Relationship carotenoids
between
R.V. Cooneya, J.S. and W. Bourneb “Cancer
Research
bQueen’s Medical
Center Center,
dietary, serum,
Bertram”,
of Hawaii,
J.H.
Hankin”,
and tissue levels of
L.N. Kolonel”,
A. Miyakeb,
Uniuersity of Hawaii, 1236 Lauhala Street, Honolulu, Street, Honohlu, Hawaii 96813 (U.S.A.)
K. Billingsb
Hawaii
96813
and
1301 Punchbowl
(Received 16 August 1991) (Accepted 16 September 1991)
Summary
Keywords: &carotene; lycopene; oids; antioxidants; buccal cells
A study was undertaken to assess the utility of the buccal scrape technique for measuring tissue levels of carotenoids in short-term intervention trials and epidemiologic studies. In 14 healthy volunteers a good correlation was found between serum P-carotene levels and
Introduction Epidemiological studies have pointed to a protective role for dietary P-carotene in the etiology of cancers of the breast [l], ovary [2], lung [3] and oral cavity [4]. Numerous animal and in vitro studies have lent support to these observations by demonstrating that P-carotene can inhibit the carcinogenic process [5 - 71. In addition, P-carotene has been shown to be effective in reversing pre-neoplastic oral leukoplakia in humans [8] and causing significant tumor regression of oral cancers in animal models [9,10]. Two competing theories have emerged to explain the observed chemopreventive and therapeutic properties of flcarotene: one involving its pro-vitamin A activity [ 1 l] and the other its antioxidant properties [12]. One often ignored conclusion of epidemiologic studies is that risk reduction best correlates with diets rich in fruits and vegetables; diets which contain many antioxidants. Included among these are diverse carotenoids which may, or may not, possess pro-vitamin A activity. For example, canthaxanthin, a carotenoid without pro-vitamin A activity in mammals, which is found in certain
recent dietary intake of @carotene as estimated from measured food records. Supplementation with 30 mg/day of P-carotene for 1 week resulted in a sixfold increase in average serum levels, while serum lycopene concentrations remained constant. Presupplementation leoels of P-carotene and lycopene in the buccal mucosa cells were not
correlated with dietary intakes or with serum levels. After supplementation, levels of both carotenoids were found to increase in buccal cells, however, most of this increase was found to be an artifact due to repeated sampling. After correcting for this artifact, P-carotene was found to increase less than twofold in tissue after supplementation.
Correspondence Lo: R. V. Cooney, Cancer Research Center of Hawaii, 1236 Lauhala Street, Honolulu, Hawaii 96813, U.S.A.
0304-3835/91/$03.50 Published and Printed
0 1991 Elsevier Scientific in Ireland
Publishers
caroten-
Ireland
Ltd
82
shellfish and is used as an approved food coloring agent [ 131, has been reported to possess cancer chemopreventive properties in vitro [6] and in vivo [14]. Lycopene, the major carotenoid in tomatoes, is a potent antioxidant [15], has shown activity in an in vitro test system [16], and has been associated with a reduced risk of cancer in epidemiologic studies [3]. Whether dietary carotenoids other than Pcarotene possess chemopreventive activity in humans and, if so, what the structural requirements are for activity, are questions that have only recently begun to be addressed. One important means of predicting the relative contribution of other carotenoids to risk reduction in humans is the determination of the relative potency of diverse carotenoids with varying antioxidant and vitamin A activities as preventive agents in experimental animal model systems of carcinogenesis. Unfortunately, the usual rodent test systems are not good models for humans in terms of absorption and distribution of carotenoids [ 171. Using a defined cell culture system, we recently reported that several carotenoids inhibit neoplastic transformation at concentrations comparable to P-carotene [16] _ This may be significant in light of the variety of carotenoids found in the diet and in the serum [18]. Although measurements of dietary intake and serum levels of carotenoids have proven useful in epidemiological assessments, it is presumed to be at the cellular level that carotenoids function. Consequently, tissue concentrations may be more indicative of protection than serum levels or dietary intake. Studies of uptake of diverse carotenoids by cells in tissue culture have demonstrated large differences in accumulation and stability of carotenoids [ 16,191. Furthermore, in tissue culture, cellular concentrations of carotenoids do not increase as a linear function of extracellular concentration [ 161, suggesting that natural limits may exist for uptake. In order to determine if the observations made in vitro extend to tissue distributions in humans, we undertook a study to compare dietary intake with serum and buccal tissue levels of fl-
carotene and lycopene in a subject population given dietary supplements of P-carotene. Materials
and Methods
Experimental The study comprised a group of 14 healthy subjects (5 male and 9 female non-smokers of mixed ethnicity ranging in age from 23 to 54) employed at either the Queen’s Medical Center or the Cancer Research Center of Hawaii. A dietary questionnaire was administered to the subjects in order to assess @carotene consumption during the week prior to the study. Blood samples (3 ml) and buccal scrapings were obtained and the subjects were provided with 7 capsules of Solatene (generously provided by Hoffman La Roche, Nutley, NJ) containing 30 mg P-carotene each. Subjects were instructed to take 1 capsule per day at the evening meal for a l-week period. During this period they measured and recorded their dietary intakes in a diary. At the end of the week, they returned to the Clinic for an additional blood sample and buccal smear and collection of the diaries for analysis. The technique described by Stich et al. [20] was used to obtain buccal cells. Cells were removed from the buccal cavity of the patient by firmly brushing the oral mucosa with a wet toothbrush. The subject then rinsed with 20 ml of water which was also used to rinse any cells remaining on the toothbrush. The cells were counted electronically, spun down at 2000 rev./min for 5 min and extracted according to the method of Rundhaug et al. [19]. We routinely collected lo6 cells by this method. Dietary assessment The dietary assessment included a weekly recall and a 7-day food record. On the first day of the study, the subject completed a selfadministered questionnaire of the frequencies and amounts of 31 major sources of carotenoids consumed during the past week. The food items included frequently consumed vegetables, fruits and juices, along with vegetable soup, beef stew, and chicken stew, which
83
are popular in Hawaii. Quantities were estimated from colored photographs showing three different serving sizes of each food. We also obtained a recall of the frequencies and amounts of beer, wine, and liquor consumed during this period. For the subsequent 7 days, during the /3carotene supplementation, subjects kept a measured record of all food items and beverages consumed. The recalls and records were analyzed from our comprehensive food composition data base. The primary source was the US Department of Agriculture’s Handbook No. 8 series [21,22], supplemented with data from several other sources. Because published values on the specific carotenoids were not available, we followed the procedures recommended by the FAO/WHO Expert Group [23], who published a percentage contribution of vitamin A from retinol, P-carotene, and other carotenes for various food groups. We then utilized the appropriate formulas for deriving the amounts of these components in each food [24]. HPLC analysis of carotenoids Cells and sera were extracted as previously described [19]. Extracts were dried under nitrogen and redissolved in 0.3 ml (0.1 ml for cell samples) of mobile phase (acetonitrile/ methylene chloride/methanol/hexane 60:20: 10:5) containing 0.025% BHT as antioxidant immediately prior to analysis. High pressure liquid chromatography (HPLC) was conducted with a Beckman System Gold with a Diode Array Spectrophotometer as detector. Twenty microliters of sample were injected onto a Spherex 5 pm reverse-phase C-18 column (4.6 x 250 mm), purchased from Phenomonex Corporation (Torrance, CA). The sample was eluted using the pre-mixed mobile phase described above at a flow rate of 1.5 ml/min. The effluent was monitored at 457 spectra and retention nm, and absorption times for each peak were compared to those of known standards. Peak areas relative to authentic standards were used to quantitate pcarotene and lycopene.
Results Pre-supplementation carotenoid profiles In addition to lycopene and P-carotene, a number of other carotenoids were found in varying extent in the sera and cells examined (Fig. 1). In all subjects the general pattern of carotenoids observed appeared similar between sera and cells, however, no quantitative correlation between cellular and serum levels was observed in individual subjects. The average ratio of P-carotene to lycopene for all subjects combined was similar between serum and cells (Table I), however, on an individual basis no correlation was observed for this ratio between serum and cells. Post-supplementation carotenoid levels As shown in Table I, average serum lycopene concentrations remained relatively constant over the week of supplementation, while serum P-carotene rose an average of sixfold (the level of supplementation represented approximately a lo-fold increase above the normal average dietary intake of P-carotene). analysis of buccal scrapings As expected, revealed a fivefold increase in average cellular P-carotene level. Cellular lycopene also increased threefold over the same period although no supplementation was given, nor was any major alteration noted in dietary intake during the observation period. Consistent with the dietary measurements, serum levels of lycopene showed no increase during this period (Table I). To determine if the increase in cellular lycopene was an artifact of the four additional insampling procedure, dividuals had their buccal mucosa scraped 1 week prior to the start of the experiment. They were then subjected to the same protocol described for the other subjects. These subjects showed no increase in cellular lycopene levels and only a modest increase in P-carotene levels after the week of supplementation (Table I). We thus conclude that the dramatic rise of cellular P-carotene and lycopene observed in individuals not subjected to a buccal pre-scrape, was, in part, an artifact
: .08- B
6.
1 .t.
.
,
,c.:.
*
t...t
.I]
_.,
z..::
4
t
0
2
4
6
8
0
2
4
6
8
0
2
4
6
8
0
2
4
6
8
TIME
(minutes)
Fig.1. Representative chromatographic profiles for Subject No.14 (no pre-scrape) of extracts from serum prior to (A) and after (B) @-carotene supplementation and cells prior to (C) and after (D) 1 week of supplementation. Inset: fivefold enhancement of the region between 4 and 7.5 min. (1) Lutein/zeaxanthin; (2) fl-cryptoxanthin; (3) lycopene; (4) OLcarotene; (5) trans-p-carotene; (6) cis-b-carotene.
resulting from the different ages of the sampled cells. There was a generally good correlation (r = 0.822) between serum levels of @-carotene and dietary intake as measured by a 1 week food record (Fig. 21, and all subjects showed increases in serum P-carotene significant after supplementation (average increase = 3.5 FM). Comparisons among individuals revealed fat intake to be positively associated (P = 0.24) and alcohol consumption negatively associated (P = 0.1) with the
observed post-supplementation increase in serum P-carotene levels, however, the effects were not statistically significant (data not shown). Cellular levels of P-carotene did not correlate with either dietary intake or serum values. Discussion The buccal scrape technique has been used previously to demonstrate that P-carotene supplementation can result in an increase in tissue
85
Table 1. Mean serum and cellular levels of p-carotene Time analyzed
Serum
and lycopene.
levels (pmol/l)
@-Carotene All subjects Day 1 Day 8
(N = 14)
Without prescrape Day 1
With prescrape Day 1 Day 8
0.7 f 0.2 4.2 * 0.5”
1.1 f 1.4 f
0.2 0.2
0.67 3.11”
0.9 f 0.2 5.0 f 0.5”
1.2 f 0.2 1.6 * 0.2
0.79 3.1gb
0.3 f 0.1 2.4 * 0.4’
0.9 f 0.1 0.8 A 0.1
0.30 2.95b
(N = IO)
Day 8 (N = 4)
Cellular levels (ng/106 All subjects Day 1 Day 8 Without prescrape Day 1 Day 8 With prescrape Day 1 Day 8
Ratio &C/Lyc
Lycopene
2.9 f 15.0 f
1 3.4’
cells)
4.9 f 14.4 f
1.5 2.7’
0.59 1.04d
1.6 +Z 0.6 18.0 f 4.4’
1.9 f 0.5 16.4 A 3.8’
0.84 1.10
6.0 f 2.4 7.7 f 3.0
12.4 f 2.5 10.0 l 1.6
0.48 0.77
Sera and buccal cells were analyzed for p-carotene and lycopene, as described in the Methods section, prior to supplementation (Day 1) and after 1 week of supplementation with 30 mg P-carotene per day (Day 8). In 4 individuals the on “P bP ‘P
buccal mucosa was pre-scraped 1 week prior to the start of the experiment the observed carotenoid levels. < 0.0001 for difference in means between Day 1 and Day 8. < 0.001 for difference in means between Day 1 and Day 8. < 0.01 for difference in means between Day 1 and Day 8. dP < 0.05 for difference in means between Day 1 and Day 8.
levels of this carotenoid [25,26]. The present study is the first to simultaneously measure the levels of P-carotene and lycopene in both buccal cells and serum. Because of the relatively constant intra-individual levels of lycopene observed in the serum, we felt that lycopene might serve as a useful internal standard for quantifying increases in serum and cellular /3-
to determine
the effect of buccal scraping
carotene. Cellular levels of P-carotene were consistent with those previously reported [26], however, the significant increases found in both P-carotene and lycopene after the week of supplementation suggested that the increase might be due in part to a sampling artifact related to repeated scraping of the buccal mucosa. Support for this hypothesis was ob-
DIETARY B-CAROTENE
(mg/day)
Fig. 2. Correlation between serum levels of @-carotene and dietary intake. All subjects combined. Serum levels were determined prior to supplementation. Dietary intake of P-carotene in the previous week was estimated from a diet history as described in Materials and Methods.
tained by pre-scraping the buccal mucosa of several individuals prior to the start of the experimental protocol (Table I). Cellular lycopene levels in these individuals remained constant while P-carotene rose slightly. One possible explanation for these observations is that the Iayer of buccal mucosa removed in the first scraping represents a much older population of cells than that obtained in a second scrape collected 1 week later (or a third scrape 2 weeks after the first). These older cells collected initially may have lost carotenoids through diffusion and/or chemical breakdown. The earlier study of Stich et al. [25] which observed an increase in buccal pcarotene over a 4-month period was probably this sampling effect. not affected by Presumably it is only in short-term supplementation studies that repeated sampling may cause this artifactual increase. The similarity in cellular concentrations of /3carotene among the subjects in our study is rather surprising in light of the wide disparity in dietary intake and serum levels. This parallels the situation observed in cell culture where we find that cellular uptake is not linearly related to the concentration in culture medium [16]. These findings suggest that mechanisms may
exist for maintaining intracellular P-carotene homeostasis or that the cells collected in the outer layer of the buccal mucosa do not reflect carotenoid concentrations found in viable tissue. All individuals increased their serum Pcarotene in response to supplementation, however, the magnitude of the increase varied between individuals. Previous studies have suggested a role for fat [27] and alcohol [28] in P-carotene uptake. As noted, our studies indicated a weak positive association between fat intake and a negative association between alcohol intake and serum levels of P-carotene. Lycopene levels showed much less inter- and intra-individual variation than did P-carotene levels in serum. The average ratio of @carotene to lycopene for all subjects prior to supplementation was approximately the same for serum (0.67) and cells (0.59). The present study in addition to pointing out limitations to the buccal scrape method for short-term studies, offers evidence that tissue levels of other carotenoids can be effectively measured in buccal tissue. In addition to /3carotene and lycopene which were quanwe have observed @titatively measured, cryptoxanthin, lutein and o-carotene in buccal tissue. The ability to detect tissue levels of these diverse carotenoids may be very useful in epidemiologic and chemoprevention studies, however, it will be essential to standardize collection techniques and the routine use of a prescrape may provide greater consistency of measurements between subjects. The accumuthat diverse carotenoids lating evidence possess cancer preventive properties calls for an increased knowledge of their presence and concentration in foods, and of their absorption and tissue distribution. Acknowledgments We wish to thank Linda Kuriyama for her assistance in manuscript preparation and Melissa Churley for technical assistance. This research was supported by grants from the University of Hawaii Research Council, the
87
American Cancer Society Queen’s Medical Center.
(No. BC 686) and
irradiated mice by the combination of retinyl palmitate canthaxanthin. Cancer Lett., 53, 27 - 31.
16
DiMascio. P.. Kaiser, S. and Sies. H. (1989) Lycopene as the most efficient biological singlet oxygen quencher. Arch. Biochem. Biophys.. 274, 532 - 538. Bertram. J.S., Pung. A., Churley. M., Kappock. T.J..
17
Wilkens, L.R. and Cooney, R.V. (1991) Diverse carotenoids protect from chemically-induced neoplastic transformation. Carcinogenesis 12. 671- 678. Bauernfeind, J.C. (1972) Carotenoid vitamin precursors
15
References 1
2
3
4
5
6
7
8
9
10
11
12 13 14
Howe, G.R.. Hirohata, T. and Hislop. T.G. et al. (1990) Dietary factors and risk of breast cancer: combined analysis of 12 case-control studies. J. Natl. Cancer Inst.. 82, 561-569. Slattery, M.L., Schuman, K.L., West, D.W., French, T.K. and Robinson, L.M. (1989) Nutrient intake and ovarian cancer. Am. J. Epidemiol., 130, 497-502. Le Marchand. L., Yoshizawa, C.N., Kolonel, L.N., Hankin. J.H. and Goodman, M.T. (1989) Vegetable consumption and lung cancer risk: a population-based casecontrol study in Hawaii. J. Natl. Cancer Inst.. 81, 1158- 1164. Winn, D.M., Ziegler, R.G., Pickle, L.W., Gridley. G., Blot, W.J. and Hoover, R.N. (1984) Diet in the etiology
18
19
of oral and pharyngeal cancer among women from the southern United States. Cancer Res., 144. 1216- 1222. Mathews-Roth, M.M. (1985) Carotenoids and cancer prevention: experimental and epidemiological studies.
20
Pure Appl. Chem., 57, 717-722. Pung, A., Rundhaug, J.. Yoshizawa. C.N. and Bertram. and canthaxanthin inhibit J.S. (1988) P-Carotene chemically- and physically-induced neoplastic transforma-
21
tion in lOT1/2 cells. Carcinogenesis, 9, 1533- 1539. Som, S., Chatterjee, M. and Banerjee. M.R. (1984) PCarotene inhibition of 7.12-dimethylbenzanthraceneinduced transformation of murine mammary cells in vitro. Carcinogenesis. 5. 937 - 940. Stich, H.F., Rosin, M.P.. Hornby, A.P., Mathew, B.. Sankararayanan. R. and Nair, M.K. (1988) Remission of oral leukoplakias and micronuclei in tobacco/betel quid chewers treated with p-carotene and with @-carotene plus vitamin A. Int. J. Cancer, 42, 195- 199. Suda, D., Schwartz, J. and Shklar, G. (1986) Inhibition of experimental oral carcinogenesis by topical p-carotene.
and
22
23
24
and analogs in foods and feeds. Agric. Food Chem.. 20. 456 - 473. Khachik, F., Beecher. G.R., Goli. M.B. and Lusby, W.R. (1991) Separation, identification and quantification of carotenoids in fruits, vegetables and human plasma by high performance liquid chromatography Pure Appl. Chem 63, 71-80. Rundhaug, J.E.. Pung, A., Read. C.M. and Bertram. J.S. (1988) Uptake and metabolism of o-carotene and retinal by C3H/lOT1/2 cells. Carcinogenesis. 9. 1541- 1545. Stich. H.F.. Hornby. A.P. and Dunn, B.P. (1986) BCarotene levels in exfoliated mucosal cells of population groups at low and elevated risk for oral cancer. Int. J Cancer, 37, 389-393. US Department of Agriculture (1972) Composition of foods: raw, processed, prepared. Data set 8-l-l. Agricultural Research Service, Washington, DC. US Department of Agriculture (1976 - 89) Composition of foods, raw, processed, prepared. Handbooks Nos. 8-l to 8-21. US Department of Agriculture, Washington, DC. FAO/WHO Expert Group (1972) Estimated distribution of sources of vitamin A activity in various foods In: Food Composition Table for Use in East Asia, p. 3. Food and Agriculture Organization and National Institutes of Health, Bethesda, MD. Food and Nutrition Board, National Research Council (1980) Recommended Dietary Allowances, 9th edn.. pp. 56-57. Natl. Acad. of Sciences, Washington D.C.
Carcinogenesis, 7, 711- 715. Schwartz, J. and Shklar, G. (1988) Regression of experimental oral carcinomas by local injection of p-carotene and canthaxanthin. Nutr. Cancer, 11, 35-40.
26
Stich, H.F.. Hornby. A.P. and Dunn, B.P. (1986) 8Carotene levels in exfoliated human mucosa cells following its oral administartion. Cancer Lett., 30. 133 - 141 Gilbert, A.M., Stich, H.F , Rosin, M.P. and Davison. A.J.
Sporn, M.B. and Newton, D.L. (1981) Retinoids and chemoprevention of cancer. In: Inhibition of Tumor fnduction and Development. pp. 71- 100. Plenum Press, New York.
27
(1990) Variations in the uptake of p-carotene in the oral mucosa of individuals after 3 days of supplementation Int. J. Cancer, 45. 855-859. Dimitrov. N.V.. Meyer, C.. Ullrey, D.E. et al (1988)
Burton, G.W. and Ingold, K.U. (1984) @-Carotene: an unusual type of lipid antioxidant. Science, 224, 569 - 573. Straub. 0. (Ed.) (1976) Key to Carotenoids. Birkhauser, Basel. Gensler, H.L., Aickin. M. and Peng, Y.M. (1990) Cumulative reduction of primary skin tumor growth in UV-
25
28
Bioavailability of p-carotene in humans Am. J. Clan. Nutr., 48, 298 - 304. Suzuki, S., Sasaki. R. and Ito. Y. et al. (1990) Changes in serum concentrations of p-carotene and changes in the dietary intake frequency of green-yellow vegetables among healthy male inhabitants of Japan. Jpn J. Cancer Res.. 81. 463-469
between
R.V. Cooneya, J.S. and W. Bourneb “Cancer
Research
bQueen’s Medical
Center Center,
dietary, serum,
Bertram”,
of Hawaii,
J.H.
Hankin”,
and tissue levels of
L.N. Kolonel”,
A. Miyakeb,
Uniuersity of Hawaii, 1236 Lauhala Street, Honolulu, Street, Honohlu, Hawaii 96813 (U.S.A.)
K. Billingsb
Hawaii
96813
and
1301 Punchbowl
(Received 16 August 1991) (Accepted 16 September 1991)
Summary
Keywords: &carotene; lycopene; oids; antioxidants; buccal cells
A study was undertaken to assess the utility of the buccal scrape technique for measuring tissue levels of carotenoids in short-term intervention trials and epidemiologic studies. In 14 healthy volunteers a good correlation was found between serum P-carotene levels and
Introduction Epidemiological studies have pointed to a protective role for dietary P-carotene in the etiology of cancers of the breast [l], ovary [2], lung [3] and oral cavity [4]. Numerous animal and in vitro studies have lent support to these observations by demonstrating that P-carotene can inhibit the carcinogenic process [5 - 71. In addition, P-carotene has been shown to be effective in reversing pre-neoplastic oral leukoplakia in humans [8] and causing significant tumor regression of oral cancers in animal models [9,10]. Two competing theories have emerged to explain the observed chemopreventive and therapeutic properties of flcarotene: one involving its pro-vitamin A activity [ 1 l] and the other its antioxidant properties [12]. One often ignored conclusion of epidemiologic studies is that risk reduction best correlates with diets rich in fruits and vegetables; diets which contain many antioxidants. Included among these are diverse carotenoids which may, or may not, possess pro-vitamin A activity. For example, canthaxanthin, a carotenoid without pro-vitamin A activity in mammals, which is found in certain
recent dietary intake of @carotene as estimated from measured food records. Supplementation with 30 mg/day of P-carotene for 1 week resulted in a sixfold increase in average serum levels, while serum lycopene concentrations remained constant. Presupplementation leoels of P-carotene and lycopene in the buccal mucosa cells were not
correlated with dietary intakes or with serum levels. After supplementation, levels of both carotenoids were found to increase in buccal cells, however, most of this increase was found to be an artifact due to repeated sampling. After correcting for this artifact, P-carotene was found to increase less than twofold in tissue after supplementation.
Correspondence Lo: R. V. Cooney, Cancer Research Center of Hawaii, 1236 Lauhala Street, Honolulu, Hawaii 96813, U.S.A.
0304-3835/91/$03.50 Published and Printed
0 1991 Elsevier Scientific in Ireland
Publishers
caroten-
Ireland
Ltd
82
shellfish and is used as an approved food coloring agent [ 131, has been reported to possess cancer chemopreventive properties in vitro [6] and in vivo [14]. Lycopene, the major carotenoid in tomatoes, is a potent antioxidant [15], has shown activity in an in vitro test system [16], and has been associated with a reduced risk of cancer in epidemiologic studies [3]. Whether dietary carotenoids other than Pcarotene possess chemopreventive activity in humans and, if so, what the structural requirements are for activity, are questions that have only recently begun to be addressed. One important means of predicting the relative contribution of other carotenoids to risk reduction in humans is the determination of the relative potency of diverse carotenoids with varying antioxidant and vitamin A activities as preventive agents in experimental animal model systems of carcinogenesis. Unfortunately, the usual rodent test systems are not good models for humans in terms of absorption and distribution of carotenoids [ 171. Using a defined cell culture system, we recently reported that several carotenoids inhibit neoplastic transformation at concentrations comparable to P-carotene [16] _ This may be significant in light of the variety of carotenoids found in the diet and in the serum [18]. Although measurements of dietary intake and serum levels of carotenoids have proven useful in epidemiological assessments, it is presumed to be at the cellular level that carotenoids function. Consequently, tissue concentrations may be more indicative of protection than serum levels or dietary intake. Studies of uptake of diverse carotenoids by cells in tissue culture have demonstrated large differences in accumulation and stability of carotenoids [ 16,191. Furthermore, in tissue culture, cellular concentrations of carotenoids do not increase as a linear function of extracellular concentration [ 161, suggesting that natural limits may exist for uptake. In order to determine if the observations made in vitro extend to tissue distributions in humans, we undertook a study to compare dietary intake with serum and buccal tissue levels of fl-
carotene and lycopene in a subject population given dietary supplements of P-carotene. Materials
and Methods
Experimental The study comprised a group of 14 healthy subjects (5 male and 9 female non-smokers of mixed ethnicity ranging in age from 23 to 54) employed at either the Queen’s Medical Center or the Cancer Research Center of Hawaii. A dietary questionnaire was administered to the subjects in order to assess @carotene consumption during the week prior to the study. Blood samples (3 ml) and buccal scrapings were obtained and the subjects were provided with 7 capsules of Solatene (generously provided by Hoffman La Roche, Nutley, NJ) containing 30 mg P-carotene each. Subjects were instructed to take 1 capsule per day at the evening meal for a l-week period. During this period they measured and recorded their dietary intakes in a diary. At the end of the week, they returned to the Clinic for an additional blood sample and buccal smear and collection of the diaries for analysis. The technique described by Stich et al. [20] was used to obtain buccal cells. Cells were removed from the buccal cavity of the patient by firmly brushing the oral mucosa with a wet toothbrush. The subject then rinsed with 20 ml of water which was also used to rinse any cells remaining on the toothbrush. The cells were counted electronically, spun down at 2000 rev./min for 5 min and extracted according to the method of Rundhaug et al. [19]. We routinely collected lo6 cells by this method. Dietary assessment The dietary assessment included a weekly recall and a 7-day food record. On the first day of the study, the subject completed a selfadministered questionnaire of the frequencies and amounts of 31 major sources of carotenoids consumed during the past week. The food items included frequently consumed vegetables, fruits and juices, along with vegetable soup, beef stew, and chicken stew, which
83
are popular in Hawaii. Quantities were estimated from colored photographs showing three different serving sizes of each food. We also obtained a recall of the frequencies and amounts of beer, wine, and liquor consumed during this period. For the subsequent 7 days, during the /3carotene supplementation, subjects kept a measured record of all food items and beverages consumed. The recalls and records were analyzed from our comprehensive food composition data base. The primary source was the US Department of Agriculture’s Handbook No. 8 series [21,22], supplemented with data from several other sources. Because published values on the specific carotenoids were not available, we followed the procedures recommended by the FAO/WHO Expert Group [23], who published a percentage contribution of vitamin A from retinol, P-carotene, and other carotenes for various food groups. We then utilized the appropriate formulas for deriving the amounts of these components in each food [24]. HPLC analysis of carotenoids Cells and sera were extracted as previously described [19]. Extracts were dried under nitrogen and redissolved in 0.3 ml (0.1 ml for cell samples) of mobile phase (acetonitrile/ methylene chloride/methanol/hexane 60:20: 10:5) containing 0.025% BHT as antioxidant immediately prior to analysis. High pressure liquid chromatography (HPLC) was conducted with a Beckman System Gold with a Diode Array Spectrophotometer as detector. Twenty microliters of sample were injected onto a Spherex 5 pm reverse-phase C-18 column (4.6 x 250 mm), purchased from Phenomonex Corporation (Torrance, CA). The sample was eluted using the pre-mixed mobile phase described above at a flow rate of 1.5 ml/min. The effluent was monitored at 457 spectra and retention nm, and absorption times for each peak were compared to those of known standards. Peak areas relative to authentic standards were used to quantitate pcarotene and lycopene.
Results Pre-supplementation carotenoid profiles In addition to lycopene and P-carotene, a number of other carotenoids were found in varying extent in the sera and cells examined (Fig. 1). In all subjects the general pattern of carotenoids observed appeared similar between sera and cells, however, no quantitative correlation between cellular and serum levels was observed in individual subjects. The average ratio of P-carotene to lycopene for all subjects combined was similar between serum and cells (Table I), however, on an individual basis no correlation was observed for this ratio between serum and cells. Post-supplementation carotenoid levels As shown in Table I, average serum lycopene concentrations remained relatively constant over the week of supplementation, while serum P-carotene rose an average of sixfold (the level of supplementation represented approximately a lo-fold increase above the normal average dietary intake of P-carotene). analysis of buccal scrapings As expected, revealed a fivefold increase in average cellular P-carotene level. Cellular lycopene also increased threefold over the same period although no supplementation was given, nor was any major alteration noted in dietary intake during the observation period. Consistent with the dietary measurements, serum levels of lycopene showed no increase during this period (Table I). To determine if the increase in cellular lycopene was an artifact of the four additional insampling procedure, dividuals had their buccal mucosa scraped 1 week prior to the start of the experiment. They were then subjected to the same protocol described for the other subjects. These subjects showed no increase in cellular lycopene levels and only a modest increase in P-carotene levels after the week of supplementation (Table I). We thus conclude that the dramatic rise of cellular P-carotene and lycopene observed in individuals not subjected to a buccal pre-scrape, was, in part, an artifact
: .08- B
6.
1 .t.
.
,
,c.:.
*
t...t
.I]
_.,
z..::
4
t
0
2
4
6
8
0
2
4
6
8
0
2
4
6
8
0
2
4
6
8
TIME
(minutes)
Fig.1. Representative chromatographic profiles for Subject No.14 (no pre-scrape) of extracts from serum prior to (A) and after (B) @-carotene supplementation and cells prior to (C) and after (D) 1 week of supplementation. Inset: fivefold enhancement of the region between 4 and 7.5 min. (1) Lutein/zeaxanthin; (2) fl-cryptoxanthin; (3) lycopene; (4) OLcarotene; (5) trans-p-carotene; (6) cis-b-carotene.
resulting from the different ages of the sampled cells. There was a generally good correlation (r = 0.822) between serum levels of @-carotene and dietary intake as measured by a 1 week food record (Fig. 21, and all subjects showed increases in serum P-carotene significant after supplementation (average increase = 3.5 FM). Comparisons among individuals revealed fat intake to be positively associated (P = 0.24) and alcohol consumption negatively associated (P = 0.1) with the
observed post-supplementation increase in serum P-carotene levels, however, the effects were not statistically significant (data not shown). Cellular levels of P-carotene did not correlate with either dietary intake or serum values. Discussion The buccal scrape technique has been used previously to demonstrate that P-carotene supplementation can result in an increase in tissue
85
Table 1. Mean serum and cellular levels of p-carotene Time analyzed
Serum
and lycopene.
levels (pmol/l)
@-Carotene All subjects Day 1 Day 8
(N = 14)
Without prescrape Day 1
With prescrape Day 1 Day 8
0.7 f 0.2 4.2 * 0.5”
1.1 f 1.4 f
0.2 0.2
0.67 3.11”
0.9 f 0.2 5.0 f 0.5”
1.2 f 0.2 1.6 * 0.2
0.79 3.1gb
0.3 f 0.1 2.4 * 0.4’
0.9 f 0.1 0.8 A 0.1
0.30 2.95b
(N = IO)
Day 8 (N = 4)
Cellular levels (ng/106 All subjects Day 1 Day 8 Without prescrape Day 1 Day 8 With prescrape Day 1 Day 8
Ratio &C/Lyc
Lycopene
2.9 f 15.0 f
1 3.4’
cells)
4.9 f 14.4 f
1.5 2.7’
0.59 1.04d
1.6 +Z 0.6 18.0 f 4.4’
1.9 f 0.5 16.4 A 3.8’
0.84 1.10
6.0 f 2.4 7.7 f 3.0
12.4 f 2.5 10.0 l 1.6
0.48 0.77
Sera and buccal cells were analyzed for p-carotene and lycopene, as described in the Methods section, prior to supplementation (Day 1) and after 1 week of supplementation with 30 mg P-carotene per day (Day 8). In 4 individuals the on “P bP ‘P
buccal mucosa was pre-scraped 1 week prior to the start of the experiment the observed carotenoid levels. < 0.0001 for difference in means between Day 1 and Day 8. < 0.001 for difference in means between Day 1 and Day 8. < 0.01 for difference in means between Day 1 and Day 8. dP < 0.05 for difference in means between Day 1 and Day 8.
levels of this carotenoid [25,26]. The present study is the first to simultaneously measure the levels of P-carotene and lycopene in both buccal cells and serum. Because of the relatively constant intra-individual levels of lycopene observed in the serum, we felt that lycopene might serve as a useful internal standard for quantifying increases in serum and cellular /3-
to determine
the effect of buccal scraping
carotene. Cellular levels of P-carotene were consistent with those previously reported [26], however, the significant increases found in both P-carotene and lycopene after the week of supplementation suggested that the increase might be due in part to a sampling artifact related to repeated scraping of the buccal mucosa. Support for this hypothesis was ob-
DIETARY B-CAROTENE
(mg/day)
Fig. 2. Correlation between serum levels of @-carotene and dietary intake. All subjects combined. Serum levels were determined prior to supplementation. Dietary intake of P-carotene in the previous week was estimated from a diet history as described in Materials and Methods.
tained by pre-scraping the buccal mucosa of several individuals prior to the start of the experimental protocol (Table I). Cellular lycopene levels in these individuals remained constant while P-carotene rose slightly. One possible explanation for these observations is that the Iayer of buccal mucosa removed in the first scraping represents a much older population of cells than that obtained in a second scrape collected 1 week later (or a third scrape 2 weeks after the first). These older cells collected initially may have lost carotenoids through diffusion and/or chemical breakdown. The earlier study of Stich et al. [25] which observed an increase in buccal pcarotene over a 4-month period was probably this sampling effect. not affected by Presumably it is only in short-term supplementation studies that repeated sampling may cause this artifactual increase. The similarity in cellular concentrations of /3carotene among the subjects in our study is rather surprising in light of the wide disparity in dietary intake and serum levels. This parallels the situation observed in cell culture where we find that cellular uptake is not linearly related to the concentration in culture medium [16]. These findings suggest that mechanisms may
exist for maintaining intracellular P-carotene homeostasis or that the cells collected in the outer layer of the buccal mucosa do not reflect carotenoid concentrations found in viable tissue. All individuals increased their serum Pcarotene in response to supplementation, however, the magnitude of the increase varied between individuals. Previous studies have suggested a role for fat [27] and alcohol [28] in P-carotene uptake. As noted, our studies indicated a weak positive association between fat intake and a negative association between alcohol intake and serum levels of P-carotene. Lycopene levels showed much less inter- and intra-individual variation than did P-carotene levels in serum. The average ratio of @carotene to lycopene for all subjects prior to supplementation was approximately the same for serum (0.67) and cells (0.59). The present study in addition to pointing out limitations to the buccal scrape method for short-term studies, offers evidence that tissue levels of other carotenoids can be effectively measured in buccal tissue. In addition to /3carotene and lycopene which were quanwe have observed @titatively measured, cryptoxanthin, lutein and o-carotene in buccal tissue. The ability to detect tissue levels of these diverse carotenoids may be very useful in epidemiologic and chemoprevention studies, however, it will be essential to standardize collection techniques and the routine use of a prescrape may provide greater consistency of measurements between subjects. The accumuthat diverse carotenoids lating evidence possess cancer preventive properties calls for an increased knowledge of their presence and concentration in foods, and of their absorption and tissue distribution. Acknowledgments We wish to thank Linda Kuriyama for her assistance in manuscript preparation and Melissa Churley for technical assistance. This research was supported by grants from the University of Hawaii Research Council, the
87
American Cancer Society Queen’s Medical Center.
(No. BC 686) and
irradiated mice by the combination of retinyl palmitate canthaxanthin. Cancer Lett., 53, 27 - 31.
16
DiMascio. P.. Kaiser, S. and Sies. H. (1989) Lycopene as the most efficient biological singlet oxygen quencher. Arch. Biochem. Biophys.. 274, 532 - 538. Bertram. J.S., Pung. A., Churley. M., Kappock. T.J..
17
Wilkens, L.R. and Cooney, R.V. (1991) Diverse carotenoids protect from chemically-induced neoplastic transformation. Carcinogenesis 12. 671- 678. Bauernfeind, J.C. (1972) Carotenoid vitamin precursors
15
References 1
2
3
4
5
6
7
8
9
10
11
12 13 14
Howe, G.R.. Hirohata, T. and Hislop. T.G. et al. (1990) Dietary factors and risk of breast cancer: combined analysis of 12 case-control studies. J. Natl. Cancer Inst.. 82, 561-569. Slattery, M.L., Schuman, K.L., West, D.W., French, T.K. and Robinson, L.M. (1989) Nutrient intake and ovarian cancer. Am. J. Epidemiol., 130, 497-502. Le Marchand. L., Yoshizawa, C.N., Kolonel, L.N., Hankin. J.H. and Goodman, M.T. (1989) Vegetable consumption and lung cancer risk: a population-based casecontrol study in Hawaii. J. Natl. Cancer Inst.. 81, 1158- 1164. Winn, D.M., Ziegler, R.G., Pickle, L.W., Gridley. G., Blot, W.J. and Hoover, R.N. (1984) Diet in the etiology
18
19
of oral and pharyngeal cancer among women from the southern United States. Cancer Res., 144. 1216- 1222. Mathews-Roth, M.M. (1985) Carotenoids and cancer prevention: experimental and epidemiological studies.
20
Pure Appl. Chem., 57, 717-722. Pung, A., Rundhaug, J.. Yoshizawa. C.N. and Bertram. and canthaxanthin inhibit J.S. (1988) P-Carotene chemically- and physically-induced neoplastic transforma-
21
tion in lOT1/2 cells. Carcinogenesis, 9, 1533- 1539. Som, S., Chatterjee, M. and Banerjee. M.R. (1984) PCarotene inhibition of 7.12-dimethylbenzanthraceneinduced transformation of murine mammary cells in vitro. Carcinogenesis. 5. 937 - 940. Stich, H.F., Rosin, M.P.. Hornby, A.P., Mathew, B.. Sankararayanan. R. and Nair, M.K. (1988) Remission of oral leukoplakias and micronuclei in tobacco/betel quid chewers treated with p-carotene and with @-carotene plus vitamin A. Int. J. Cancer, 42, 195- 199. Suda, D., Schwartz, J. and Shklar, G. (1986) Inhibition of experimental oral carcinogenesis by topical p-carotene.
and
22
23
24
and analogs in foods and feeds. Agric. Food Chem.. 20. 456 - 473. Khachik, F., Beecher. G.R., Goli. M.B. and Lusby, W.R. (1991) Separation, identification and quantification of carotenoids in fruits, vegetables and human plasma by high performance liquid chromatography Pure Appl. Chem 63, 71-80. Rundhaug, J.E.. Pung, A., Read. C.M. and Bertram. J.S. (1988) Uptake and metabolism of o-carotene and retinal by C3H/lOT1/2 cells. Carcinogenesis. 9. 1541- 1545. Stich. H.F.. Hornby. A.P. and Dunn, B.P. (1986) BCarotene levels in exfoliated mucosal cells of population groups at low and elevated risk for oral cancer. Int. J Cancer, 37, 389-393. US Department of Agriculture (1972) Composition of foods: raw, processed, prepared. Data set 8-l-l. Agricultural Research Service, Washington, DC. US Department of Agriculture (1976 - 89) Composition of foods, raw, processed, prepared. Handbooks Nos. 8-l to 8-21. US Department of Agriculture, Washington, DC. FAO/WHO Expert Group (1972) Estimated distribution of sources of vitamin A activity in various foods In: Food Composition Table for Use in East Asia, p. 3. Food and Agriculture Organization and National Institutes of Health, Bethesda, MD. Food and Nutrition Board, National Research Council (1980) Recommended Dietary Allowances, 9th edn.. pp. 56-57. Natl. Acad. of Sciences, Washington D.C.
Carcinogenesis, 7, 711- 715. Schwartz, J. and Shklar, G. (1988) Regression of experimental oral carcinomas by local injection of p-carotene and canthaxanthin. Nutr. Cancer, 11, 35-40.
26
Stich, H.F.. Hornby. A.P. and Dunn, B.P. (1986) 8Carotene levels in exfoliated human mucosa cells following its oral administartion. Cancer Lett., 30. 133 - 141 Gilbert, A.M., Stich, H.F , Rosin, M.P. and Davison. A.J.
Sporn, M.B. and Newton, D.L. (1981) Retinoids and chemoprevention of cancer. In: Inhibition of Tumor fnduction and Development. pp. 71- 100. Plenum Press, New York.
27
(1990) Variations in the uptake of p-carotene in the oral mucosa of individuals after 3 days of supplementation Int. J. Cancer, 45. 855-859. Dimitrov. N.V.. Meyer, C.. Ullrey, D.E. et al (1988)
Burton, G.W. and Ingold, K.U. (1984) @-Carotene: an unusual type of lipid antioxidant. Science, 224, 569 - 573. Straub. 0. (Ed.) (1976) Key to Carotenoids. Birkhauser, Basel. Gensler, H.L., Aickin. M. and Peng, Y.M. (1990) Cumulative reduction of primary skin tumor growth in UV-
25
28
Bioavailability of p-carotene in humans Am. J. Clan. Nutr., 48, 298 - 304. Suzuki, S., Sasaki. R. and Ito. Y. et al. (1990) Changes in serum concentrations of p-carotene and changes in the dietary intake frequency of green-yellow vegetables among healthy male inhabitants of Japan. Jpn J. Cancer Res.. 81. 463-469
