![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
The Journal of Nutrition Vol. 128 No. 11 November 1998,
pp. 1933-1936
-Carotene and 
-Carotene in Women Are Correlated1,2,3
Department of Family and Preventive Medicine, University of California, San Diego, La Jolla, CA 92093-0901 USA and * Department of Food Science and Technology, Ohio State University, Columbus, OH 43210-1096 USA
 |
ABSTRACT |
|---|
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Results from epidemiologic studies suggest that a carotenoid-rich diet may reduce risk for cervical cancer, possibly by inhibiting the progression of cervical intraepithelial neoplasia, a preneoplastic lesion of the cervical tissue. Laboratory studies suggest that the mechanism may be linked to the metabolism of carotenoids to retinoic acid or retinoic acid-like compounds, which has been hypothesized to occur in the cervical tissue. The purpose of this study was to demonstrate the presence of provitamin A carotenoids in biopsied samples of this peripheral tissue in human subjects and to examine the relationship between baseline concentrations of these carotenoids in plasma and normal cervical tissue in subjects who were being evaluated for possible participation in a diet intervention trial. Subjects were 13 women aged 19-41 y. With the use of HPLC methodology, plasma concentrations of 
-carotene, 
-carotene and -cryptoxanthin were determined with UV/visible light detection for plasma and electrochemical detection for cervical tissue. Relationships between plasma and cervical tissue were evaluated with Pearson correlation analysis. Adjusted for plasma cholesterol concentration, plasma -carotene and -carotene were correlated with cervical tissue concentrations (r = 0.91, P < 0.001; r = 0.90, P < 0.001; respectively). Adjusted for plasma cholesterol concentration, plasma -cryptoxanthin tended to be correlated with cervical tissue concentrations (r = 0.62, P = 0.058). These findings suggest that plasma concentrations of -carotene and -carotene are good predictors of cervical tissue concentrations of these compounds in human subjects and describe a first step toward demonstrating a biological link between provitamin A carotenoids and cervical cancer in vivo.
The development of cervical cancer is characterized by a progression of normal cervical epithelium to preinvasive cervical lesions, and ultimately, to in situ or invasive carcinoma (Mitchell et al. 1995 Human papilloma virus (HPV) is now believed to be a major cause of cervical cancer (Schiffman and Brinton 1995 The purpose of this study was to demonstrate the presence of provitamin A carotenoids in biopsied cervical tissue from human subjects, for which only microsamples can be obtained for analysis, and to examine the relationship between concentrations of these carotenoids in plasma and normal cervical tissue in the subjects.
Subjects.
Women in this study were consecutive subjects undergoing baseline evaluation for possible participation in a clinical trial to test the efficacy of a carotenoid-rich diet in the treatment of cervical dysplasia. Inclusion criteria were as follows: >18 y of age, recent abnormal cytology results suggesting the possibility of CIN II and premenopausal status. Exclusion criteria included the following: pregnancy, lactation or postmenopausal status. None of the subjects reported use of Procedures.
After recruitment, subjects were scheduled for a clinical examination, at which time a blood sample and biopsies of the cervical lesion for histopathological assessment and of normal cervical tissue for carotenoid analysis were obtained. Blood samples were collected by venipuncture into heparin-treated tubes and were protected from light during processing and handling. Plasma was separated by centrifugation at 2300 × g at 4°C for 10 min. Plasma aliquots were stored at Plasma carotenoid and cholesterol analysis.
Plasma carotenoids were separated and quantified by using the HPLC method of Bieri et al. (1985) Cervical tissue analysis.
Cervical tissue samples were thawed and rinsed with 1.0 mL fresh saline solution (8.5 g sodium chloride/L). The tissue was rinsed with deionized water, weighed and placed in an 8-mL vial containing 1.8 mL of PBS with 1000 units of collagenase and 0.2 mL of PBS with 2.5 g/L ascorbic acid. The sample was ground by mechanical homogenization for no more than 60 s using a variable speed rotor/blade-type homogenizer (Tissue Tearor, Whatman Lab Sales, Portland, OR) equipped with a Dremel (Racine, WI) motor and incubated at 37°C for 1 h. The homogenate was rinsed in 2 mL ethanol, and the rinsate was added to the sample vial after incubation. Carotenoids were extracted successively with three 2.0-mL aliquots of hexane containing 0.2 g/L butylated hydroxytoluene. The solution was vortexed for 30 s, and the hexane layer was collected and saved. The combined hexane layers were evaporated under nitrogen.
Statistical analysis.
Initially, descriptive statistics were calculated on all variables, and distributions were examined for normality. Plasma carotenoid concentrations were adjusted for total plasma cholesterol concentrations (as carotenoid:cholesterol) in statistical analysis. Because the distributions for tissue and plasma carotenoid concentrations were skewed and not normally distributed, appropriate power transformations (e.g., log, square root) were investigated for ability to transform the distributions to more closely approximate normality, so that parametric statistics could be computed. Square-root transformation was found to best improve normality; thus this was done before statistical analysis. Correlations between the plasma and cervical tissue concentrations of Subjects recruited were 13 women aged 30 ± 2 y (range 19-41 y). Table 1 lists the medians and ranges of
Demonstrating a causative relationship between dietary constituents associated with cancer risk in epidemiologic studies, such as carotenoids and cervical cancer, and elucidating the underlying mechanism are challenging tasks in clinical studies. However, establishing this biological link is important and necessary (Greenwald 1996
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). Cervical cancer is an important health problem worldwide. In the U.S., 13,700 new cases of invasive cervical cancer are predicted in 1998 with 4900 deaths attributable to this disease (1.8% of all cancer-related deaths in women) (Landis et al. 1998), but these figures do not accurately reflect the extent of the problem. For every case of invasive cervical cancer, there are ~50 cases of abnormal cervical smears that require monitoring and follow-up (Franco 1997), and millions of ablative procedures are performed each year as an approach to treatment.
). Results from numerous epidemiologic studies suggest that nutritional factors, particularly carotenoids, are also among the determinants of risk for cervical cancer (Potischman and Brinton 1996), possibly by influencing the progression through the multistep process of cervical carcinogenesis. Topical retinoic acid has been shown to increase the rate of regression of cervical intraepithelial neoplasia (CIN) II (moderate dysplasia) in clinical trials (Meyskens et al. 1994), and all-trans-retinoic acid at physiologic concentrations (1 nmol/L) inhibits the proliferation of HPV-infected cells in culture (Creek et al. 1994). In cervical dysplasia-derived cell lines, the presence of -carotene (10 µmol/L) was found to induce apoptosis in cervical dysplastic cells via down-regulation of epidermal growth factor receptor protein (Muto et al. 1995). A protective effect of carotenoids against cervical cancer may be mediated by vitamin A-like activities in the affected cervical epithelial cells. Metabolism of -carotene to retinal has been shown to occur in other peripheral tissues, such as human adipose tissue, primate lung and kidney, and bovine corpus luteum (Schweigert et al. 1988, Wang et al. 1991), in addition to intestinal mucosal cells and hepatocytes. However, a difficult aspect of investigating a possible mechanism in vivo is that only very limited amounts of this target tissue can be biopsied and measured in observational studies and clinical trials.
MATERIALS AND METHODS.
Abstract
Introduction
Methods
Results
Discussion
References
-carotene supplements. Demographic data, including age, were obtained from the subjects during recruitment interviews. Procedures for this study were approved by the Human Subjects Committee of the University of California, San Diego, School of Medicine.
70°C in cryogenic tubes until lipid extraction and analysis. The cervical tissue biopsies were obtained from the exocervix with the squamocolumnar junction during a standard gynecologic examination using a cervical biopsy punch forceps (Sklar Mini Tischler Delicate Punch, McKesson General Medical, San Diego, CA). Colposcopy was used to visualize the tissue, and the cervix was swabbed with acetic acid solution (50 g/L) and examined to identify areas of normal tissue and CIN lesions to be biopsied. The lesion biopsy was sent to the pathology laboratory for analysis and grading of the dysplasia. The normal tissue sample was rinsed with normal saline (8.5 g sodium chloride/L), and stored at 80°C in normal saline until analysis.
, as modified by Craft et al. (1988), with further modifications to reduce oxidative loss and improve recovery of compounds during analysis (Craft 1992). HPLC analysis was conducted with a Varian Star 9010, 9050 system with variable wavelength UV/visible light detector (Varian Analytical Instruments, Walnut Creek, CA) with wavelength set at 450 nm. The mobile phase was acetonitrile/methanol/methylene chloride (70:10:20, v/v/v), with triethylamine (0.13 mL/L acetonitrile) and ammonium acetate (0.1 g/L methanol) modifiers used to enhance recovery. The column used was a Supelco (Bellefonte, PA) Supelcosil LC-18 (25 cm × 4.6 mm × 5 µm). This analytical method measures 90% of the plasma total carotenoids present and permits quantification of the predominant carotenoids. Accuracy was assessed by periodic analysis of National Institute of Standards and Technology Standard Reference Material SRM 986, Fat-Soluble Vitamins, and a pooled plasma reference sample was analyzed concurrently with batches of study samples to monitor analytical precision, with day-to-day CV <10%.
). Standard reference materials from the manufacturer were used to validate analytical precision of these procedures. Plasma cholesterol concentrations were used in the evaluation of carotenoid data because the levels of the cholesterol-carrying lipoproteins are important determinants of the circulating pool of these compounds (Rock 1997). Carotenoids are transported primarily in low density lipoproteins (Johnson and Russell 1992), which also carry the major proportion of cholesterol in the plasma. Thus, plasma concentrations of the provitamin A carotenoids are a function of the cholesterol-carrying lipoprotein concentrations.
). The column used was a 4.6 mm i.d. × 150 mm, 5-µm C30 column, obtained from YMC (Wilmington, NC) and a guard column packed with C:18 stationary phase. Detection was accomplished with an ESA Model 5600 Coularray electrochemical detector (Chelmsford, MA), equipped with eight channels in series; the potential settings from channels 1-8 were 100-520 mV in 60-mV increments. The chromatographic data were collected and integrated using the ESA Coularray software and data management system. Separations were achieved using gradient elution with different concentrations of methanol:methyl-tert-butyl ether:ammonium acetate in reservoirs A (95:3:2) and B (20:78:2). The following gradient was used: 1-5 min, 85% solvent A, 15% solvent B; 5-25 min, linear gradient to 60% solvent A, 40% solvent B; hold 5 min. Peak identifications were confirmed by injection of authentic standards. We previously demonstrated successful application of this methodology in analysis of various extracts and biological tissues, including a sample of cervix tissue (Ferruzzi et al. 1998). Concentrations obtained with electrochemical and UV/visible light detection have been previously found to be correlated (MacCrehan and Schonberger 1987), but detection limits for the carotenoids are substantially improved with the new technology (Ferruzzi et al. 1998).
-carotene, -carotene and -cryptoxanthin were then examined using Pearson product-moment correlation analysis. All analyses were performed using the SPSS for Windows (version 6.0, SPSS, Chicago, IL). Values are means ± SEM.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
-carotene, -carotene and -cryptoxanthin concentrations in plasma and cervical tissue of the subjects. Amounts of cervical tissue that were available for the carotenoid analysis ranged from 5.6 to 62.4 mg; half of the samples weighed 12 mg, reflecting the inherent variability in the amount of tissue that is comfortably removed during a clinical procedure. Total plasma cholesterol concentrations were 5.04 ± 0.26 mmol/L. Pearson correlation analysis revealed significant correlations between cervical tissue and plasma concentrations, adjusted for plasma cholesterol concentrations, for -carotene (r = 0.91, P < 0.001) and -carotene (r = 0.90, P < 0.001), as shown in Figure 1. The correlation between -cryptoxanthin concentrations of cervical tissue and plasma, adjusted for plasma cholesterol concentrations, was marginally significant (r = 0.62, P = 0.058).
View this table:
Table 1.
-Carotene, -carotene and -cryptoxanthin concentrations in plasma and normal cervical tissue biopsies in
study subjects1
View larger version (16K):
[in a new window]
Fig 1.
Plots of square root-transformed cervical tissue concentrations vs. square root-transformed plasma concentrations (adjusted for plasma cholesterol as carotenoid:cholesterol) for (A) -carotene (r = 0.91, P < 0.001), and (B) -carotene (r = 0.90, P < 0.001) in women.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
, Weinstein 1991). Biological action depends on the presence of the protective compound in the target tissue at the crucial point in the pathophysiologic process, which is the focus of this study. We found the provitamin A carotenoids to be measurable in very small cervical tissue biopsy samples using new detection methodology. Also, we found -carotene and -carotene to be present in cervical tissue at concentrations that were highly correlated with the concentrations observed in plasma samples.
) and exfoliated cervicovaginal cells (Manetta et al. 1996, Mikhail et al. 1994, Palan et al. 1992). These approaches permit the use of conventional HPLC detection methodology (i.e., UV/visible light) to quantify carotenoids because relatively large amounts of tissue and cells can be obtained. Clearly, surgical removal of the uterine cervix would not be appropriate in a clinical trial because this does not permit serial analyses to examine effects of intervention. Exfoliated cell collection has limited tissue site specificity, which is necessary in the investigation of etiologic mechanisms in a prospective study. The amount of cells that can be removed directly from the exocervix with cytologic scraping or brushing is limited, which necessitates the collection of vaginal epithelial cells as well; thus the sample is described more accurately as cervicovaginal epithelial cells (Manetta et al. 1996). The small amount of tissue that is typically biopsied for histopathologic examination of cervical tissue has historically limited the ability to quantify carotenoids, until the availability of electrochemical detection methods (Ferruzzi et al. 1998).
collected colorectal tissue via colposcopy in 18 subjects with and without colonic polyps or cancer and reported -carotene concentrations at recruitment into an intervention trial to average 0.03-0.07 nmol/g in that tissue across their study subgroups. Oral mucosal samples were obtained by punch biopsy in 28 subjects in a study by Brandt et al. (1994), in which -carotene concentration was found to average 2.00 nmol/g. Peng et al. (1995) obtained skin biopsies from 96 subjects via punch biopsies and observed -carotene concentrations in that tissue to average 0.10 -0.18 nmol/g across their study subgroups.
, serum -carotene was found to account for only 38% of the variance in mucosal tissue concentrations of biopsied samples, suggesting a substantially weaker relationship between circulating and peripheral pools than was observed in this study. In comparison, good correlations between plasma and skin biopsy -carotene concentrations (r = 0.75) were found by Peng et al. (1995). A high correlation for -carotene was also observed in the study by Peng at al. (1995) (r = 0.66), and a somewhat lower correlation (although significant) between plasma and skin -cryptoxanthin (r = 0.59) concentrations, similar to our observations. The present study group was comprised entirely of premenopausal women; thus greater homogeneity of the population may have contributed to the improved correlations between plasma and peripheral tissue concentrations that were observed.
-carotene and -carotene were highly correlated, suggesting that plasma concentrations of these compounds are predictive of amounts in the target tissue.
| |
FOOTNOTES |
|---|
Manuscript received 8 April 1998. Initial reviews completed 24 June 1998. Revision accepted 12 July 1998.
| |
LITERATURE CITED |
|---|
|
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
-carotene.
Biochem. Med. Metab. Biol.
1994;
51:55-60[Medline]-carotene among lipoproteins in healthy men.
Am. J. Clin. Nutr.
1992;
56:128-135-tocopherol, and -carotene in serum by liquid chromatography with absorbance and electrochemical detection.
Clin. Chem.
1987;
33:1585-1592-Carotene treatment of cervical intraepithelial neoplasia: a Phase II study.
Cancer Epidemiol. Biomark. Prev.
1996;
5:929-932[Abstract]-carotene through down-regulation of epidermal growth factor receptor.
Am. J. Clin. Nutr.
1995;
62 (suppl.):1535S-1540S-carotene and -tocopherol levels in breast and gynecologic cancers.
Gynecol. Oncol.
1994;
55:72-77[Medline]-Carotene levels in exfoliated cervicovaginal epithelial cells in cervical intraepithelial neoplasia and cervical cancer.
Am. J. Obstet. Gynecol.
1992;
167:1899-1903[Medline]-tocopherol) and tissue (carotenoids) levels after supplementation with -carotene in subjects with precancerous and cancerous lesions of signoid colon.
Eur. J. Clin. Nutr.
1997;
51:661-666[Medline]-carotene into -apocarotenals and retinoids by human, monkey, ferret, and rat tissues.
Arch. Biochem. Biophys.
1991;
285:8-16[Medline]This article has been cited by other articles:
|
|
 |
|
  C. L. Rock, L. Natarajan, M. Pu, C. A. Thomson, S. W. Flatt, B. J. Caan, E. B. Gold, W. K. Al-Delaimy, V. A. Newman, R. A. Hajek, et al. Longitudinal Biological Exposure to Carotenoids Is Associated with Breast Cancer-Free Survival in the Women's Healthy Eating and Living Study Cancer Epidemiol. Biomarkers Prev., February 1, 2009; 18(2): 486 - 494. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Thomson, N. R. Stendell-Hollis, C. L. Rock, E. C. Cussler, S. W. Flatt, and J. P. Pierce Plasma and Dietary Carotenoids Are Associated with Reduced Oxidative Stress in Women Previously Treated for Breast Cancer Cancer Epidemiol. Biomarkers Prev., October 1, 2007; 16(10): 2008 - 2015. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Demark-Wahnefried, E. C. Clipp, I. M. Lipkus, D. Lobach, D. C. Snyder, R. Sloane, B. Peterson, J. M. Macri, C. L. Rock, C. M. McBride, et al. Main Outcomes of the FRESH START Trial: A Sequentially Tailored, Diet and Exercise Mailed Print Intervention Among Breast and Prostate Cancer Survivors J. Clin. Oncol., July 1, 2007; 25(19): 2709 - 2718. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A Ello-Martin, L. S Roe, J. H Ledikwe, A. M Beach, and B. J Rolls Dietary energy density in the treatment of obesity: a year-long trial comparing 2 weight-loss diets Am. J. Clinical Nutrition, June 1, 2007; 85(6): 1465 - 1477. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Natarajan, S. W. Flatt, X. Sun, A. C. Gamst, J. M. Major, C. L. Rock, W. Al-Delaimy, C. A. Thomson, V. A. Newman, J. P. Pierce, et al. Validity and Systematic Error in Measuring Carotenoid Consumption with Dietary Self-report Instruments Am. J. Epidemiol., April 15, 2006; 163(8): 770 - 778. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Rock, S. W. Flatt, L. Natarajan, C. A. Thomson, W. A. Bardwell, V. A. Newman, K. A. Hollenbach, L. Jones, B. J. Caan, and J. P. Pierce Plasma Carotenoids and Recurrence-Free Survival in Women With a History of Breast Cancer J. Clin. Oncol., September 20, 2005; 23(27): 6631 - 6638. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. E. Castle and A. R. Giuliano Chapter 4: Genital Tract Infections, Cervical Inflammation, and Antioxidant Nutrients--Assessing Their Roles as Human Papillomavirus Cofactors J Natl Cancer Inst Monographs, June 1, 2003; 2003(31): 29 - 34. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. McEligot, C. L. Rock, S. W. Flatt, V. Newman, S. Faerber, and J. P. Pierce Plasma Carotenoids Are Biomarkers of Long-Term High Vegetable Intake in Women with Breast Cancer J. Nutr., December 1, 1999; 129(12): 2258 - 2263. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
