Micronutrients in Cervical Dysplasias and Cancer Table 4 Effects of cigarette smoking on plasma micronutrient levels in controls and women with CIN and cervical cancer Micronutrient Smoking status NormaP CIN a ' b Cancer

Epidemiological studies continue to identify an association of dietary antioxidant micronutrients in cancer prevention. A number of case-control and cohort studies have demonstrated a relationship between high intake of foods rich in carotenoids, tocopherols, and vitamin C with a reduced risk of certain human malignancies. The purpose of this study was to investigate the comparative plasma levels of a profile of known dietary antioxidants, namely, [5-carotene, lycopene, canthaxanthin, retinol, c~-tocopherol, and x-tocopherol. The target population was women with a histopathological diagnosis of cervical intraepithelial neoplasia (CIN) or cervical cancer and a control group. All women resided in the same catchment area (Bronx Borough, New York City) and were of similar inner-city socioeconomic backgrounds representing a fairly homogenous population group. A cross-sectional sample of 235 women was recruited with informed consent. Plasma nutrient levels were measured by reverse-phase high pressure liquid chromatography under study codes. The mean plasma levels of carotenoids ([3-carotene, lycopene, and canthaxanthin), as well as ot-tocopherol, were significantly lower in women with CIN and cervical cancer. In contrast, the mean plasma level of x-tocopherol was higher among patients with CIN, while the mean plasma level of retinol was comparable among the groups. There were significant linear trends for all three carotenoids and quadratic trends for o~and x-tocopherol with the degree of cervical histopathology. Plasma 13-carotene concentrations in cigarette smokers were significantly lower regardless of cervical pathology, whereas plasma lycopene and canthaxanthin levels were significantly lower in smokers with CIN. The findings of a decrease in all plasma antioxidant nutrient levels except x-tocopherol in women with CIN and cancer suggest a potential role for antioxidant deficiency in the pathogenesis of CIN and carcinoma of the cervix, which requires further investigation. Received 5/26/95; revised 9/1/95; accepted 9/11/95. To whom requests for reprints should be addressed, at the Department of Obstetrics and Gynecology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: (718) 430-3374; Fax: (718) 430-8758. I N T R O D U C T I O N The incidence of some human cancers have been reported to be reduced in patients with increased dietary intake of vitamins E and C and [3-carotene (1-5). Limited objective information is available in support of epidemiological reports concerning plasma and/or tissue concentrations of essential antioxidant nutrients in women with precancerous and cancerous lesions of the uterine cervix (6-10). The purpose of this study was to investigate the relative plasma concentrations of three potent antioxidant carotenoids: 13-carotene, lycopene, canthaxanthin as well as retinol, cx-tocopherol, and "r-tocopherol in coded peripheral venous samples using standardized HPLC 2 technology. M A T E R I A L S AND M E T H O D S Subject Population. We studied a cross-sectional sample of 235 nonpregnant patients attending the Family Planning Clinics, Colposcopy Clinics, and/or admitted to the Weiler Hospital of the Albert Einstein College of Medicine (Bronx, NY). The study protocol was approved by the Institutional Review Board, and all subjects were recruited with informed consent. Cases consisted of patients who had abnormal Pap smears and a biopsy-confirmed histopathological diagnosis of CIN I-III or cervical cancer. In addition, nonpregnant women attending regular gynecological clinics for routine care who had three consecutive normal Pap smears, were not using p.o. contraceptives, and had normal menstrual cycles constituted the control group. All study subjects came from the same catchment area (Bronx Borough, New York City) with similar inner-city socioeconomic backgrounds representing a fairly homogenous group. All subjects were on Medicaid or received some form of public assistance. The majority of the study population was African-American (41%) and Hispanic (46%). An epidemiological questionnaire including age, date of last menstrual period, vitamin supplementation, and smoking history was completed by each participant. Recent dietary intake was recorded. Subjects were excluded from the study if they reported a history of current vitamin supplementation. Venous Blood Samples. A single peripheral venous blood sample (10 ml) was obtained from each participant, including cancer patients, prior to any therapeutic intervention. No dietary restrictions were imposed upon any of the subjects. Coded blood samples, wrapped in aluminum foil, were transported promptly to the laboratory. The plasma was separated and rapidly frozen at -70°C in a light-protected container until assayed. All of the assays were completed within a period of 1 week without any knowledge of the patient's clinical history. 2 The abbreviations used are: HPLC, high-pressure liquid chromatography; CIN, cervical intraepithelial neoplasia; Pap, Papanicolaou. Research. on October 16, 2017. © 1996 American Association for Cancer clincancerres.aacrjournals. org Downloaded from 182 Micronutrients in Cervical Dysplasias and Cancer HPLC Analysis of Plasma Micronutrients. Processing of samples was carded out under subdued or gold light. Plasma 13-carotene, retinol, and a-tocopherol levels were measured using HPLC attached to a Waters 490E multiwavelength detector, as previously described (6). In brief, samples were extracted by shaking a mixture of 0.3 ml plasma, 0.3 ml ethanol, and 3.0 ml n-hexane containing 0.25 g [3-hydroxy-toluene/liter. After centrifugation, the entire hexane layer was separated and evaporated under nitrogen stream. The residue was redissolved in 0.3 ml ethanol containing 0.25 g [3-hydroxytoluene/liter. For the [3-carotene assay, 0.03 ml of freshly extracted samples were injected onto a Ultramax reversed-phase C18 5 u column (4.6 mm internal diameter × 150 mm) and was eluted with acetonitrile:methylene chloride:methanol (75:20:5) at a flow rate of 1.0 ml/min, with monitoring at 450 nm. Lycopene and canthaxanthin levels were simultaneously measured in the same extracted plasma samples with monitoring at 472 nm. For oL-tocopherol, "r-tocopherol, and retinol, 0.03 ml of each previously extracted plasma samples were injected onto another C18 column preequilibrated with a mixture of methanol:water (97.5: 2.5) and eluted at a flow rate of 1.5 ml/min, monitoring at 292 and 325 nm for respective nutrients. All determinations were carried out in duplicate. Results were calculated using the peak area of the compound over the peak area of the internal standard. The mean interassay coefficient of variation was <8%. The described HPLC system with a four-channel detector allows the separation of many carotenoids in the same run. There was no interference from lutein or zeaxanthin in the detection of canthaxanthin in our system. Histopathological Diagnoses and Statistical Analysis. Tissue specimens were submitted to staff pathologists who were blinded to subjects antioxidant status. Statistical analyses for differences between two means were calculated using the twosample t tests, and for comparing multiple groups, one-way ANOVA was applied. The pairwise comparisons were carried out using the Scheffe test, and smoking data were analyzed using the X 2 test. Measures of the linear, quadratic, and cubic trends for each micronutrient were carried out using a contrast test following the ANOVA using the SAS system.