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Juliet VanEenwyk,2 Faith G. Davis, and Neville Colman recommendations, and both of these types of foods are
Department of Epidemiology and Biostatistics, School of Public Health,
good sources of folates.
University of Illinois at Chicago, Chicago, Illinois 6061 2 [1. V., F. G. D.];
and VA Medical Center, Bronx, New York 10468, and Center for Clini-
cal Laboratories, Mount Sinai Medical Center, New York, New York
Introduction
10029 [N. C.] Folic acid, a B-group vitamin, is necessary for normal cell
replication, and cells grown in folate-deficient media
manifest chromosomal abnormalities which correspond
Abstract to those found in many types oftumor cells (1). In relation
A case-control study was designed to assess the to cervical cancer, it has been noted that folate deficiency
relationship between cervical intraepithelial neoplasia can lead to cervical cellular changes which resemble
(CIN) and folate in serum, red blood cells, and diet. neoplastic change (2) and that preneoplastic cervical
The association between CIN and dietary vitamin C cellular changes among users of oral contraceptives me-
gness with folate supplementation (3). Three case-control
was also investigated. Cases were selected from women
studies of folate consumption and cervical cancer (4-6)
with biopsy-confirmed CIN. Controls were age-,
have found little evidence of an association between
race-, and clinic-matched women with normal cervical
folate intake and disease.
(Pap) smears. Study participants completed self-
This investigation assesses the relationship between
administered food frequency (n = 100 matched pairs)
folic acid and CIN.3 The premalignant condition was
and health (n = 102 matched pairs) questionnaires.
chosen to attenuate physiological changes which may
Fasting venous blood samples were collected for serum
ensue from rather than foreshadow disease. It was hy-
(n = 98 matched pairs) and red cell (n 68 matched
pothesized that higher levels of serum and red cell folate
pairs) folate assays. Conditional logistic regression
and higher dietary intake of folate would be associated
models were used to estimate crude odds ratios and
with a reduction in disease. Because folates and vitamin
odds ratios adjusted for smoking, income, number of
C are found in many of the same foods and because
sexual partners, frequency of cervical smear, use of
vitamin C protects folates from oxidative cleavage, die-
spermicidal contraceptive agents, history of genital
tary intake of vitamin C was also assessed.
warts, and Quetelet index. Dietary intake variables
were adjusted for total energy intake prior to logistic
regression. A protective effect of red cell folate was Materials and Methods
evident with adjusted odds ratios (95% confidence Details of case and control selection, serum and dietary
intervals) of 0.1 (0.0-0.4), 0.6 (0.2-2.0), and 0.5 (0.2- measurement procedures, measurement of confounding
1.9) for those in quartiles 4 (highest), 3, and 2 variables, and statistical analysis have been previously
compared to quartile 1 (lowest). Supporting evidence documented (7, 8). These topics are reviewed below.
for the protective effect of folate was provided by Case and Control Selection. Participants were recruited
inverse associations between CIN and folate in both from clinics at Cook County Hospital and University of
serum and diet. An inverse association was also found Illinois Hospital between April 1987 and June 1 989. Cases
between CIN and dietary vitamin C with adjusted odds (n = 102) were selected from women aged 18 to 49 years
ratios (95% confidence intervals) of 0.2 (0.0-0.7), 0.6 with biopsy-confirmed CIN I, II, or Ill. Age- and race-
(0.2-1.6), and 0.6 (0.2-1.8) for those in quartiles 4, 3, matched controls were selected from women who at-
and 2, respectively, compared to quartile 1. These tended the same clinics as the cases and whose Pap
findings support dietary recommendations, such as smears showed no abnormality of a severity greaten than
those of the American Cancer Society, the National or equal to benign atypia. Women who had been preg-
Cancer Institute, and the U.S. Dietary Guidelines, nant or lactating within the past year were excluded from
which allow for adequate intake of folate and vitamin the study because of the potential for folate depletion
C, both of which are found in good quantity in fruits under these circumstances. In this population, pregnant
and vegetables. Increased consumption of legumes and and postpartum women may also be at increased risk for
whole grains is also in accord with current dietary a diagnosis of CIN, since pregnancy brings women into
the clinic where cervical smears are obtained as part of
prenatal care. Women with epilepsy on sickle cell anemia
were also excluded, since these conditions are associated
with low blood folate and with bringing women into the
Re eived 4/t 6/91.
1 Funded in part by a grant froni the American Canc er Society, Illinois
Division, and I)y the State Cancer Plan of the Illinois Cancer Council.
2 To whom requests for reprints should be addressed, at Illinois Depart-
ment of Public Health, Division of Health Statistics and Policy Develop- ‘ The abbreviations used are: CIN, cervical intraepithelial neoplasia; OR,.
ment, 1 01) West RailoIph, Suite 6-600, Chicago, IL 60601. ( rude odds ratio; OR,,, adjuste(l odds ratios; CI, ci)ntidenc(’ interval.
120 Folate, Vitamin C, and Cervical lnlraepithelial Neoplasia
medical system where they are likely to have cervical Measurement of Confounders. RIrti( pants (ompleted a
smears. Women with diabetes were exduded due to the self-administered questionnaire whi h asked aI)out back-
requirenient for ci 1 0-h f,ist. ground, health and pregnin y history, smoking, and sex-
All eligible wonien were requested to participate in ual behavior. Inforniation mroni this questionnaire was
the study. ofeligible
166 cases, 102 were enrolled, used to assess independent contributors to risk of CIN in
yielding a panticipation rate of approximately 61%. To this sample and to control for confounders of the disease-
enroll an equal number of controls, 195 eligible women exposure relationship. Confounders were defined as van-
were approached, giving a participation rate of 52%. ables which have been reported as risk factors in previous
Measurement of Exposure. The food frequency portion studies and variables whose inclusion led to a change of
of the Health Habits and History Questionnaire of the more than 20% in the adjusted odds ratio for the nu-
National Cancer Institute, Division of Cancer Prevention trients of interest.
and Control, version 2. 1 , was used to assess dietary intake Statistical Analysis. OR, and OR,, and 95% CIs were
of folate and vitamin C (9). Participants were asked to estimated using the MCSTRAT program (15), which per-
complete this questionnaire prior to the clinic visit. The forms an iterative conditional maximum-likelihood fit of
conversion of foods on the food frequency questionnaire a logistic regression model. Quartiles for the hematolog-
to nutrients was accomplished via the microcomputer cal measures and calorie-adjusted nutrient intake were
software version 2.2, August 1989, provided by the Na- defined from the distribution of the controls. Those in
tional Cancer Institute, Division of Cancer Prevention the lowest quartile (quartile 1) served as the comparison
and Control (9). The measure of dietary vitamin C ob- group. Adjusted models included independent contrib-
tamed from this procedure includes vitamin C from both utors to risk in this sample, as well as potential confoun-
food and vitamin supplements. There is no provision for dens of the disease-exposure relationship. Tests of trend
including supplemental folates in the dietary folate mea- were achieved by entering quartiles of a given nutrient
sure. Adjustment for total energy intake using the regres- into the logistic model as different values of a single
sion procedure of Willett and Stampfer (10) was used to ordinal variable.
control for over- and underreporting of dietary intake. Pearson product-moment correlation coefficients for
Two participants failed to adequately complete the food the correlation between the natural log of the hemato-
frequency questionnaire, resulting in 100 matched pairs logical measures and calorie-adjusted nutrient and food
available for analysis. intake measures were generated using SAS procedures.
Fasting venous blood samples were collected for Confounding due to intenassay variability was as-
radioassay of serum and red cell folate. Red cell hemo- sessed by including a dichotomous variable in the logistic
lysates were prepared on site by the method of Gutcho models. This variable was (Treated by calculating the
(11). Serum was also aliquoted on site, and all blood mean value for the quality control samples and charact-
samples were stored at -70#{176}C until shipment to the enizing assay groups according to whethen their quality
laboratory on dry ice every 6 to 10 weeks. Assays for control samples were above on below the mean.
serum and red cell folate were conducted by modiuica-
tion of the methods of Waxman et a!. (12) and Longo
and Herbert (13), respectively, using Becton Dickinson Results
Simultrac kits as reagents (14). For the folate assays, the The distribution of cases and controls on demographic
intraassay coefficient of variation was 1 .4-4.6%, and the and nondietary risk factors associated with CIN have
intenassay coefficient of variation was 3.8-8.2% for con- been presented (7, 8). Table 1 shows the OR,,s and 95%
trol samples at the limits of sensitivity of the assay. Low CIs for the nonnutnient variables included in the multi-
levels were associated with the highest coefficients of vanial)le conditional logistic regression models. Increased
variation. All laboratory personnel were unaware of case OR,,s were associated with current smoking status, more
or control status of the blood samples. than t’te year between cervical smears, any use of con-
Failure to withdraw blood from four women resulted traceptive spermicidal foams or gels, and a self-reported
in serum measures for 98 matched pains. Inadequate on- history of genital warts. An inverse association was ob-
site preparation of the red cell hemolysates resulted in served between OddS of disease and monthly income
unreliable data from the first 28 cases and 14 controls, bracket in $400 increments to $2000. Quartile of Que-
leading to the exclusion of 28 matched pairs from the telet index (kg/m2) and number of sexual partners were
final red cell folate analyses. Failure to collect a lavender- not independent contributors to risk after adjustment for
top tube from one woman and missing red cell data from the other variables. However, these variables were me-
one woman resulted in the exclusion of an additional tamed in the final model, because they were considered
two pairs, leaving 68 matched observations for the red to be potential confoundens of the disease-exposure
cell folate analyses. relationship. Use of oral contraceptives and parity have
Because more cases were enrolled at the beginning been reported to relate to both folate status and risk for
of the study and more controls enrolled toward the end CIN. However, since these factors were not independent
of the study period, there was a disparity in the allocation contributors to risk in this sample and their inclusion in
of case and control blood samples to the laboratory assay the logistic models did not alter the adjusted estimates,
groups. To enable control for confounding due to be- they were not included in the final models. Excessive
tween-nun variability, two quality control samples of alcohol consumption is associated with lowered blood
pooled blood were included in 1 1 of the 16 shipments. folates. Controlling for this variable (lid not alter the
These 1 1 batches accounted for 88% of the serum and findings and it is not included in the adjusted models.
100% of red cell samples included in the logistic models. Table 2 shows the quartiles of serum and red (Tell
Laboratory personnel were unaware of the inclusion of folate. The number of cases and controls in each quartile
these samples. and the percentage with deficiencies are also presented.
Cancer Epidemiology, Biomarkers & Prevention 121
Serum folate level )ng/ml) 1.3-3.4 3.5-4.4 4.5-6.3 6.4-21.2 <3.0 (15%io) <5.0(41%”)
Controls’ 26 22 25 25 14.3% 61.2%
Cases#{176} 36 23 25 14 23.5% 67.4%
Red cell folate level )ng/ml) 57-126 127-149 150-190 191-325 <140 (13%’) <160)20%’)
Controls’ 17 17 17 17 41.2% 60.3%
Cases’ 26 18 18 6 51.5% 73.5%
a Deficiency levels are those defined by the laboratory.
b The percentages of women from the Second National Health and Nutrition Evaluation Survey who were below deficiency levels (16) are in parentheses.
C Women aged 20-44 years.
d Estimated for women aged 20-64 years (Ref. 16, p. 37).
e The number of cases and controls in each quartile and the percentage below the specified deficiency level are presented.
I Estimated for women aged 20-64 years (Ref. 16, p. 38).
122 Folate, Vitamin C, and Cervical lntraepithelial Neoplasia
Table I Odds ratios, 95% CIs, and tests ot trend for serum and red cell
folate T,il)le 4 Odds ratios, 95% CIs, ,uicl lists of trend for dietary intake of
Icilate .uil vit.iniin C
Quartile Test i)f trend
Nutrient Qu artile Test of trend
1 (low) 2 3 4 (high) P N utrient
1 low) 2 3 4 high) P
Serum folate
OR, 1.0 0.9 0.8 0.4 0.04 Dietary folate
95%CI 0.4-2.0 0.3-1.7 0.2-0.9 OR. 1 .0 1. 1 1)6 0.5 0.03
95% CI 0.5-2.3 0)3 1 .5 01.2- 1.1
()R, 1.0) 0.9 1.1 0.3 0.15
95%CI 0.3-2.6 0.4-3.2 0.1-1.1 OR, 1.0 0.8 0.7 0.4 0.07
95%CI 0.3-1.9 01.2-2.0) 0.1-1.1
Red cell folate
OR, 1.0 0.7 0.8 0.2 0.03 Dietary vitamin C
95%CI 0)3-1.7 0.3-1.9 0.1-0.7 OR, 1 .0) 0.7 0.6 0.2 0.00”
95% CI 0.3 1 .6 0.3 1 .3 O).1 0.5
OR, 1.0 0.5 0.6 0.1 0.01
.,Adtusted
qu(’ncy
95%
of cervical
CI
for current
sniear
smoking
(annual
status,
0.2- 1 .9
monthly
versus less often),
0.2-2.0
personal
00b_04
income.
any use ot sperniicidal
Ire-
.,Adjusted
OR;’
95% Cl
br calories prior
1 .0
to Iogistb
0.2
(1.6
-1 .8
regression
0.2
using
01.6
1 .6
Iine,ir
0.0)’
0.2
regression
- 0.7
0.03
contraceptive agents, sell-reported history of genital warts, quartile of procedures ) 1 0). The logistic moh’I inc luck’s acljustnient for current
Quetelet index, and natural log of number ot sexual partners. smoking status, monthly personal incoilic’, frequency of cervical smear
b <0.05. annual versus less (ifteii), any use ut sI)ernliciclal cc)ntraceptive agents.
self-reported history of genital s’arts, qu,irtile of Quetelet index, and
natural log of number of sexual p.irtic’rs.
,‘ <0.005.
( <0.05.
reflect red cell folate, the findings for red cell folates may CIN. Additionally, the estimates of effect for both serum
be overstated. and dietary folate offer supporting evidence for the role
Folate deficiency has been reported in patients with of inadequate folate nutritional status in the development
a diversity of malignancies (1 7). The question of whether of CIN.
this is a cause or a consequence of cancer remains Some of the difference in the findings for the folate
unanswered. However, it has been suggested that be- variables may be attributable to characteristics of the
cause tumors exhibit rapid cell multiplication, those with measurements. Greater variability is expected in the
cancer may be at increased risk for folate deficiency (18). serum compared to the red cell folate measure, because
Although CIN may represent an early stage of the neo- serum folate indicates short-term changes in folate bal-
plastic process, the length of time required for the pro- ance, while red cell folate reflects changes over several
gression from CIN I and II to cancer (19) argues against months (16). For folates, there is a greater potential for
rapid cell multiplication at this stage of the disease. Since misclassification from dietary intake measures compared
79% of the cases were diagnosed with CIN I or II, it to laboratory data because of inaccuracies in reporting
seems unlikely that the elevated ORs for those in the and converting food to nutrients, the destruction of fo-
Cancer Epidemiology, Biomarkers & Prevention 123
recommendations, and both of these types of foods are 1 1. Longo. D. L., and Herbert, V. Raclioassav for s(’rum and red ( elI
folate. I. Lab. Clin. Med., 87: 138-151, 1976.
good sources of folates.
14. laiob, F., CoIiian, N., and HerI’rt, V. Evaluation of siniult,ineous
radioassav for two vit,imins: folate and vitaiiin B- 1 2. Am. I. Clin. Nutr.,
Acknowledgments 30:616, 1977.
1 5. Naessans, I. M., Offord, K. P., S ott, W. F., md l)aoocl, S. I . The
The authors wish Ic) acknowledge the support c)f Stanley Gall at the
MCSTRAT procedure. In: R. P. Hastings ed.), SUGI Supplemental Library
University of Illinois Hospital, Michael Makii at Cook County Hospital.
User’s Guide, Version 5 Ed., ip. 307- 328. Can,’, NC: SAS Institute, In.,
Eileen McAleer at the Bronx V.A. Hospital, and Ray Murphy at the Illinois
1986.
Department of Public Health. They have appreciated the advice and
(oiiiments of Phyllis Bowen, lack GOIdE)erg, William Haenszel, Frederick 16. Sc’nti, F. R., .10(1 t’ilch, S. lvi. Assessnient (ii tl’ Folate Nutritional
Kviz, and Victoria Persky at the University of Illinois at Chicago. Status of the U.S. Population Based on Data Collected in the Sec ond
National Health and Nutrition Examination Survey 1976- 1 98L). Bethesda,
MD: Life Sciences Research Offices, FASEB, 1984.
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