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Human Nutrition and Metabolism

Correlation between Carotenoid Concentrations in Serum and Normal Breast Adipose Tissue of Women with Benign Breast Tumor or Breast Cancer14
Kyung-Jin Yeum,*5 Sei-Hyun Ahn, Sergio Alberto Rupp de Paiva,* ** Yang Cha Lee-Kim, Norman I. Krinsky* and Robert M. Russell*
*Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA; Department of General Surgery, College of Medicine, University of Ulsan, Ulsan, Korea; **Faculdade de Medicina de Botucatu, UNESP, Botucatu, Sao Paulo, Brazil; Department of Food and Nutrition, Yonsei University, Seoul, Korea; and Department of Biochemistry, School of Medicine, Tufts University, Boston, MA.
ABSTRACT To evaluate the relationship between carotenoid concentrations in serum and breast tissue, we measured serum carotenoid concentrations and endogenous carotenoid levels in breast adipose tissue of women with benign breast tumor (n 46) or breast cancer (n 44). Before extraction, serum was digested with lipase and cholesterol esterase, and breast adipose tissue was saponied. Serum and tissue carotenoids were extracted with ether/hexane and measured by using HPLC with a C30 column. Serum retinoic acid was extracted with chloroform/ methanol and measured using HPLC with a C18 column. There were no signicant differences in serum carotenoids [lutein, zeaxanthin, cryptoxanthin (both - and -), -carotene, all-trans -carotene, 13-cis -carotene and lycopene], retinoids (retinol, all-trans and 13-cis retinoic acids), and - and - tocopherol concentrations between benign breast tumor patients and breast cancer patients. A substantial amount of 9-cis -carotene was present in adipose tissue and was the only carotenoid that had a signicantly lower level in benign breast tumor patients than in breast cancer patients. Correlations between carotenoid concentrations in serum and in breast adipose tissue were determined by combining the data of the two groups. Concentrations of the major serum carotenoids except cryptoxanthin showed signicant correlations with breast adipose tissue carotenoid levels. When the concentrations of serum carotenoids were adjusted for serum triglycerides or LDL, correlations between serum carotenoid concentrations and breast adipose tissue carotenoid levels markedly increased, including that of cryptoxanthin (P 0.001). The strong correlation between serum carotenoid concentrations and endogenous breast adipose tissue carotenoid levels indicate that dietary intake inuences adipose tissue carotenoid levels as well as serum concentrations, and that adipose tissue is a dynamic reservoir of fat-soluble nutrients. J. Nutr. 128: 1920 1926, 1998. KEY WORDS:

carotenoids

adipose tissue

correlation

humans

breast cancer

Data from epidemiologic studies consistently indicate that increased intakes of fruits and vegetables (Freudenheim et al. 1996, La Vecchia et al. 1998) and high serum concentrations of carotenoids are associated with a decreased risk of breast

1 Presented at Experimental Biology 98, April 1998, San Francisco, CA [Yeum, K.-J., Ahn, S.-H., Paiva, S.A.R., Lee-Kim, Y. C., Krinsky, N. I. & Russell, R. M. (1998) Correlation between carotenoid concentrations in serum and in normal breast tissues of women with benign breast tumor or breast cancer. FASEB J. 12: A5595 (abs.)]. 2 The contents of this publication do not necessarily reect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products or organizations imply endorsement by the U.S. government. 3 Supported in part by Federal funds from the U.S. Department of Agriculture, under contract number 53-3K06-5-10 and by National Institutes of Health grant 5RO1 CA66914-02. 4 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 USC section 1734 solely to indicate this fact. 5 To whom correspondence and reprint requests should be addressed.

cancer (Hunter and Willet 1993, Potischman et al. 1990, Rohan et al. 1988). In a large prospective study, it was also found that the consumption of both preformed vitamin A and carotenoids appeared to reduce the risk of breast cancer (Hunter et al. 1993). In addition to acting as antioxidants (Palozza and Krinsky 1992), carotenoids may exert anticarcinogenic properties through conversion to retinoids, specically retinoic acid (Wang et al. 1992). It is generally accepted that serum carotenoid concentrations are markers of recent fruit and vegetable intake (Drewnowski et al. 1997, Polsinelli et al. 1998, Yeum et al. 1996), whereas tissue carotenoid levels are indicators of longer-term carotenoid consumption patterns (Parker 1989). Several human studies have shown the kinetics of uptake of carotenoids into the circulation from foods rich in carotenoids (Bowen et al. 1993, Martini et al. 1995, Yeum et al. 1996). However, very few direct observations have been made on the absorption and tissue distribution of carotenoids from either

0022-3166/98 $3.00 1998 American Society for Nutritional Sciences. Manuscript received 4 May 1998. Initial review completed 1 June 1998. Revision Accepted 12 July 1998. 1920

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dietary intake or supplementation. -Carotene concentration in adipose tissue as well as in serum was shown to be increased after supplementation with a 120-mg single oral dose of -carotene (Johnson et al. 1995) or after 30 mg -carotene supplementation for 6 mo (Kardinaal et al. 1995). Kardinaal et al. (1995) found a signicant correlation between -carotene concentrations in serum and in adipose tissue obtained from the buttock. Because adipose tissue is quantitatively the most important site of storage of fat-soluble nutrients (Parker 1993), adipose tissue carotenoid levels might be relatively stable indicators of body status. However, Zhang et al. (1997) could not nd any correlation between breast adipose tissue concentrations of several carotenoids and the dietary intake of these carotenoids. Therefore, it is important to determine the strength of the correlation of carotenoid concentrations between the two different pools because these two pools, serum and adipose tissue, are thought to be inuenced by short-term and longer-term dietary intakes, respectively. In this study, fasting blood and normal breast adipose tissue were obtained from women with breast cancer or benign breast tumor, which is a risk factor for breast cancer (Colditz 1993). We measured fat-soluble antioxidant nutrients, carotenoids, retinoids and tocopherols in serum and endogenous carotenoid levels in normal breast adipose tissue. Furthermore, the correlations between serum carotenoid concentrations and breast adipose tissue carotenoid levels were determined. MATERIALS AND METHODS
Subject and sample collection. Two groups of women (benign breast tumor patients, n 46; breast cancer patients, n 44), who were rst diagnosed with a benign breast tumor or breast cancer at the Breast Clinic at Asan Medical Center in Seoul, Korea, were enrolled in this study. Overnight fasting blood samples were collected for routine preoperative blood tests, liver function tests, lipid analyses and carotenoid analyses. Serum samples were protected from light and centrifuged for 15 min (800 g, 4C) within 1 h of collection. Aliquots of serum were stored at 70C until analyzed. Normal breast adipose tissue that would have been otherwise discarded was obtained either from benign breast tumor patients during the surgical resection or from cancer patients during mastectomy or breast quadrantectomy. Normal breast adipose tissue was obtained at least 1 cm away from a benign tumor of patients with benign breast disease or at least 5 cm away from malignant tissue of patients with breast cancer. Tissue samples were promptly frozen in liquid nitrogen in polypropylene freezer tubes, wrapped with foil and then stored at 70C. Serum and tissue samples were analyzed within 1 y of being frozen. The study protocol was approved by the Human Investigation Review Committee of Tufts University and the New England Medical Center, and the Institutional Review Board of the Asan Medical Center, Seoul, Korea. Serum and breast adipose tissue carotenoid analyses. All-trans -carotene (type IV), -carotene, lycopene, retinol, all-trans retinoic acid, -tocopherol, -tocopherol, triglyceride hydrolase (Chromobacterium viscosum) and cholesterol esterase (Pseudomonas species) were purchased from Sigma Chemical (St. Louis, MO). Lutein was purchased from Kemin Industries (Des Moines, IA). Zeaxanthin, cryptoxanthin, 13-cis -carotene, 9-cis -carotene, echinenone, 13-cis retinoic acid and TMMP [all-trans-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraenoic acid] were gifts from Hoffmann-La Roche (Nutley, NJ). Solutions of carotenoids and retinoids were prepared under red light before use. A Bond Elut aminopropyl column (500 mg/2.8 mL) and a Vac Elut vacuum elution apparatus were obtained from Analytichem International (Harbor City, CA). All HPLC solvents were obtained from J. T. Baker Chemical (Philipsburg, NJ) and were ltered though a 0.2- m membrane lter before use. Serum carotenoids, retinol and tocopherols were extracted using a modied enzyme extraction method reported earlier (Yeum et al. 1996). Echinenone, retinyl acetate and tocol were added as internal

standards for the analysis of carotenoids, retinoids and tocopherols, respectively. Breast adipose tissue ( 40 mg) was incubated with 100 L of 12% pyrogallol in ethanol, 200 L of 30% KOH in water and 1 mL ethanol for 2 h at 37C (Qin et al. 1997). After saponication, 1 mL H2O and 100 L of echinenone in ethanol (internal standard) were added. The samples were extracted with 3 mL of ether/hexane (2:1, stabilized with 1% ethanol) twice, and diluted with 2 mL H2O and 2 mL ethanol. After centrifugation for 5 min at 800 g , the organic layer was collected. The extract was evaporated under N2 in a 40C water bath, resuspended with 100 L of ethanol, and 50 L was injected onto the HPLC system. The extracted sample was analyzed for carotenoids, retinoids and tocopherols for serum, and carotenoids for adipose tissue using a reverse-phase, gradient HPLC system. The HPLC system consisted of a Series 410 LC pump (Perkin-Elmer, Norwalk, CT), a Waters 717 plus autosampler (Millipore, Milford, MA), a C30 carotenoid column (3 m, 150 4.6 mm, YMC, Wilmington, NC), an HPLC Column Temperature Controller (Model 7950 Column Heater/Chiller, Jones Chromatography, Lakewood, CO) and a Waters 840 Digital 350 data station. The Waters 994 programmable photodiode array detector was set at 450 and 475 nm for carotenoids and 340 nm for retinoids. A uorescence detector (excitation at 292 nm, emission at 330 nm; Waters 470; Millipore) was connected in series for tocopherol analysis. The HPLC mobile phase was methanol/methyl-tert-butyl ether/water (83:15:2, v/v/v, with 1.5% ammonium acetate in the water; solvent A) and methanol/methyl-tert-butyl ether/water (8:90:2, v/v/v, with 1% ammonium acetate in the water; solvent B). The gradient procedure at a ow rate of 1 mL/min (16C) was as follows: 1) 100% solvent A for 1 min; 2) a 7-min linear gradient to 70% solvent A; 3) a 5-min hold at 70% solvent A; 4) a 9-min linear gradient to 45% solvent A; 5) a 2-min hold at 45 % solvent A; 6) a 10-min linear gradient to 95% solvent B; 7) a 4-min hold at 95% solvent B; and 8) a 2-min gradient back to 100% solvent A. Using this method, lutein, zeaxanthin, cryptoxanthin ( -cryptoxanthin and -cryptoxanthin eluted as a single peak), -carotene, 13-cis -carotene, all-trans -carotene, 9-cis -carotene and trans- and cis-lycopenes were adequately separated. 9-cis -Carotene was monitored at both 450 and 475 nm and conrmed through diode-array spectra because 9-cis -carotene coelutes with -carotene ( max 400) in our system. Typical HPLC chromatograms of carotenoids in serum and adipose tissue are shown in Figure 1. Carotenoids, retinol and tocopherols were quantied by determining peak areas in the HPLC chromatograms, calibrated against known amounts of standards. The lower limits of detection were 0.2 pmol for carotenoids, 2.0 pmol for retinol and 2.7 pmol for tocopherols. Serum retinoic acid analysis. Serum retinoic acids were extracted by using a modied extraction method reported earlier (Tang and Russell 1990). TMMP in methanol was added as an internal standard for the analysis of retinoic acid. The HPLC system consisted of a Waters 996 pump (Millipore), a Waters 717 plus autosampler (Millipore), a Pecosphere-3 C18 column (3 m, 83 4.6 mm, PerkinElmer), a Millenium 2010 chromatography manager PDA software option version 2.15, and a Waters 996 photodiode array detector. The detector was set at 340 nm. The HPLC mobile phase was methanol/ water (75:25, v/v, with 1% ammonium acetate in the water; solvent A) and methanol (solvent B). The gradient procedure at a ow rate of 1 mL/min was as follows: 1) 100% solvent A for 25 min; 2) a 2-min linear gradient to 100% solvent B; 3) a 5-min hold at 100% solvent B; and 4) a 2-min gradient back to 100% solvent A. With the use of this method, all-trans, 13-cis and 9-cis retinoic acids were adequately separated. The lower limit of detection was 0.2 pmol for retinoic acid. Laboratory analyses. Cholesterol and triglycerides were analyzed by enzymatic reaction kits (Boehringer Mannheim reagent, Mannheim, Germany) and measured photometrically (Hitachi 747200 Automatic Analyzer, Tokyo, Japan). HDL cholesterol was directly analyzed (without pretreatment) by enzymatic reaction kits (Kyowa, Tokyo, Japan) and also measured photometrically (Hitachi). LDL cholesterol was calculated by using the formula of Friedewald et al. (1972). Laboratory data were not available for two subjects. Statistics. When there was a normal distribution, comparisons between two groups were made by Students t test. When the data showed a nonnormal distribution, the Mann-Whitney U Test was used. The relationship of the values of the breast tissue to the serum

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TABLE 1
Characteristics of study subjects1
Benign breast tumor (n 44) Age, y Wt, kg Ht, cm BMI,2 kg/m2 Albumin, g/L Hemoglobin, g/L RBC, 1012/L WBC, 109/L GOT, IU/L GPT, IU/L Triglycerides, mmol/L Total Cholesterol, mmol/L LDL Cholesterol, mmol/L HDL Cholesterol, mmol/L Smoker, n 37.8 54.6 160.3 21.4 45.3 125.6 4.2 5.3 17.4 15.3 2.2 4.5 2.2 1.3 1 1.4 1.1 0.5 0.4 0.4 1.7 0.04 0.2 0.8 1.3 0.2 0.1 0.1 0.05 Breast cancer (n 44) 44.3 57.6 157.6 23.1 42.7 120.5 4.0 5.5 17.8 16.8 2.5 4.7 2.3 1.2 3 1.3 1.4 0.8 0.6 0.5 2.2 0.06 0.3 1.0 0.9 0.2 0.2 0.2 0.05

1 Values are means SEM. 2 BMI, body mass index; WBC, white blood cell; GOT, glutamicox-

aloacetic transaminase; GPT, glutamicpyruvic transaminase.

FIGURE 1 HPLC chromatograms of the major carotenoids in human serum (A) and human breast adipose tissue (B) obtained from a benign breast tumor patient. 1: lutein; 2: zeaxanthin; 3: cryptoxanthin; 4: echinenone (internal standard); 5: 13-cis -carotene; 6: -carotene; 7: all-trans -carotene; 8: 9-cis -carotene/ -carotene; 9: lycopene.

variables was examined by using Spearman correlation coefcients. Serum carotenoid concentrations were adjusted for cholesterol, triglyceride, HDL cholesterol and LDL cholesterol to evaluate the correlation of carotenoid concentrations between serum and tissue. Statistical signicance was set at the P 0.05 level. All values are presented as means sem. Data analysis was carried out with Sigma Stat 2.0 for Windows (Jandel Scientic software, San Rafael, CA).

and breast cancer patients, were cryptoxanthin, -carotene and lutein, which were 51.4, 28.5 and 10.8%, respectively, of total carotenoids (Table 4). In the breast adipose tissue, cryptoxanthin (35.9%), -carotene (26.1%) and lutein (19.3%) were also the major carotenoids in all subjects, as shown in Table 4. Correlations between serum carotenoids and breast adipose tissue carotenoids in all subjects including benign breast tumor patients and breast cancer patients are presented in Table 5. Lycopene, all-trans -carotene, 13-cis -carotene, -carotene, lutein and zeaxanthin concentrations showed signicant correlations between serum and breast adipose tissue. However, serum cryptoxanthin showed no correlation with TABLE 2
Concentrations of carotenoids, retinoids and tocopherols in serum of women with benign breast tumor or with breast cancer
Benign breast tumor (n 46) Nutrient Mean
SEM

RESULTS Characteristics of benign breast tumor or breast cancer patients are shown in Table 1. Clinical laboratory test values, such as hemoglobin, RBC, white blood cells (WBC), glutamicoxaloacetic transaminase (GOT), glutamicpyruvic transaminase (GPT), albumin, cholesterol and triglycerides did not differ between the two groups and were normal in all patients. There were no signicant differences in serum carotenoids (lutein, zeaxanthin, cryptoxanthin, -carotene, all-trans -carotene, 13-cis -carotene and lycopene), retinoids (retinol, all-trans retinoic acid and 13-cis retinoic acid), and tocopherols ( - and -tocopherols) between benign breast tumor patients and breast cancer patients (Table 2). There was a trace amount of a mixture of -carotene and 9-cis -carotene in the serum, but substantial amounts of 9-cis -carotene in breast adipose tissue, as shown in the HPLC chromatograms (Fig. 1) and their spectra (Fig. 2), taken from the diode-array detector during HPLC analyses. There were also no signicant differences in tissue major carotenoid concentrations between breast tumor patients and breast cancer patients except for 9-cis -carotene (Table 3). 9-cis -Carotene in breast adipose tissue was signicantly lower in benign breast tumor patients than in breast cancer patients. The predominant serum carotenoids in all subjects, including benign breast tumor patients

Breast cancer (n 44) Mean


SEM

Median

Median

mol/L Lutein Zeaxanthin Cryptoxanthin -Carotene trans -Carotene 13-cis -Carotene trans Lycopene Retinol -Tocopherol -Tocopherol 0.79 0.30 4.62 0.19 2.11 0.12 0.09 2.02 18.19 3.82 0.05 0.02 0.43 0.02 0.14 0.06 0.01 0.10 1.95 0.40 0.69 0.29 3.73 0.17 1.85 0.11 0.07 1.88 20.95 3.78 0.75 0.26 3.78 0.17 2.15 0.12 0.10 2.09 19.78 3.77 0.05 0.02 0.36 0.01 0.13 0.01 0.01 0.11 2.09 0.45 0.67 0.24 3.33 0.16 1.99 0.11 0.08 2.03 20.43 3.10

nmol/L all-trans RA1 13-cis RA


1 RA, retinoic acid.

5.92 6.09

0.21 0.28

5.63 5.80

5.97 5.39

0.26 0.28

5.96 5.08

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FIGURE 2 The spectra of peak A-8 (from serum) and B-8 (from adipose tissue) in the chromatogram of Figure 1, taken from the diodearray detector during HPLC analysis. (A) Serum peak consists of a mixture of 9-cis -carotene and -carotene; (B) adipose tissue peak consists of 9-cis -carotene exclusively. The inset is the absorbance spectra of the 9-cis -carotene (solid line) and -carotene (dashed line) standards.

breast cancer patients. Our clinical data indicate that subjects in both groups were healthy except for the recent diagnosis of benign or malignant breast tumor. Women who are diagnosed as having either benign breast tumor or breast cancer undergo surgery as soon as possible in Korea. Because most of the cancer patients in this study were at an early stage of the disease, it would not be expected that the disease process would affect the serum concentrations of nutrients. Mean carotenoid concentrations in adipose tissue from sites adjacent to or distant from a breast malignancy have been reported to be similar (Rautalahti et al. 1990). In our study, normal breast adipose tissue was obtained at least 1 cm away from a benign tumor or 5 cm away from a malignant tumor. Major serum carotenoids in this Asian study population were cryptoxanthin, -carotene and lutein. Serum lycopene concentrations were 1.4% of total carotenoid concentrations in our subjects. Because plasma carotenoids are markers of recent fruit and vegetable intake (Drewnowski et al. 1997, Marchand et al. 1994, Polsinelli et al. 1998, Yeum et al. 1996), the relatively high serum concentration of cryptoxanthin, -carotene and lutein and extremely low concentration of lycopene in this study population probably reect the higher consumption of tangerines, yellow and green leafy vegetables and the lower intake of tomatoes and their products. This agrees with the Korean National Nutrition Survey (Korea Institute of Food Hygiene 1994), which revealed extremely low tomato and tomato product consumption in this population. Mean serum retinol concentration in this Korean population was similar to that reported in other populations (Ascherio et al. 1992). Lower serum -tocopherol concentrations and higher serum -tocopherol levels were found compared with Western populations (Ascherio et al. 1992, Vogel et al. 1997). Both serum vitamin A and vitamin E levels were in the range found in Western populations. Serum concentrations of all-trans retinoic acid and 13-cis retinoic acid in these subjects were similar to what has been reported earlier in a study in the U.S. (Tang and Russell 1990). We were not able to detect 9-cis retinoic acid in our subjects serum, even though Gundersen et al. (1997) reported the existence of 9-cis retinoic acid in human serum. It is not surprising to see relatively high triglyceride levels and lower cholesterol concentrations in our subjects compared with the U.S. women because carbohydrate is the major energy source in this study population (Carughi

breast adipose tissue cryptoxanthin levels. When the serum concentrations of carotenoids were adjusted for triglycerides, the correlations between serum and tissue carotenoids were strikingly increased for all carotenoids (Table 5). After adjustment, serum concentrations of cryptoxanthin also showed a signicant association with tissue levels of cryptoxanthin (P 0.001). When the concentrations of carotenoids in serum were adjusted for LDL, the correlations between serum and breast adipose tissue carotenoid concentrations were also markedly increased as shown in Table 5. DISCUSSION A reduction in risk of breast cancer in women who consume a diet rich in fruits and vegetables or who have higher serum levels of carotenoids has been reported frequently (Freudenheim et al. 1996, La Vecchia et al. 1998, Longnecker et al. 1997, Zhang et al. 1997). In this study, there were no differences in serum concentrations of carotenoids, retinoids or tocopherols between benign breast tumor patients and

TABLE 3
Concentrations of carotenoids in breast adipose tissue of women with benign breast tumor or breast cancer
Benign breast tumor (n 46) Mean
SEM

Breast cancer (n 44) Mean


SEM

Median

Median

mol/kg Lutein Zeaxanthin Cryptoxanthin -Carotene trans -Carotene 13-cis -Carotene 9-cis -Carotene trans Lycopene 1.78 0.40 3.47 0.21 2.34 0.57 0.37 0.09 0.14 0.04 0.32 0.02 0.25 0.05 0.03 0.01 1.72 0.32 2.25 0.16 1.87 0.49 0.32 0.07 1.69 0.41 3.67 0.22 2.75 0.67 0.47 0.09 0.12 0.04 0.31 0.02 0.27 0.06 0.04* 0.01 1.91 0.39 3.48 0.22 2.34 0.66 0.42 0.07

* Signicantly different between the groups, P ney U Test).

0.05 (Mann-Whit-

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TABLE 4
Concentrations of carotenoids in serum and in breast adipose tissue of women with breast disease (benign breast tumor or breast cancer)
Serum (n Nutrient Mean
SEM

90) % % (0.69) (0.25) (3.47) (0.16) (1.92) (0.11) (0.07) 10.8 3.8 51.4 2.5 28.5 1.6 1.4 1.74 0.40 3.57 0.21 2.54 0.63 0.42 0.09 0.09 0.03 0.22 0.01 0.18 0.04 0.03 0.01 Mean
SEM

Tissue (n

90) % % (1.80) (0.35) (2.26) (0.19) (2.23) (0.56) (0.34) (0.07) 19.3 4.4 35.9 2.3 26.1 6.6 4.4 1.0

(Median) mol/L

Median mol/kg

Lutein Zeaxanthin Cryptoxanthin -Carotene trans -Carotene 13-cis -Carotene 9-cis -Carotene trans Lycopene

0.77 0.28 4.21 0.18 2.13 0.12 0.09

0.04 0.01 0.28 0.01 0.09 0.01 0.01

and Hooper 1994, Johnson et al. 1993, Mensink and Katan 1987). Also, presurgical stress might induce mobilization of adipose tissue triglycerides (Nonogaki et al 1995). In accordance with the ndings of Stahl et al. (1993), trace amounts of 9-cis -carotene were detected in serum, whereas a substantial amount of 9-cis -carotene was measured in breast tissue. It is possible that breast tissue efciently takes up 9-cis -carotene from the circulation as suggested by Stahl et al. (1995). The HPLC chromatogram also illustrated the presence of highly polar carotenoids in breast adipose tissue, which are low or absent in serum, a nding that is in agreement with that of Parker (1993). 9-cis -Carotene, which has a higher antioxidant potency than that of the all-trans isomer (Ben-Amotz and Levy 1996, Levin and Mokady 1994), was the only carotenoid that showed a signicant difference in the concentration of breast adipose tissue in benign breast tumor patients vs. breast cancer patients in whom the level was elevated. It has been reported that 9-cis retinoic acid can be formed from 9-cis -carotene in vitro (Wang et al. 1994) and in vivo (He buterne et al. 1995). The predominant tissue carotenoids in our subjects were cryptoxanthin, -carotene and lutein, which mirrors the major serum carotenoids. Again, breast adipose tissue lycopene was present in the lowest concentration among tissue carotenoids. It has been reported that serum carotenoid concentrations

are readily increased by fruit and vegetable intakes (Drewnowski et al. 1997, Polsinelli et al. 1998, Yeum et al. 1996), and that adipose tissue carotenoid levels also can be increased by dietary and supplemental carotenoids (Johnson et al. 1995, Kardinaal et al. 1995). Therefore, we thought it would be useful to study the strength of the correlation between carotenoid concentrations in these two different pools, serum and adipose tissue. In agreement with Kardinaal et al. (1995), we found a strong correlation of carotenoid concentrations between serum and breast adipose tissue. Higher serum carotenoid concentrations would be thought to reect higher adipose tissue carotenoid levels. It is also possible that fat acts as a dynamic reservoir of carotenoids that can supply carotenoids to the circulation when circulatory levels drop. It should be noted that plasma -carotene shows a stronger association with concurrent carotene intake, estimated by the diet record, than with long-term carotene intake estimated by food-frequency questionnaire (Ascherio et al. 1992). Indeed, Kardinaal et al. (1995) could not nd any correlation between dietary intake as assessed by food-frequency questionnaire and either serum carotenoid concentrations or those in adipose tissue. Also, Zhang et al. (1997) were not able to nd any correlation between breast adipose tissue concentrations of carotenoids and the intake of dietary carotenoids. This may be due to the wide variation in concentrations in small adipose

TABLE 5
Correlation coefcients between serum carotenoids or serum carotenoids adjusted for cholesterol or triglyceride and breast adipose tissue carotenoids in women with breast disease (benign breast tumor or breast cancer)1,2
Adjustment Cholesterol Nutrient Lutein Zeaxanthin Cryptoxanthin -Carotene all-trans -Carotene 13-cis -Carotene trans Lycopene None 0.340** 0.394*** 0.133 0.235* 0.428*** 0.334** 0.614*** Triglyceride 0.637*** 0.599*** 0.444*** 0.552*** 0.873*** 0.604*** 0.683*** Total 0.342** 0.354** 0.170 0.255* 0.485*** 0.367*** 0.584*** HDL 0.344** 0.388*** 0.090 0.242* 0.432*** 0.337** 0.587*** LDL 0.463*** 0.427*** 0.339*** 0.382*** 0.550*** 0.484*** 0.554***

1n 88 2 Spearman Rank Order Correlation, *P

0.05; **P

0.005; ***P

0.001.

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tissue biopsies, or to recall bias in the food-frequency questionnaires that they employed (Zhang et al. 1997). Large interand intra-individual variations in intake and plasma concentrations of carotenoids have been reported by Scott et al. (1996); these are due in part to the seasonal variation in serum/plasma carotenoid concentrations (Cooney et al. 1995, Olmedilla et al. 1994, Rautalahti et al. 1993). However, the strong correlation of carotenoid concentrations between serum and adipose tissue suggests that fasting serum carotenoid levels may be equally as good as adipose tissue levels in reecting body carotenoid status. Note that cryptoxanthin was the only carotenoid that did not show a signicant correlation between serum and adipose tissue. In contrast to lutein, which comprises 11% of total carotenoids in serum and 20% in adipose tissue, cryptoxanthin comprised over 51% of total carotenoids in serum and 36% in adipose tissue, respectively. It is possible that the inux, i.e., disposition, of each carotenoid from the circulation into tissue might be different. The correlation between serum carotenoid concentrations and breast adipose tissue carotenoid levels was markedly increased after adjustment of serum carotenoid concentrations for serum triglyceride levels. Presurgical stress might have induced mobilization of adipose tissue triglycerides, along with adipose tissue carotenoids (Nonogaki et al. 1995), which would result in a transient increase in the correlation between serum and adipose tissue carotenoids, after adjusting for triglycerides. However, the stronger correlation after adjustment for triglycerides might not be generalizable to a healthy nonstressed population. It is generally accepted that nonpolar carotenoids such as -carotene, -carotene and lycopene are transported primarily in LDL particles, whereas polar carotenoids such as lutein and zeaxanthin are equally distributed between HDL and LDL (Parker 1989, Romanchik et al. 1995). When the serum concentration of carotenoids were adjusted for LDL cholesterol, the correlation between serum and adipose tissue carotenoids improved for both nonpolar and polar carotenoids, as would be expected (Krinsky et al. 1958, Parker 1989, Romanchik et al. 1995). However, when we adjusted the serum concentration of carotenoids for HDL cholesterol, the correlations between serum and adipose tissue for the more polar carotenoids such as lutein, zeaxanthin and cryptoxanthin were not improved. This may indicate that tissue carotenoids in a stress situation are transported not by HDL but by VLDL, reecting increased lipid ux from adipose tissue through the liver, secreted as VLDL (Parker 1996). Our study clearly indicates that serum carotenoid concentrations strongly correlate with carotenoid levels in breast adipose tissue. It is probable that adipose tissue is a dynamic reservoir of carotenoids that reects the circulating carotenoid concentrations, and that carotenoid concentrations in adipose tissue as well as in serum can be elevated by the consumption of high fruit and vegetable diets. LITERATURE CITED
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