistry, Vol. 69, No. 10, May 15, 1997

although at much lower concentrations. Typical HPLC profiles from an extract of serum of a lactating mother on a C18 reversedphase column (HPLC system 1, eluent A) and a nitrile-bonded column (HPLC system 1, eluent B) are shown in Figures 1 and 2, respectively. Carotenoids separated by HPLC-photodiode array detection were also simultaneously monitored by particle beam mass spectrometry using electron capture negative ionization (ECNI). Therefore, the identity of carotenoids was established by HPLC-UV/visible-MS as shown in Table 1. In addition, the HPLC-UV/visible-MS data of each carotenoid were further confirmed by comparison with a standard sample, prepared by partial synthesis or isolated from various natural sources. For chemical structures of serum and milk carotenoids, see Figure 3. Several non-carotenoid components such as piperine (major component of black pepper) and caffeine were also shown to be present in the extracts from serum and milk. BHT, which was added to the serum and milk samples as an antioxidant at the beginning of the extraction, could also be separated by HPLC (Figure 1). The identification of these non-carotenoid components will be described later in this text. As shown in Figure 1, following the elution of retinol, polar carotenoids such as lutein, zeaxanthin,

Figure 1. Typical HPLC profile of carotenoids from an extract of serum of a lactating mother on a C18 reversed-phase column (eluent A). The carotenoid profile of enzymatically hydrolyzed milk is identical to that of serum. Ethyl β-apo-8′-carotenoate (shown in broken line) was employed as an external standard. Conditions are described in the text. For peak identification, see Table 1.

Figure 2. Typical HPLC profile of carotenoids from an extract of serum of a lactating mother on a silica-based, nitrile-bonded column (eluent B). The carotenoid profile of enzymatically hydrolyzed milk is identical to that of serum. Conditions are described in the text. For peak identification, see Table 1.

and their oxidative metabolites as well as their geometrical isomers (1-17, see Table 1) appear as several unresolved HPLC peaks, while monohydroxycarotenoids and carotenes (18-34) are well separated. Additionally, the HPLC separation of serum carotenoids on a nitrile-bonded column, as shown in Figure 2, results in complete separation of the polar carotenoids (1-17) while failing to separate monohydroxycarotenoids and carotenes (18-34). Therefore, it is essential to employ both of these HPLC columns to separate all 34 serum and milk carotenoids listed in Table 1. There is some HPLC peak overlap between a number of analytes, as shown in Figures 1 and 2. These are (1) (Z)-βcryptoxanthin (peak 22) and γ-tocopherol, (2) ζ-carotene (peak 27) and R-carotene (peak 28), and (3) 3′-epilutein (peak 11) and caffeine. These HPLC peak overlaps are of no concern since the absorption maxima of these compounds are sufficiently different to allow the identification and quantification of these compounds (see Table 1). In the following, we describe the separation and identification of the various components of serum and milk, which have been classified as (1) dietary carotenoids, vitamin A, and

vitamin E, (2) lutein and zeaxanthin metabolites, (3) lycopene metabolites, and (4) non-carotenoids of dietary origin. (1) Dietary Carotenoids and Vitamins A and E. Most dietary carotenoids, as well as vitamins A and E, are best separated by reversed-phase HPLC as shown in Figure 1. However, specific dietary carotenoids such as lactucaxanthin (peak 6), lutein (peak 8), and zeaxanthin (peak 10), as well as their geometrical isomers (peaks 12-17) can be more effectively separated by normal-phase HPLC on a nitrile bonded column as shown in Figure 2. The dietary source of lactucaxanthin is limited to Romaine lettuce (Lactuca sativa), where we have detected low concentration by HPLC (unpublished results). We have previously reported the separation and structural elucidation of the geometrical isomers of lutein and zeaxanthin in human plasma.11 Recently, for the first time, we have identified a di-Z (cis) isomer of lutein in the extracts of human serum, which has been identified as (13Z,13′Z,3R,3′R,6′R)lutein (peak 7). We have also detected low concentrations of (13Z,13′Z)-lutein in several green vegetables including spinach, broccoli, and kale (Brassica oleracea var. acephala). (13Z,13′Z)Analytical Chemistry, Vol. 69, No. 10, May 15, 1997

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Figure 3. Chemical structures of carotenoids separated by HPLC from extracts of serum and milk of a lactating mother. With the exceptions of phytofluene and phytoene, only the all-E isomers are shown. Only the absolute configuration rather than the planar structure for those carotenoids with confirmed chirality have been depicted. An asterisk indicates that only the relative and not the absolute configuration at C-2, C-5, and C-6 is known. Where possible, the common names for certain carotenoids have been used. For complete nomenclature, see Table 1.

Lutein in the extracts of serum and milk was identified by comparison of its HPLC-UV/visible-MS profile with that of the authentic compound isolated from kale. The authentic sample of (13Z,13′Z)-lutein was fully characterized by 1H- and 13C-NMR as well as UV/visible and MS. The details of isolation and identification will be described in a future publication. The chromatogram in Figure 1 depicts the separation of dietary monohydroxycarotenoids R-cryptoxanthin (peak 20), β-cryptoxanthin (peak 21), and cis- or (Z)-β-cryptoxanthin (peak 22) from γ- and R-tocopherol. 1878 Analytical Chemistry, Vol. 69, No. 10, May 15, 1997

The hydrocarbon carotenoids separated by reversed-phase HPLC (Figure 1) from serum and milk extracts were lycopene (peak 23), (Z)-lycopene (peak 24), neurosporene (peak 25), γ-carotene (peak 26), ζ-carotene (peak 27), R-carotene (peak 28), (all-E)-β-carotene (peak 29), (9Z)-β-carotene (peak 30), (13Z)-βcarotene (peak 31), phytofluene and its Z (cis) isomer (peaks 32 and 33), and phytoene (peak 34). The peak appearing following lycopene (peak 24 in Figure 1) with λmax ) 466 nm and intense Z (cis) peaks at 348 and 362 nm is a mixture of several geometrical isomers of lycopene which are probably of dietary origin. The

most likely dietary source of these geometrical isomers is thermally processed (cooked) tomatoes and tomato-based products. The geometrical isomers of (all-E)-β-carotene were identified by comparing their HPLC-UV/visible-MS profiles with those of reference samples obtained from Hoffmann-La Roche (Basel, Switzerland). Both (9Z)-β-carotene and (13Z)-β-carotene exhibited Z (cis) peaks at 340 nm, but the Z peak was markedly more intense in the absorption spectrum of the latter isomer. The HPLC peak of phytofluene in extracts from serum and milk appeared as an unresolved doublet (peaks 32 and 33, Figure 1), indicating the presence of a geometrical isomer. However, since the absorption spectra of both compounds show maxima at 334, 350, and 368 nm, it is not possible to determine which one of the two HPLC peaks is associated with the (all-E)-phytofluene. This HPLC pattern of elution for phytofluene has also been observed in several extracts from fruits and vegetables.22 Because of the abundant presence of a number of cholestryl esters in serum and milk extracts, which coeluted with the HPLC peaks of phytofluene and phytoene, the molecular parent ion of these carotenoids was not observed by HPLC-MS. Among these esters, only cholestryl oleate is shown in Figure 1, since its close elution to phytoene could result in misidentification. (2) Lutein and Zeaxanthin Metabolites. Among the metabolites of lutein which can be separated on a C18 reversed-phase column are (3R,6′R)-3-hydroxy-3′,4′-didehydro-β,γ-carotene (peak 18, Figure 1) and (3R,6′R)-3-hydroxy-2′,3′-didehydro-β,-carotene (peak 19, Figure 1). These dehydration products of lutein have recently been prepared by partial synthesis from lutein, and their structures have been established by NMR spectroscopy and circular dichroism (CD).12 These metabolites are presumably formed as a result of dehydration of lutein under acidic conditions, similar to the pH of the human stomach. In our opinion, the most interesting metabolites of carotenoids in serum and milk are the oxidative metabolites of lutein and zeaxanthin; these can only be separated by HPLC using a nitrile-bonded column (Figure 2). These consist of a number of monoketocarotenoids (peaks 2, 4, and 5), a diketocarotenoid (peak 1), and a dihydroxycarotenoid (peak 11) known as 3′-epilutein or (3R,3′S,6′R)-lutein. The identification of these carotenoids has been described previously.9 It is important to point out that conventional alkaline hydrolysis of breast milk extracts with KOH resulted in total destruction of ketocarotenoids. Likewise, Buchecker and Eugster23 previously demonstrated that monoketocarotenoids such as 3-hydroxy-β,caroten-3′-one (peak 4, Figure 2) are extremely sensitive to 1% KOH in methanol. Therefore, breast milk samples were hydrolyzed enzymatically to preserve the integrity of the oxidative metabolites of lutein and zeaxanthin. For detailed discussion of the metabolic pathways of lutein and zeaxanthin which lead to the formation of the oxidation products of these compounds, see the publications by Khachik et al.18,20 (3) Lycopene Metabolites. Two lycopene metabolites with a novel five-membered-ring end group, i.e., 3 and 9 (see Table 1 and Figure 3), in serum and milk extracts were identified by comparison of their HPLC-UV/visible-MS profile with those of synthetic compounds. Compounds 3 (major component) and 9 (minor component) are best separated on a nitrile-bonded column (Figure 2). These carotenoids are believed to result from (22) Khachik, F.; Beecher, G. R.; Lusby, W. R. J. Agric. Food Chem. 1989, 37, 1465-1473. (23) Buchecker, R.; Eugster, C. H. Helv. Chim. Acta 1979, 62, 2817-2824.

metabolic oxidation of lycopene to lycopene 5,6-epoxide, which, due to its instability, undergoes rearrangement to form an epimeric mixture of 2,6-cyclolycopene 1,5-epoxides.19,20 The enzymatic or acidic hydrolysis of these epoxides may then yield an epimeric mixture of 2,6-cyclolycopene-1,5-diol I (3, Figure 3) and 2,6cyclolycopene-1,5-diol II (9, Figure 3) as major and minor components, respectively. While the epimeric 2,6-cyclolycopene 1,5-epoxides are not detected in the extracts from serum or milk, their hydrolysis products are present. We have recently proposed possible metabolic pathways for oxidation of lycopene in humans.20 However, the detailed structural elucidation of the oxidative metabolites of lycopene, based on extensive 1H- and 13C-NMR spectroscopic studies, will be published in the future. The metabolites of lycopene (3 and 9) consist of three asymmetric centers at C-2, C-5, and C-6 (Figure 3). Although the relative configurations at C-2, C-5, and C-6 for these carotenoids have been determined from extensive NMR studies, the absolute configurations are not established. The true metabolic formation of 2,6cyclolycopene-1,5-diols (3 and 9) from lycopene in humans remains tenuous, as minute quantities of these carotenoids have been tentatively identified in the extracts of tomatoes and tomatobased products.21 (4) Non-Carotenoids of Dietary Origin. Among the noncarotenoid components of human serum and breast milk, the presence of piperine is intriguing. Piperine occurs in concentrations of ∼3-5% in ground pepper used for culinary purposes. Freshly picked pepper berries (Piper nigrum L.) are “green pepper”, becoming “black pepper” on sun drying and “white pepper” when the dried outer shell of the berries is removed from the black variety. Extracts of serum and milk of the lactating mothers examined by reversed-phase (Figure 1) and normal-phase (Figure 2) HPLC showed the presence of piperine; however, the concentration of this compound in breast milk was about 10-15fold lower than that of serum. To establish the identity of piperine, it was isolated from an extract of serum and milk by preparative HPLC; its HPLC-UV/visible-MS profile was compared with that of a commercially available standard. The UV/visible absorption spectra of piperine (λmax ) 342 nm) in the HPLC solvents (eluents A and B) and ethanol were identical. The mass spectrum (ECNI) of piperine at the ion source temperature used for the analysis of carotenoids by LC-MS (250 °C) consisted of molecular parent ion at m/z 285. However, at lower ion source temperatures of 150-200 °C, the detection sensitivity for piperine increased significantly. During the previous several years, we have measured the concentration of piperine in the serum of 66 subjects and have observed that the serum levels of this compound vary over the range of 200-10 000 µg/dL. Piperine possesses some very interesting biological properties, and the metabolic disposition of this compound in rats has been extensively studied.24 Extraction and HPLC procedures for determination of piperine in black and white pepper have been reported by several researchers.25 However, to our knowledge, this is the first report to identify piperine in human serum and milk. Another non-carotenoid of dietary origin in serum and milk is caffeine, which was identified by comparison of its HPLC-UV/ visible-MS profile with that of a commercially purchased standard (λmax ) 274 nm, m/z 194). (24) Ganesh Bhat, B.; Chandrasekhara, N. Toxicology 1987, 44, 99-106. (25) Weaver, K. M.; Neale, M. E. J. Assoc. Off. Anal. Chem. 1988, 71, 53-55.

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Table 2. Distribution of Carotenoids in Serum and Breast Milka concentration, µg/dL (nmol/L) serum

milk

ratio serum/milk

carotenoidsb

1

2

3

1

2

3

1

2

3

7 + 8 + 12 + 13 + 14 10 + 15 + 16 + 17 11 18 + 19 20 21 + 22 23 + 24 27 28 29 + 30 + 31 32 + 33 34

17.9 (315.0) 1.9 (33.5) 1.5 (26.4) 1.7 (30.9) 3.1 (56.2) 15.0 (271.7) 37.3 (695.9) 10.8 (200.0) 11.7 (218.3) 24.4 (455.2) 7.8 (143.9) 1.9 (34.9)

19.5 (343.3) 2.0 (35.2) 1.3 (22.9) 1.6 (28.4) 2.7 (48.9) 12.7 (230.1) 32.5 (606.3) 5.1 (95.0) 6.0 (111.9) 22.1 (412.3) 6.7 (123.6) 3.3 (60.7)

12.2 (214.8) 2.4 (42.3) 0.8 (14.1) 2.7 (49.1) 4.3 (77.9) 10.6 (192.0) 39.4 (735.1) 1.1 (20.0) 1.6 (29.9) 12.5 (233.2) 13.8 (254.6) 1.9 (34.9)

3.3 (58.1) 0.5 (8.8) 0.3 (5.3) 0.4 (7.3) 0.4 (7.3) 2.7 (48.9) 1.9 (35.5) 0.5 (10.0) 0.9 (16.8) 2.4 (44.8) 1.4 (25.8) 0.1 (1.8)

2.3 (40.5) 0.3 (5.3) 0.1 (1.8) 0.2 (3.6) 0.3 (5.4) 1.6 (29.0) 0.6 (11.2) 0.2 (3.0) 0.3 (5.6) 1.0 (18.7) 0.4 (7.4) 0.1 (1.8)

2.1 (37.0) 0.5 (8.8) 0.1 (1.8) 0.3 (5.5) 0.4 (7.3) 1.2 (21.7) 0.7 (13.1) 0.05 (1.0) 0.1 (1.9) 0.6 (11.2) 0.8 (14.8) 0.1 (1.8)

5.4 3.8 5.0 4.3 7.8 5.6 19.6 20.0 13.0 10.2 5.6 19.0

8.5 6.7 13.0 8.0 9.0 7.9 54.2 31.6 20.0 22.1 16.8 33.0

5.8 4.8 8.0 9.0 10.8 8.8 56.3 20.0 16.0 20.8 17.3 19.0

a

Three different lactating females (1-3). b For identification of carotenoids, see Table 1 (peak numbers correspond to compound numbers).

The HPLC separation of serum and milk carotenoids on a C18 reversed-phase column at the monitoring wavelength of 276 nm revealed the presence of butylated hydroxytoluene (BHT). BHT was added as an antioxidant at the beginning of the extraction of the serum and milk samples to prevent the possible oxidation of carotenoids. BHT was identified by comparison of its HPLCUV/visible-MS profile with that of a reference sample (λmax ) 276-278 nm, m/z 220). (B) Quantitative Distribution of Carotenoids in Serum and Breast Milk. Although vitamin A and vitamin E (γ- and R-tocopherol) could also be quantified by the HPLC methods employed, only the quantification of carotenoids is described here. The carotenoids in the extracts of serum and milk were quantified from the HPLC response factors of the isolated or synthetic reference compounds at five or six different concentrations, employing HPLC system 1 (eluent B) and HPLC system 2. The relative standard deviation for the calibration curves (i.e., area response at various concentrations) of each of the reference samples was less than 5%. Only the dietary carotenoids and their geometrical isomers in serum and milk extracts of the lactating mothers were quantified. With the exception of lutein, zeaxanthin, and 3′-epilutein (metabolite, not of dietary origin), which were quantified on HPLC system 1 (eluent B), the rest of the dietary carotenoids were quantified on HPLC system 2, with a more sensitive detector. Although in this study large volumes of the serum and milk samples were collected to determine the qualitative distribution of carotenoids and their metabolites, only about 1 mL of serum and about 15 mL of breast milk samples are required for routine quantitative analysis by HPLC system 2. The HPLC peak areas of Z isomers of carotenoids were combined with those of their all-E counterparts. Therefore, it has been assumed that the response factors of the Z isomers of carotenoids are reasonably close to that of their (all-E)-carotenoids. The quantitative distribution of carotenoids in serum and breast milk of three lactating mothers is shown in Table 2. As shown in Table 2, depending on the nature of carotenoids, the concentrations of these compounds in breast milk are much lower than those in serum and are comparable to those reported elsewhere by us.1 With regard to the concentration of certain carotenoids in milk, there also appear to be large variations among the three individuals. The mean concentrations of major dietary carotenoids in serum and milk from three lactating mothers who participated 1880 Analytical Chemistry, Vol. 69, No. 10, May 15, 1997

Figure 4. Comparison of the mean concentration of major dietary carotenoids in serum and milk from three lactating mothers. For quantitative values of carotenoids for each subject, see Table 2.

in the present study are compared in Figure 4. Although the concentrations of three major serum carotenoids in these lactating mothers appear to be in the order of lycopene > β-carotene > lutein, the most abundant carotenoids in their breast milk are lutein and zeaxanthin. There is significant interindividual and intraindividual variability in serum and milk carotenoid concentrations.1 Therefore, the data shown in Table 2 should not be considered representative of the concentration of carotenoids in the serum and breast milk of the large human populations, as these data were obtained from only three lactating mothers with diets fairly rich in fruits and vegetables. The data do show that our HPLC-UV/visible-MS procedures can be used for identification and quantification of carotenoids and their metabolites that have not been previously reported. CONCLUSION In this report, we have demonstrated that 34 carotenoids, including 13 of their geometrical isomers and eight metabolites, are present in both human serum and breast milk. This finding emphasizes the importance of dietary carotenoids and their

metabolites in addition to the provitamin A carotenoids, e.g., Rand β-carotene. For nearly 2 decades, it has been suggested that one mechanism by which carotenoids exerted biological activity in disease prevention was by functioning as antioxidants. In 1992, for the first time, we reported detection of several oxidation products of carotenoids in human plasma.9,10 More recently, we demonstrated the human in vivo oxidation of specific carotenoids, e.g., lutein, zeaxanthin, and possibly lycopene.18,20 However, based on results from several recent studies, including our own unpublished data, it appears that carotenoids, in addition to their antioxidant mechanism of action, may also exert their potential biological activity in prevention of certain diseases by other mechanisms. These are (1) enhancement of the activity of the cellular communication,26 (2) stimulation of the activity of the phase II enzymes (detoxification enzymes), and (3) anti-inflammatory/immune-related properties. These mechanisms have also revealed that various carotenoids and their metabolites, depending on their chemical structures, exhibit different degrees of activity. Furthermore, in some cases, these activities are more pronounced with carotenoid metabolites than with dietary carotenoids. We postulate that this wide spectrum of carotenoids and metabolites functions in concert; as a result, their presence in serum and breast milk promotes the health of the mother and infant. Based on our findings, it appears prudent that diets of lactating mothers should include a variety of fruits and vegetables to supply a wide spectrum of bioavailable carotenoids to mother and infant. However, in many instances, because of certain health-related problems, mothers are unable to breast-feed and rely on infant formula to provide adequate nutrition for their child. Currently, most infant formula is fortified with vitamins, nutrients, and, in some cases, β-carotene. We believe, since most of the prominent carotenoids described in this report are currently available from natural sources, the infant formula can be modified to include as many dietary carotenoids as possible. The selection of a mixture of carotenoids for human studies should be carefully designed (26) King, T. J.; Khachik, F.; Bortkiewicz, H.; Fukushima, L. H.; Morioka, S.; Bertram, J. S. Pure Appl. Chem. in press. (27) Khachik, F.; Nir, Z.; Ausich, R. L. Distribution of carotenoids in fruits and vegetables as a criterion for the selection of appropriate chemopreventive agent. Proceedings of the Second International Conference on Food Factors: Chemistry and Cancer Prevention; Ohigashi, H., Ed.; Springer-Verlag: Tokyo, 1997 (in press).

and should closely resemble the relative distribution of these compounds in human serum.27 Future studies should concentrate on elucidating the bioavailability, metabolism, function, interaction, and efficacy of serum and breast milk carotenoids as well as their metabolites. NOMENCLATURE For convenience, the trivial names of certain carotenoids have been used throughout this text. The trivial and correct systematic names for these carotenoids have been presented in Table 1. The trivial names of 2,6-cyclolycopene-1,5-diols I and II have been assigned to the lycopene metabolites with a novel five-memberedring end group. The terms all-E and -Z isomers of carotenoids refer to all-trans and cis isomers of carotenoids, respectively. For in-chain geometrical isomers of carotenoids, the terms all-trans and cis, which have been used with the old nomenclature, are no longer appropriate. ACKNOWLEDGMENT The work described in this text has been presented in part at the Eleventh International Symposium on Carotenoids, Leiden, The Netherlands, August 1996. Partial support by the National Institutes of Health, Grant No. RO1-HD-26715, is acknowledged. The Swiss group thanks the Swiss National Foundation and F. Hoffmann-La Roche, Ltd. (Basel, Switzerland) for financial support. The authors thank Professor Conrad Hans Eugster (Institute of Organic Chemistry, University of Zu¨rich) for his proposed trivial names for the metabolites of lycopene. We also thank Eleanor Neilson (Department of Biochemistry, University of Arizona) for technical assistance. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.

Received for review October 23, 1996. Accepted February 21, 1997.X AC961085I X

Abstract published in Advance ACS Abstracts, April 1, 1997.

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