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Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, United Kingdom, and Norfolk and Norwich Hospital, Brunswick Road, Norwich, NR1 3SR, United Kingdom
2 To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • fish oil • lipids • colon • crypt • cancer • proliferation • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Although the total intake of fat appears not to be a strong risk factor
(World Cancer Research Fund 1997
), the pattern of lipids
consumed may be important because fatty acid intake influences the
lipid composition of cellular membranes, and hence many aspects of
cellular function (Brasitus and et al. 1989
).
Consumption of polyunsaturated fatty acids (PUFA) is difficult to
measure accurately in epidemiological studies (Erickson 1998). However some reports described a weak adverse effect of
a high intake of PUFA on risk of CRC (Goldbohm et al. 1994
) while others reported a slight protective effect
(Bostick et al. 1994
). There is stronger evidence
that the balance of (n-3) and (n-6) PUFA in the diet may play an
important etiological role, however. For example it was recently
reported that some coastal populations have a low incidence of CRC
compared to urban dwellers, which may be associated with high levels of
(n-3) fatty acids in plasma derived from a high intake of marine fish
(Schloss et al. 1997
).
Patients with colorectal neoplasia have an abnormal rate and
distribution of colorectal crypt cell turnover (Terpstra et al. 1987
) and are at increased risk of developing further
colorectal malignancies. Anti et al. (1992)
reported
that relatively high doses of fish oil (FO) reduced the rate of
proliferation in the normal rectal mucosa of patients with previously
resected sporadic adenomata. In a subsequent report (Anti et al. 1994
) they showed that adenoma patients given as little as
2.5 g of FO per day showed some normalization of rate and spatial
distribution of mitosis compared to controls given a placebo. The
changes in proliferation were largely confined to those patients with
grossly abnormal proliferation at entry into the trial, but they were
associated with an increase in the levels of eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA) and a reduction in arachidonic
acid in the rectal mucosal tissue. Although other groups also obtained
evidence for suppression of proliferation by FO, the relationship
between these effects and the incorporation of PUFA into cellular
lipids is unclear. For example Bartram et al. (1993)
fed
11.0 g FO per day to 12 healthy subjects for two 4-wk periods in a
double-blind crossover trial and observed small changes in crypt
cell proliferation but no incorporation of PUFA into rectal mucosal
fatty acids. In a more recent study (Bartram et al. 1995
), the same group conducted a similar experiment in healthy
subjects given a high-fat diet with a low (n-3)/(n-6) ratio and
observed no effect on proliferation, and again no incorporation of
(n-3) fatty acids into mucosal lipids. In the present study we explored
the effect of FO supplementation on the fatty acid composition of the
colorectal mucosa and the associated mesenteric adipose tissue, and on
rectal crypt cell proliferation in patients about to undergo surgery
for left-sided carcinoma of the large bowel.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Patients requiring surgery for left-sided carcinoma of the colon were invited to take part in the study. Volunteers were asked to complete a food frequency questionnaire, and a lifestyle questionnaire which included a question on the habitual use of dietary supplements including marine oils. A total of 51 patients (26 male, 25 female; mean age 71.8 ± 1.1 y) were randomly allocated to FO supplementation or placebo groups by the hospital pharmacy using computer-generated random numbers. The allocation of patients to groups remained unknown to the experimenters prior to final analysis of data. At surgery, tissue samples were collected as described below. A hospital pathologist's report was obtained for each resected tumor, and the modified Duke's stage for each subject was recorded. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Norwich District Research Ethics Committee.
Lipid supplementation.
During the period prior to surgery (7–21 d), the volunteers received capsules containing either "super strength" FO (Boots, Nottingham, United Kingdom; 2 g b.d.) containing 1.4 g of EPA and 1.0 g of DHA per day (FO group), or safflower oil as a control (2 g b.d.; placebo group). Once the patients had resumed a full diet the capsules were recommenced for a period of 8 to 12 wks. The absence of EPA and DHA in the safflower oil was confirmed by analysis using gas–liquid chromatography as described below.
Collection of tissue samples.
Two rectal biopsies were taken using a rigid sigmoidoscope at the presurgical examination. One biopsy was fixed in 3:1 ethanol/glacial acetic acid for determination of crypt cell proliferation as described below, and the other was snap-frozen on dry ice and stored at -80°C. At surgery, two mucosal biopsies were obtained from the descending colon and rectal regions of the resected bowel, at least 5 cm from the margin of the tumor, together with a small sample of mesenteric adipose tissue. One biopsy from each site was fixed as described above and the other, together with the adipose tissue, was snap-frozen immediately after collection. At the postsurgical clinic, two further rectal biopsies were collected and treated as before. Snap-frozen samples were coded and stored at -80°C. Fixed specimens were coded and stored at +1°C.
Crypt cell proliferation.
Crypt cell proliferation was assessed using whole microdissected crypts
(Matthew et al. 1994
). Approximately half of each fixed
biopsy (2–4 mg of fresh tissue) was rehydrated by passage through 50%
ethanol and pure distilled water and then stained with Feulgen's
reagent. Following transfer to aqueous acetic acid (7.9 mol/L) for a
minimum of 16 h, small rows of crypts were microdissected under a
low-power microscope, placed under a coverslip, and slightly
flattened to display individual crypts. The length of each crypt was
estimated by comparison with a calibrated eyepiece graticule, the
numbers of dividing cells per crypt were counted, and the positions of
all nuclei in prophase, metaphase, anaphase, or telophase were noted.
This information was collected from 10 crypts per biopsy and used to
calculate mean numbers of dividing cells per crypt, and the
distribution of dividing cells between five equally spaced longitudinal
compartments.
Epithelial and adipose tissue lipid profiles.
Samples of the intestinal mucosa and mesenteric adipose tissue, taken
from the descending colon of the resected specimen at surgery (50–100
mg fresh weight) were homogenized in chloroform/methanol/water
(2:2:1.8), and lipid extraction was carried out using the
Bligh-Dyer technique (Bligh and Dyer 1959
). The
extracted lipid was derivatized by a cold methylation technique prior
to capillary gas chromatography (CP Sil-88 column; Chrompak,
Middelburg, The Netherlands) to separate the fatty acid methyl esters
(Brown et al. 1998
).
Statistics.
Data are expressed as means with SEM. Student's paired or unpaired t-test was used to compare differences within or between groups of subjects. A logarithmic transformation of the data was carried out where there was a gross difference in variance between groups. In some cases Spearman's correlation coefficient was calculated in order to examine associations between variables. All statistical analyses were carried out using the Minitab statistical package (State College, PA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The average duration of capsule consumption prior to surgery was 12.3 ± 0.5 d for all patients, 12.1 ± 1.0 d for the placebo group, and 12.5 ± 0.5 d for the FO group. The average FO consumption in the FO group was 29.6 ± 1.3 g. Colonic tissue samples were collected at surgery from all patients in both groups, but as one had an abdomino-perineal resection, collection of rectal samples was possible for 25 patients in the FO group and 23 in the placebo group. Of these patients, 17% had tumors classified as Modified Duke's stage A, 46% as B and 37% as C/D. Rectal biopsies were obtained 8–12 wk postoperatively from 11 FO patients and 9 patients in the placebo group. Reasons for withdrawal from the study included abdomino-perineal resection, postoperative chemotherapy or radiotherapy, death or critical illness, or poor compliance.
Incorporation of (n-3) PUFA into the colonic epithelium.
One patient in the placebo group was observed to have a level of EPA in
adipose tissue lying more than 3 SD above the group mean.
This apparent outlier is included in the statistical analysis of
mucosal lipids described below, but it was confirmed that its exclusion
did not materially alter the findings of the study. There were no
significant differences in the proportions of palmitic acid (16:0, data
not shown), stearic acid (18:0), or linoleic acid (18:2) present in the
FO group compared to the placebo group, but the level of oleic acid
(18:1) was significantly lower in the FO group (P
< 0.02; Fig. 1
A). The principal difference observed was that the FO patients had
substantially higher levels of EPA (20:5) in the colonic mucosa than
the patients receiving the placebo (P < 0.001) and the level of eicosatrienoic acid (20:3) was
significantly lower (P < 0.05; Fig 1
B). Both the ratio of EPA + DHA to linoleic acid
(P < 0.001) and the overall ratio of
(n-3) to (n-6) fatty acids (P < 0.05)
in the mucosa was significantly higher in the FO patients than in the
placebo group (Fig 1
C).
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In contrast to the significant incorporation of (n-3) fatty acids into epithelial tissue, there was no evidence that supplementation with FO prior to surgery led to any difference in the level of (n-3) PUFA in the mesenteric adipose tissue. The quantity of EPA expressed as a proportion of the total fatty acids present was 0.074 ± 0.017 g/100 g in the placebo group; this value did not differ significantly from the FO group (0.064 ± 0.007 g/100 g). One patient in the placebo group had a level of EPA in adipose tissue lying more than 3 SD above the group mean. When this value was excluded from the analysis, the corrected mean, though smaller (0.058 ± 0.0065%), did not differ significantly from the FO group.
In a secondary analysis, the data from both FO and placebo groups were combined to determine whether self-reported habitual use of marine oil supplements during the 12 mo prior to surgery was associated with differences in the level of PUFA in adipose tissue. The single patient with abnormally high levels of EPA in adipose tissue reported long-term use of FO but was treated as an outlier and excluded from the analysis. Even so, those subjects who reported habitual use of FO within the 12 mo prior to entering the study (n = 11) had a significantly higher proportion of EPA in the adipose tissue (0.085 ± 0.009 g/100 g) than those who did not (0.057 ± 0.005 g/100 g; P < 0.001), and a higher ratio of (n-3) fatty acids to (n-6) fatty acids (0.010 ± 0.004 g/100 g) compared with those reporting no use of FO (0.0042 ± 0.0004 g/100 g; P < 0.001).
Effect of FO on crypt cell turnover and spatial localization.
The average frequency of mitosis per rectal crypt in the placebo group (14.6 ± 1.2) did not differ from that for the FO group (11.4 ± 1.3) at entry to the study, nor at surgery (13.1 ± 1.5 vs. 11.6 ± 1.0), or at the postsurgical clinic (10.8 ± 1.3 vs. 12.5 ± 2.0). The percentage of mitoses per crypt falling into the upper (luminal) 40% of the total crypt length was 8.81% ± 1.1 in the FO group compared with 10.33% ± 1.2 in the placebo group at entry to the study (P > 0.05). At surgery the equivalent figures for the FO group (8.81% ± 1.0) did not differ from the initial value, nor from the placebo group (8.71% ± 1.2), and similar figures were obtained at the postsurgical clinic (FO: 9.96% ± 1.6; placebo: 8.34% ± 1.5; P > 0.05).
Since there was no effect of treatment on adipose tissue lipid profile, the groups were pooled to identify possible correlations between adipose tissue fatty acid level and crypt cytokinetics. There was no significant relationship between crypt cell proliferation or the proportion of mitoses in the upper crypt and EPA in the adipose tissue or previous FO use, but there was a weak inverse relationship between the proportion of mitoses in the upper crypt at entry and the proportion of linoleic acid in the adipose tissue fatty acids (r = -0.321; P = 0.04). There was also a trend in the same direction at the final time point (r = -0.367; P = 0.085). There was no evidence of a relationship between Duke's stage and any aspect of crypt cell proliferation, nor with previous use of FO or level of EPA in the adipose tissue. However there was a weak but significant correlation between Duke's stage and the proportion of linoleic acid in the adipose tissue (r = 0.387; P = 0.015).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were the first to report any
extensive studies on the incorporation of (n-3) fatty acids into the
colorectal mucosal lipids of human subjects. Patients with inflammatory
bowel disease were given a daily dietary supplement of 18 g of FO
for 12 wk. After 3 wk there was a 7-fold increase in EPA concentration
and 50% increase in DHA in colonic biopsies, and these levels remained
constant throughout the study. Levels of arachidonic acid and
prostaglandin production fell as (n-3) fatty acid levels rose. In the
studies on crypt cell proliferation of Anti et al. (1994)
, patients with a history of colorectal polyps received
2.5 g, 5.1 g, or 7.7 g of FO per day for 30 d or,
2.5 g of FO per day for 6 mo. In both the short- and the
long-term studies, there was ~2-fold increase in mucosal EPA and
DHA at all levels of supplementation and a significant fall in
arachidonic acid concentrations. In contrast, Bartram et al. (1993)
fed 11.0 g of FO per day, containing 4.4 g of
(n-3) fatty acids, to 12 healthy subjects for 4 wk but observed no
change in mucosal (n-3) fatty acids. In the present study a relatively modest supplementation with FO led to a measurable incorporation of EPA into the colonic mucosa of patients with CRC within 7–21 d. The percentage contribution of EPA to the mucosal fatty acid profile was approximately doubled, and there was a significant decline in oleic acid (18:0), though not of linoleic acid (18:2). We were also able to investigate the effect of dietary (n-3) PUFA supplementation on mesenteric adipose tissue for the first time. There was no measurable effect of FO within the short period prior to surgery, but self-reported habitual use of FO supplements was associated with a measurable difference in the EPA content of adipose lipids compared to self-reported nonusers. This finding is consistent with the adipose tissue lipid pool having a relatively slow rate of fatty acid turnover compared to the mucosa.
Anti et al. (1992)
reported that relatively high
doses of FO reduced the rate of proliferation as assessed by
autoradiographic analysis of [3H]thymidine incorporation
in the flat rectal mucosa of patients with previously resected sporadic
adenomata. In a subsequent study (Anti et al. 1994
) they
reported that in 15 adenoma patients receiving 2.5 g of FO per day
both the frequency and spatial distribution of labeled cells were
normalized compared to control patients given a placebo. Huang et al. (1996)
fed Ca. 6.1 g of EPA and DHA per
day to patients after resection of colorectal tumors and observed a
reduction in the proliferative activity of crypts in rectal mucosa as
assessed by bromodeoxyuridine incorporation. As in the studies of
Anti et al. (1994)
, the normalization of DNA synthesis
was observed only in patients with a high level of proliferative
activity at entry into the study. Incorporation of fatty acids into
mucosal tissue was not measured, but a reduction in the plasma
(n-6):(n-3) fatty acid ratio was observed in patients whom rectal crypt
cell proliferation was reduced by FO.
In the present study both the frequency and spatial distribution of mitotic cells within the crypt remained stable in patients before and after surgery for CRC. We found no evidence of any effect of supplementation with FO on these parameters, despite the clear evidence of incorporation of EPA into epithelial cells, and there was no evidence of a relationship between crypt mitosis and self-reported use of FO supplements during the previous 12 mo. Our study therefore provides no additional evidence to support the hypothesis that consumption of FO at levels acceptable to patients or compatible with a normal diet, could provide a means of correcting abnormal crypt cell proliferation in subjects at risk of neoplasia. One important difference between this and previous studies is that whereas other groups measured incorporation of labels into the DNA of crypt cells incubated ex vivo, we measured the frequency and position of mitotic profiles in the mucosa immediately after recovery of the biopsy. It is interesting to speculate that the distribution of DNA synthesis which occurs during a postsurgical incubation in vitro, as is usually measured, may not necessarily reflect the true distribution of cells which enter mitosis in the native colorectal mucosa, which is measured here. This issue deserves further investigation.
Whereas diets rich in EPA were found to inhibit metastasis and cell
proliferation in animal models of bowel cancer (Iwamoto et al. 1998
), linoleic acid was shown to enhance metastasis of mammary
tumors (Hubbard and Erickson 1987
). Concern was
expressed that high levels of linoleic acid consumption may increase
the risk of some types of human cancer, and although the evidence for
this remains tenuous (Zock and Katan 1998
), the
possibility that PUFA may influence metastasis deserves further
investigation (Erickson and Hubbard 1990). In this
context, the observed positive relationship between and the level of
linoleic acid in mesenteric adipose tissue and the Duke's Stage
classification of the resected tumour may be important. Caution is
needed in interpreting this association because Duke's classification
is a pathological staging system, which does not necessarily reflect
biological activity. However the possibility of an association between
the rate of tumor progression and the availability of linoleic acid
from endogenous fatty acids cannot be ruled out. In view of the
increased intake of linoleic acid from the diet which has occurred in
many Western countries over the last 10 y, further attention to
this issue seems warranted.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CRC, colorectal cancer; DHA,
docosahexaenoic acid; EPA, eicosapentanoic acid; FO, fish oil; PUFA,
polyunsaturated fatty acid.
Manuscript received February 25, 1999. Initial review completed April 12, 1999. Revision accepted June 7, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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