- Email: [email protected]
Altered plasma phospholipid fatty acids and nutritional status in patients with uterine cervical cancer
Clinical Nutrition (2008) 27, 371e377
available at www.sciencedirect.com
http://intl.elsevierhealth.com/journals/clnu
ORIGINAL ARTICLE
Altered plasma phospholipid fatty acids and nutritional status in patients with uterine cervical cancer Adriana Q. Lisboa a, Marilia Rezende b, Maria I. Muniz-Junqueira c, Marina K. Ito a,* a
Department of Nutrition, Faculty of Health Sciences, University of Brasilia, Brasilia, DF, Brazil Radiotherapy Service, Brası´lia Districtal Hospital, Brası´lia, DF, Brazil c Laboratory of Cellular Immunology Pathology, Faculty of Medicine, University of Brasilia, Brasilia, DF, Brazil b
Received 16 November 2007; accepted 18 March 2008
KEYWORDS Uterine cervical cancer; Nutritional status; Fatty acid status
Summary Background & aims: Little is known about the fatty acid status of uterine cervical neoplasm patients. Therefore, the aim of this study was to investigate the changes in the plasma phospholipid fatty acid (FA) profile along with nutrition status of cervical cancer patients. Methods: A controlled cross-sectional study with uterine cervical cancer patients virgin of treatment was conducted. Nutritional status, nutrient intake and plasma phospholipid FA were evaluated. Results: Patients had lost weight in relation to their weight 6 months prior (5.1 8.8%) (p < 0.01). Compared to the control group and their usual intake, patients ingested less energy (p < 0.01). Patients also ingested less protein (p < 0.01) and 18:3n-3 FA (p < 0.05), compared to the control group. Patients plasma concentrations of 18:0, 24:0 FA and the 18:0/18:1 FA ratio were higher (p < 0.001), and 16:0 (p Z 0.001) and 18:2n-6 (p Z 0.02) concentrations were lower than that of the control group. Conclusions: The weight loss and low nutrient intake among patients suggest the importance of nutrition intervention at an early stage of the diagnosis and plasma 18:0/18:1 FA ratio may be an indirect marker of FA metabolism disregulation in these patients. ª 2008 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
* Corresponding author. Programa de Po ¸a ¸a ´s-graduac ˜o em Nutric ˜o Humana, Faculdade de Cie ˆncias da Sau ´de, Universidade de Brası´lia, Brası´lia, DF, 70910-900, Brazil. Tel.: þ55 61 3307 2548; fax: þ55 61 3273 3676. E-mail address: [email protected] (M.K. Ito). 0261-5614/$ - see front matter ª 2008 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2008.03.006
372
Introduction Cancer of the cervix is the second leading cancer affecting women worldwide, most of them in developing countries. In Brazil, estimates indicate that about 20,000 women were diagnosed with uterine cervical neoplasm in 2006.1 Human papillomavirus infection is the single most important risk factor for this type of cancer, but together with other relevant cofactors, nutrients and foods may play a role in cervical carcinogenesis,2 protecting against or stimulating its development. Among food factors, dietary polyunsaturated fatty acids of the n-3 series (n-3 PUFA) have shown a protective effect against many cancers.3e5 Tumor initiation, i.e. DNA adduct formation and promotion of colon cancer were prevented when rats were fed n-3 PUFA from fish oil.6 and in mice bearing human breast cancer xenograft and fed on a 10% n-3 PUFA diet, the tumor growth rate was significantly slower than the n-6 PUFA diet.7 In cancer patients, the supplementation of diets with n-3 PUFA inhibited weight loss8 and increased life span.9 Parallel to these PUFA protective effects on tumor cells, elevated fatty acid synthase (FAS) activity has been identified in human breast cancer cells,10 particularly in aggressive types, and many other malignancies.11 The enzyme FAS is responsible for de novo synthesis of medium and long chain saturated fatty acids and its overexpression was shown to increase the ratio of saturated to unsaturated fatty acids in tumor tissues.11 Under normal physiological circumstances, FAS activity is inhibited by long chain fatty acids.12 Therefore, this alteration has been implicated with cancer proliferative properties as it reflects the activity of several genes involved in lipid metabolism.13 The increased expression of the FAS gene and its activity have been hypothesized to play a central role in the neoplastic transformation of cells in the early stages of cancer development.14 Additionally, Menendez et al.15 demonstrated the existence of a strong inhibitory effect of 18:3n-6 (gamma-linolenic acid) and 18:3n-3 (alpha linolenic acid, ALA) fatty acids on human breast cancer associated tumor cell FAS, in vitro. Thus, the understanding of the mechanism involved in the inhibition of tumor cell FAS activity is of great interest due to its potential cancer treatment target.11 However, studies reporting the fatty acid status of cancer patients are rare. Women with higher 18:0/18:1 fatty acid ratio in the blood were found to be at higher risk for breast cancer16 and Leeuwen et al.,17 studying patients with lung, esophageal or pancreatic cancer, found reduced plasma concentration of n-3 PUFA in pancreatic patients compared to the control group and suggested that plasma fatty acids may vary among different cancers. Thus, considering the lack of information on fatty acid status in uterine cervical cancer patients and given the potential therapeutic possibilities of these essential nutrients, the aim of this study was to investigate the plasma phospholipid fatty acid profile, along with nutrition status and intake parameters, of untreated uterine cervical cancer patients, in order to examine their relationship with the established disease.
Materials and methods Study outline A cross-sectional controlled study was conducted with 29 women with uterine cervical cancer during initial diagnosis
A.Q. Lisboa et al. and before the initiation of treatment and a control group of 25 healthy women. During the period from January 2002 to March 2003 all patients at the radiotherapy service of the Brası´lia Districtal Hospital, in Brasilia, who fulfilled the inclusion criteria, were invited to participate in the study. The inclusion criteria were: histopathologically confirmed invasive epidermoid carcinoma of the cervix with indication for radiotherapy with or without sensitizing chemotherapy as initial treatment, without co-morbidities, except for nutritional and psychological ones. Patients with malignant neoplasia of a histological type differing from uterine cervix epidermoid carcinoma or a second neoplasia or a distant metastasis were excluded. Other criteria of exclusion were: local recidivation after surgery and patients under 18 or above 65 years of age. Patients were classified according to their disease stage, following the International Federation of Gynecology and Obstetrics (FIGO) staging classification.18 This research was approved by the Research Ethics Committees of the Federal District State Health Secretariat and of the Faculty of Medicine of the University of Brası´lia and all participants signed a written consent before entering the study.
Anthropometry The nutritional status of the participants was determined by the body mass index (BMI) calculated from body weight (Soehnle Alpha electronic scale, 0.1 kg) divided by the square of the height (Dexter height scale, 0.1 cm), in kg/ m2. The usual weight (UW), consisting of weight 6 months prior to the interview and the weight change (WC) during that period were informed by the patient. The percentage of weight change (%WC) was calculated based on the UW.
Food intake A semi-quantitative food frequency questionnaire (FFQ) based on foods consumed by the Brazilian population19 was used to determine usual food intake. To assess recent consumption, the 24-h recall method was used. Illustrative material was used to help people identify the portions consumed. Daily ingestion of calories and the energy percentages from macronutrients were calculated. Additionally, total PUFAs and the fatty acids: linoleic (LA) (18:2 n-6), alpha-linolenic (ALA) (18:3 n-3), arachidonic (AA) (20:4 n-6), eicosapentaenoic (EPA) (20:5 n-3), and docosahexaenoic (DHA) (22:6 n-3) in the diet were calculated. The nutritional composition of food questionnaires was calculated using the NutriSurvey for Windows software program. The participants were questioned about the presence of symptoms related to dietary intake, such as constipation, appetite, nausea and/or vomiting and diarrhea.
Plasma phospholipid fatty acids Blood samples were collected from the arm vein into a heparinized test tube. The plasma was separated by centrifugation and stored at 80 C until analysis. Total lipids were extracted from plasma (1 ml) by the Folch et al.,20 method. The extracted lipid was evaporated under N2 and resuspended in 20 ml of chloroformemethanol 2:1
FA and nutritional status of uterine cervical neoplasm patients
373
(v/v) for phospholipids separation using silica gel high performance thin-layer chromatography (HPTLC) plates (Merck). The solvent system for HPTLC separation was hexane:diethyl ether:glacial acetic acid (80:20:2, v/v/ v).21 The phospholipid band was removed from the plate and methylated with KOH at 0.5 M in methanol, in the presence of 12% BF3 in methanol.22 Fatty acid methyl ester samples were analyzed by gas chromatography (Shimadzu, model 17A) equipped with a flame ionization detector and DB-Wax capillary column (J&W Scientific), 30 m 0.25 mm 0.25 mm. Injector and detector were kept at 250 C. The oven temperature was a gradient of 150 C initial temperature for 1 min, increments of 5 C/min up to 220 C which was maintained for 12 min. The sample was injected in split mode (1:50) and hydrogen was used as carrier gas. Fatty acids were identified by comparing the individual retention times to external standards of fatty acids (Sigma). Results were expressed as percentage of fatty acid in relation to the total peak area of fatty acids identified in chromatograms.
patients had a significant weight loss (3.6 5.6 kg) in relation to their weight 6 months prior to the interview (p < 0.01) and in relation to the weight change of the control group (þ0.2 2.2 kg) during the same period (p < 0.01).
Statistical analysis
Food intake and related symptoms
Student’s t-test was used to compare independent unpaired data from patients and control group. For correlation analyses, Pearson or Spearman coefficients were used, according to the sample normality distribution. The statistical software package used was the SAS version 8.2. Differences with a two-tailed value of p < 0.05 were considered statistically significant.
Among the gastrointestinal symptoms, 58.6% of patients reported intestinal constipation, compared to 11.8% of the control group. Furthermore, 44.8% of patients reported anorexia, 34.5% nausea and/or vomiting and 3.4% diarrhea, even before initiating the treatment. In terms of usual intake (Table 3), the FFQ showed no statistical difference in the energy intake between the groups. Patients had lower usual protein intake (p < 0.01), of 59.4 23.4 g/day, compared to the control group (78.9 22.0 g/day). Patients’ lower protein intake was counterbalanced by a higher intake of carbohydrates (p < 0.05). Patients also had higher than usual intake of 20:4n-6 compared to the control group (p < 0.01). However, the recent calorie intake (1087 496 kcal/ day) of the patients was significantly reduced compared to their usual intake assessed by the FFQ (p < 0.01) and to the control group’s recent intake (1493 471 kcal/day) (p < 0.01). Consequently, a shift from a normocaloric diet of 24 8 kcal/kg BW, to a hypocaloric one of 19 9 kcal/ kg BW (p < 0.01), was observed among patients. Recent protein intake was also lower among patients (53 31 g/ day) than the control group (72 33 g/day) (p < 0.05). In terms of the fatty acids ingested, the most striking difference was observed in the ALA intake of the patients, which decreased from their usual intake of 1.04 0.48 g/day to 0.71 0.36 g/day (p < 0.01). This recent intake was lower than that of the control group (p < 0.05). In addition, compared to their usual ingestion, patients had reduced their recent carbohydrate (p < 0.05), PUFA (p < 0.01) and LA (p < 0.05) intake. In the control group, habitual and recent intakes were similar (Table 3).
Results The patients’ and control groups’ mean SD ages were 46 8.7 years and 44 8.7 years, respectively, with no significant difference between them (Table 1). As for the clinical staging, 48% of patients were in stage IIa or IIb and 52% of them in stage IIIb. The cancer patients showed a significantly higher mean number of parity than the control group (p < 0.05).
Nutrition status Patients and control groups were similar in weight, with mean BMI ranging from 24.3 to 24.7 kg/m2, respectively (Table 2). However, compared to their usual weight,
Table 1 Clinical characteristics of the cervical cancer patients and control group Variables
Control
Patients
n Age (years) Parity (no.) Clinical staging IIa IIb IIIb
25 44 (28e63) 2 (0e12)
29 46 (29e60) 4 (1e12)a
e e e
4 10 15
a
p < 0.05.
Table 2 Anthropometric data (mean SD) of untreated cervical cancer patients and the control group
Usual weight (kg) Weight (kg) BMI (kg/m2) WC (kg) WC (%)
Control (n Z 24)
Patients (n Z 29)
59.8 10.7 59.8 10.4 24.7 4.9 0.2 2.2 0.4 3.2
61.8 14.8 58.1 13.2 24.3 4.8 3.6 5.6a 5.1 8.8a
BMI, body mass index; WC, weight change. a p < 0.01.
Plasma phospholipid fatty acids In both groups, the fatty acids with highest concentrations were, in decreasing order: LA, AA, stearic acid (18:0), palmitic acid (16:0), oleic acid (18:1n-9) and DHA. These fatty acids represented more than 80% of the total fatty acids identified (Table 4).
374 Table 3
A.Q. Lisboa et al. Nutrient composition of habitual (FFQ) and recent (24 h recall) diets of cancer patients and control group Control (n Z 23)
Calories (kcal/day) kcal/kg Protein (g/day) Carbohydrates (%) Lipid (%) Polyunsaturated (g) Fatty acids (g) 18:2 n-6 20:4 n-6 18:3 n-3 20:5 n-3 22:6 n-3
Patients (n Z 29)
FFQ
24 h recall
FFQ
24 h recall
1641 563 28 9 78.9 22.0 50.4 8.6 27.4 6.9 8.1 4.1
1493 471 25 10 72.5 33.0 49.6 13.4 29.7 10.4 7.8 5.9
1446 470 24 8 59.4 23.4d 56.9 6.6d 26.0 5.8 8.1 4.0
1087 496b,d 19 9b,c 53.7 31.5c 52.0 8.0a 27.5 7.6 5.5 2.9b
6.26 3.60 0.08 0.04 1.13 0.49 0.03 0.04 0.10 0.11
6.14 5.36 0.14 0.20 1.10 0.67 0.06 0.10 0.14 0.16
6.13 3.42 0.23 0.22d 1.04 0.48 0.03 0.06 0.15 0.17
4.21 2.29a 0.16 0.15 0.71 0.36b,c 0.03 0.05 0.09 0.12
FFQ, food frequency questionnaire; kcal/kg, kilocalories ingested per kilogram of body weight; protein (g/kg), grams of ingested protein per kilogram of body weight. a Significantly different from food frequency questionnaire (p < 0.05). b Significantly different from food frequency questionnaire (p < 0.01). c Significantly different from control group (p < 0.05). d Significantly different from control group (p < 0.01).
Patients showed a striking higher 18:0/18:1 ratio (2.27) than the control group (1.84) (p < 0.001) (Table 4), as a result of changes in the saturated and in the monounsaturated fatty acid concentrations. Patients had higher percentages of 18:0 (16.39%) (p < 0.001) and tetracosanoic acid (24:0) (2.92%) (p < 0.001) and lower percentages of 16:0 (12.94) (p Z 0.001) than the control group. Of the monounsaturated fatty acids, a non-significant reduction (p < 0.06) of 18:1 and increases in 20:1 and 22:1 were observed. Among n-3 PUFA, patients had a tendency toward reduced EPA (p Z 0.06) and no difference was observed in other n-3 PUFA or in their total value. Among the n-6 PUFA, patients had lower concentration of LA compared to the control group (p Z 0.02). A positive correlation between the LA intake and its plasma concentration was seen (r Z 0.46; p Z 0.04). The EPA intake was positively correlated to its plasma phospholipids concentration (r Z 0.50; p Z 0.02) as well as with plasma ALA concentration (r Z 0.54; p Z 0.01).
Discussion Weight loss and altered 18:0/18:1 ratio in the plasma phospholipid of uterine cervical cancer patients were the two most relevant results observed in the present study. The significant weight loss before the initiation of the treatment (5.1 8.8%) seen in the patients of this study (Table 2) was similar to that observed by Leeuwen et al.17 in patient with lung cancer (4.8 2.0%) and esophageal cancer (5.7 0.9%). Similar weight loss was also reported by Orr et al.23 for patients with Stage III and IV uterine cervical neoplasm and the observed weight loss was not correlated to age or race. Tunca24 found a slight weight loss (0.5 5.6 kg) among patients with cancer of the cervix (n Z 21) but considered it not nutritionally significant. In our study, the side effects, such as anorexia, reported by
almost 50% of patients, and nausea and/or vomiting seen in 34.5% of them, were unexpected and may partly explain the reduction in calorie intake (p < 0.01) of the patients (Table 3) which, in turn, might be related to the observed weight loss. Although not commonly seen in uterine cervical neoplasms, decreased body weight of oncologic patients is a multi-causal phenomenon. In advanced stages of the disease, cancer cachexia may develop due to a series of interactions between the advanced disease, host metabolic alterations and the prescribed anti-tumor treatment.25 In the early stages of the disease, however, cancer of gastrointestinal origin and surrounding organs is known to lead to early weight loss as a consequence of reduced food intake.26 Psychological causes of weight loss may also be present, reflecting the patients’ anxiety about cancer, its possible progression, depression and anticipatory phenomena.27 In fact, Rezende28 had observed psychological deterioration in the patients of the present study. According to Wilson,25 these factors may lead to reduction in food intake and, therefore, may partly explain the weight loss observed in our patients. This finding underscores the impact of the cancer diagnosis on the patients’ psychological status and calls attention to the importance for healthcare providers to deliver appropriate psychological and dietary interventions in the early stages of treatment to help patients cope with this common symptom.25,26 The higher plasma ratio of 18:0/18:1 fatty acids in cancer patients of this study (Table 4) is in accordance with similar changes observed in surgically removed colonic adenocarcinoma cells.11 To our knowledge, this is the first report of such a change observed in uterine cervical cancer patients. In their study, compared to normal colonic mucosa, Rakheja et al.11 observed that 18:0 in malignant cell biopsies was the only fatty acid that was significantly increased, resulting in a significantly higher 18:0/18:1 ratio of 6.44, in comparison to a ratio of 3.92 for normal colonic mucosa. Similar to our results in the plasma, the ratio of
FA and nutritional status of uterine cervical neoplasm patients Table 4 Mean SD of the composition of plasma phospholipid fatty acids of the control group and of untreated cervical cancer patients, as the percentage of total area Fatty acids Saturated 16:0 17:0 18:0 19:0 20:0 21:0 22:0 24:0 Total Monounsaturated 16:1n-7 18:1n-9 20:1 22:1n-9 Total Polyunsaturated n-6 18:2n-6 20:2n-6 20:3n-6 20:4n-6 22:2n-6 Total n-6 Polyunsaturated n-3 18:3n-3 20:5n-3 22:3n-3 22:6n-3 Total n-3 Total polyunsaturated Ratio n6/n3 18:0/18:1
Control (n Z 24)
Patients (n Z 20)
p-value
15.15 1.72 0.38 0.07 14.56 1.33 0.06 0.07 0.17 0.09 4.97 1.13 0.12 0.06 2.28 0.58 38.00 2.01
12.94 2.24 0.37 0.06 16.39 1.41 0.10 0.09 0.17 1.10 5.37 1.83 0.16 0.12 2.92 0.44 38.76 3.01
0.001 0.63 <0.001 0.15 0.88 0.40 0.15 <0.001 0.34
0.34 0.10 8.15 1.50 0.23 0.06 0.12 0.08 8.85 1.63
0.28 0.15 7.36 1.25 0.30 0.14 0.23 0.23 8.17 1.27
0.13 0.06 0.06 0.06 0.13
22.97 3.10 0.74 0.19 0.36 0.22 16.97 2.92 2.35 2.35 43.39 1.94
20.46 4.05 0.71 0.29 0.25 0.19 18.45 3.21 4.00 4.82 43.88 4.49
0.02 0.69 0.08 0.11 0.17 0.65
0.25 (0.14) 1.20 (0.73) 1.15 (0.37) 7.16 (1.84) 9.76 (2.17) 53.15 (2.44)
0.19 (0.12) 0.88 (0.34) 1.14 (0.39) 7.26 (2.12) 9.47 (2.17) 53.35 (3.34)
0.19 0.06 0.89 0.86 0.65 0.82
4.95 (1.19) 1.84 (0.39)
4.96 (1.15) 2.27 (0.36)
0.50 <0.001
16:0/16:1 did not change between adenocarcinoma and normal cells.11 Nevertheless, the increased ratio of 18:0/ 18:1 observed here suggests the possibility of the plasma fatty acid ratio being a surrogate marker of the overexpressed FAS and possibly of the altered fatty acid metabolism, as seen in other cancer cells. Except for lactating breast and cyclic endometrium, in well-nourished humans the FAS enzyme activity is low.12 Therefore, the increased FAS activity seen in mammary, prostatic, endometrial, ovarian, gastric, esophageal, pulmonary, colonic, among other common human cancers,11 might be occurring in cervix cancer cells as well. FAS expression was seen particularly in aggressive varieties of some of these cancers.13 Although the mechanism involved in the overexpression of this enzyme in tumor cells has not been fully elucidated, it is thought to play an important role in the growth of malignant cells since FAS inhibition was cytotoxic to cancer cells in vitro.10,13,15 The demonstration of the prominent
375 inhibitory effect of the fatty acids ALA and DHA on breast cancer-associated FAS, as opposed to AA, by Menendez and co-workers,15 helps explain some of the mechanisms involved in the protective effect of n-3 PUFA against tumor cells.3e9 Furthermore, because the FAS enzyme synthesizes de novo medium and long chain saturated fatty acids, it has been hypothesized that saturated fatty acids might be more abundant than PUFA in cancer cells, being a potential therapeutic target, due to, for example, the different freezing temperature of these FA.11 Results on the 18:0/ 18:1 FA ratio from Leeuwen et al.’s17 study of pancreatic, lung and esophageal cancer patients and Mosconi et al.’s29 patients with untreated esophageal (n Z 7), stomach (n Z 3) and pancreatic (n Z 2) cancer do not match the present study. Aside from the fact that no fatty acid or food intake information was provided in their studies, which could have influenced their results, part of the explanation may come from the fact that their patients’ tumor histology varied and the results of 18:1 FA in plasma were the sum of 18:1n-7 and 18:1n-9 FA.17 Nevertheless, the increased 18:0/ 18:1 ratio seen in the present study calls for further investigation of its possible relationship with changes in FAS activity in tumor cells and of the mechanisms involved in such changes in uterine cervical neoplasms. In terms of the fatty acid intake as a whole, our plasma phospholipid results are in line with other studies that correlate intake with plasma and/or red blood cell fatty acids. Habitual fat intake of control and patient groups (Table 3) were in accordance with the usual intake of Brazilian women.19,30 In general, cooking oil based on soybean is the main source of fat in the Brazilian diet.30 In comparison to their usual intake and that of the control group, patients had reduced ingestion of 18:3n-3 fatty acid (p < 0.01), which is the precursor of EPA. Plasma EPA concentration was positively correlated to its intake, as well as to 18:3n-3 intake. Patients were also ingesting less 18:2n-6 (p < 0.05) and their plasma18:2n-6 was lower (p < 0.05), compared to the control group. Some recent publications have established plasma phospholipid fatty acids as a marker for dietary fat intake.31,32 In one study, the decreased plasma 18:2n-6 was considered the strongest predictor of a low fat intake.32 Thus, if we consider plasma phospholipid fatty acids as biochemical markers of intake, our results on the patients’ diet indicating no change between habitual and recent intake (26.0% from FFQ and 27.5% from R24) may be overestimating the patients’ recent fat intake. The concept of a biochemical marker of fat intake needs further studies. Likewise, patients’ lower plasma 18:2n-6 concentration strengthens the results of Mosconi et al.29 in malnourished cancer patients, as well as those from pancreatic17 and lung33 cancer patients. These authors found decreased 18:2n-6 fatty acids in plasma and/ or red blood cell membrane phospholipids of patients in comparison to healthy subjects. Although a study that analyzed the composition of fatty acids of lysophosphatidic acid and lysophosphatidylinositol in plasma of ovarian, endometrial and uterine cervical cancer patients detected higher unsaturated fatty acids, such as 18:2n-6, 18:1 and 20:4 fatty acids, in most advanced cases34 Qi et al.,35 who investigated the influence of dietary 18:2n-6 fatty acid on cancer development using human papillomavirus 16 transgenic mice fed on diet rich
376 in corn oil, concluded that in contrast to skin cancer, high dietary fat had no effect on the development of uterine cervical cancer. It is interesting to note that while skin cancer growth was enhanced with the diet, estrogen was the cofactor needed for cervical cancer development,35 indicating the involvement of different cellular and tissue specific factors mediating tumor cell proliferation. Therefore, the low level of plasma 18:2n-6 in patients of the present study is suggestive of a lowered intake rather than disease related metabolic alterations. There are several limitations in this study. Results from cross sectional studies can only infer about the association between results. The food intake assessment methods used in the present study are known for their intrinsic limitations. The FFQ and R24 intake methods must rely on patients’ memory with its corresponding intra- and inter-subject and observer measurement errors, therefore the differences observed need to be evaluated with caution. However, assuming that the intra-observer errors were uniform throughout the study because the same person carried out all the interviews, we were able to observe important differences between patients and healthy volunteers. In conclusion, in our study we observed weight loss in uterine cervical cancer patients even before the initiation of the treatment, associated with a reduction in calorie intake and presence of symptoms such as anorexia, nausea and vomiting. The weight loss and low nutrient intake of cervical cancer patients present at the time of disease diagnosis suggest the importance of nutrition intervention at an early stage of the disease treatment. The increased ratio of plasma 18:0/18:1 fatty acids may be an indirect marker of FA metabolism dysregulation in these patients.
Conflict of interest statement There are no financial conflicts of interest for any of the authors of this work.
Acknowledgements Contributors: A.L. and M.R. carried out the sample and data analyses. A.L. drafted the manuscript. M.M. and M.I. conceived the study, participated in the design and the coordination of the study, assisted in the statistical analyses and were responsible for the final writing of the manuscript.
References 1. Estimativa 2006, Incide ˆncia de Ca ˆncer no Brasil. Brasil. Ministe ¸˜ ao a ´rio da Sau ` Sau ´de. Secretaria de Atenc ´de, Instituto Nacional de Caˆncer. Rio de Janeiro: INCA, 2005 [accessed November 1, 2007, at, http://www.inca.gov.br/estimativa/2006/ versaofinal.pdf]. 2. Garcia-Closas R, Castellsaque X, Bosch X, Gonzalez CA. The role of diet and nutrition in cervical carcinogenesis: a review of recent evidence. Int J Cancer 2005;117:629e37. 3. Robinson LE, Clandinin T, Field CJ. R3230AC rat mammary tumor and dietary long-chain (n-3) fatty acids change immune cell composition and function during mitogen activation. J Nutr 2001;131:2021e7.
A.Q. Lisboa et al. 4. Boudreau MD, Sohn KH, Rhee SH, Lee SW, Hunt JD, Hwang DH. Suppression of tumor cell growth in nude mice and in culture by n-3 polyunsaturated fatty acidy: mediation through cycooxygenase-independent pathways. Cancer Res 2001;61:1386e91. 5. Kato T, Hancock RL, Mohammadpour H, McGregor B, Manalo P, Khaiboullina S, et al. Influence of omega-3 fatty acids on the growth of human colon carcinoma in nude mice. Cancer Lett 2002;187:169e77. 6. Davidson LA, Nguyen DV, Hokanson RM, Callaway ES, Isett RB, Turner ND, et al. Chemopreventive n-3 polyunsaturated fatty acids reprogram genetic signatures during colon cancer initiation and progression in the rat. Cancer Res 2004;64:6797e804. 7. Hardman WE. (n-3) Fatty acids and cancer therapy. J Nutr 2004;134:S3427e30. 8. Barber MD, Ross JA, Preston T, Shenkin A, Fearon KCH. Fish oil-enriched nutritional supplement attenuates progression of the acute-phase response in weight-losing patients with advanced pancreatic cancer. J Nutr 1999;129:1120e5. 9. Gogos CA, Ginopoulos P, Salsa B, Apostolidou E, Zoumbos N, Kalfarentzos F. Dietary omega-3 polyunsaturated fatty acids plus vitamin E restore immunodeficiency and prolong survival for severely III patients with generalized malignancy. A randomized control trial. Cancer 1998;82:395e402. 10. Kuhajda FP, Jenner K, Wood FD, Henningar RA, Jacobs LB, Dick JD, et al. Fatty acid synthesis: a potential selective target for antineoplastic therapy. Proc Natl Acad Sci USA 1994;91: 6379e83. 11. Rakheja D, Kapur P, Hoang MP, Roy LC, Bennett MJ. Increased ratio of saturated to unsaturated C18 fatty acids in colonic adenocarcinoma: implications for cryotherapy and lipid raft function. Med Hypotheses 2005;65:1120e3. 12. Volpe JJ, Vagelos PR. Saturated fatty acid biosynthesis and its regulation. Annu Rev Biochem 1973;42:21e60. 13. Kuhajda FP, Pizer ES, Li JN, Mani NS, Frehywot GL, Townsend CA. Synthesis and anti-tumor activity of a novel inhibitor of fatty acid synthase. Proc Natl Acad Sci USA 2000; 97:3450e4. 14. Menendez JA, Colomer R, Lupu R. Why does tumor-associated fatty acid synthase (oncogenic antigen-519) ignore dietary fatty acids? Med Hypotheses 2005;64:342e9. 15. Menendez JA, Ropero S, Mehmi I, Atlas E, Colomer R, Lupu R. Overexpression and hyperactivity of breast cancer-associated fatty acid synthase (oncogenic antigem-519) is insensitive to normal arachidonic fatty acid-induced suppression in lipogenic tissues but it is selectively inhibited by tumoricidal alpha-linolenic and gamma-linolenic fatty acids: a novel mechanism by which dietary fat can alter mammary tumorigenesis. Int J Oncol 2004;24:1369e83. 16. Chaje `s V, Hulte ´n K, Kappel ALV, Winkvist A, Kaaks R, Hallmans G, et al. Fatty-acids composition in serum phospholipids and risk of breast cancer: an incident case-control study in Sweden. Int J Cancer 1999;83:585e90. 17. Leeuwen SDZ, Heijden MSVD, Rietveld T, Berg JWOVD, Tilanus HW, Burgers JA, et al. Fatty acids composition of plasma lipids in patients with pancreatic, lung and oesophageal cancer in comparison with healthy subjects. Clin Nutr 2002;21: 225e30. 18. Percorelli S, Benedet JL, Creasman WT, Sheperd JH. FIGO staging of gynecologic cancer. 1994e1997 FIGO Committee on Gynecologic Oncology. International Federation of Gynecology and Obstetrics. Int J Gynecol Obstetr 1999;65:5e10. 19. Sichieri R, Everhart JE. Validity of a Brazilian food frequency questionnaire against dietary recalls and estimated energy intake. Nutr Res 1998;18:1649e59. 20. Folch J, Lees M, Stanley GHS. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226:497e509. 21. Christie WW. HPTLC and lipids. A practical guide. 2nd ed. 1987.
FA and nutritional status of uterine cervical neoplasm patients 22. Ito MK, Simpson KL. The Biosynthesis of u3 fatty acids from 18: 2 n-6 in Artemia ssp. Comp Biochem Physiol 1996;115B:69e76. 23. Orr JW, Wilson K, Bodiford C, Cornwell A, Soong SJ, Honea KL, et al. Nutritional status of patients with untreated cervical cancer. Am J Obstet Gynecol 1985;151:625e31. 24. Tunca JC. Nutritional evaluation of gynecologic cancer patients during initial diagnosis of their disease. Am J Obstet Gynecol 1983;147:893e6. 25. Wilson RL. Optimizing nutrition for patients with cancer. Clin J Oncol Nurs 2000;4:23e8. 26. Nitenberg G, Raynard B. Nutritional support of the cancer patient: issues and dilemmas. Crit Rev Oncol Hematol 2000; 34:137e68. 27. Lesko LM. Psychosocial issues in the diagnosis and management of cancer cachexia and anorexia. Nutrition 1989;5:114e6. 28. Rezende, M. Influe ˆncia da radioterapia sobre a capacidade fagocita ´ria e o estado psicolo ´gico em mulheres com carcinoma epidermo ´ide de colo uterino. Master’s dissertation, Faculdade de Cie ˆncias da Sau ´de, Universidade de Brası´lia; 2003. 29. Mosconi C, Agradi E, Gambetta A, Bozzetti F, Galli C. Decrease of polyunsaturated fatty acids and elevation of the oleic/stearic acid ratio in plasma and red blood cell lipids of malnourished cancer patients. J Parenter Enteral Nutr 1989;13:501e4.
377 30. Cunha J, Costa THM, Ito MK. Influences of maternal dietary intake and suckling on breast milk lipid and fatty acid composition in low-income women from Brasilia, Brazil. Early Hum Dev 2005;81:303e11. 31. Raatz SK, Bibus D, Thomas W, Kris-Etherton P. Total Fat intake modifies plasma fatty acid composition in humans. J Nutr 2001; 131:231e4. 32. King IB, Lemaitre RN, Kestin M. Effect of a low-fat diet on fatty acid composition in red cells, plasma phospholipids and cholesterol esters: investigation of a biomarker of total fat intake. Am J Clin Nutr 2006;83:227e36. 33. Prisco D, Paniccia R, Coppo M, Filippini M, Francalanci I, Brunelli T, et al. Platelet activation and platelet lipid composition in pulmonary cancer. Prostaglandins Leukot Essent Fatty Acids 1995;53:65e8. 34. Shen Z, Wu M, Elson P, Kennedy AW, Belinson J, Casey G, et al. Fatty acid composition of lysophosphatidic acid and lysophosphatidynositol in plasma from patients with ovarian cancer and other gynecological diseases. Gynecol Oncol 2001;83: 25e30. 35. Qi M, Chen DZ, Liu K, Auborn KJ. N-6 polyunsaturated fatty acids increase skin but not cervical cancer in human papillomavirus 16 transgenic mice. Cancer Res 2002;62:433e6.
available at www.sciencedirect.com
http://intl.elsevierhealth.com/journals/clnu
ORIGINAL ARTICLE
Altered plasma phospholipid fatty acids and nutritional status in patients with uterine cervical cancer Adriana Q. Lisboa a, Marilia Rezende b, Maria I. Muniz-Junqueira c, Marina K. Ito a,* a
Department of Nutrition, Faculty of Health Sciences, University of Brasilia, Brasilia, DF, Brazil Radiotherapy Service, Brası´lia Districtal Hospital, Brası´lia, DF, Brazil c Laboratory of Cellular Immunology Pathology, Faculty of Medicine, University of Brasilia, Brasilia, DF, Brazil b
Received 16 November 2007; accepted 18 March 2008
KEYWORDS Uterine cervical cancer; Nutritional status; Fatty acid status
Summary Background & aims: Little is known about the fatty acid status of uterine cervical neoplasm patients. Therefore, the aim of this study was to investigate the changes in the plasma phospholipid fatty acid (FA) profile along with nutrition status of cervical cancer patients. Methods: A controlled cross-sectional study with uterine cervical cancer patients virgin of treatment was conducted. Nutritional status, nutrient intake and plasma phospholipid FA were evaluated. Results: Patients had lost weight in relation to their weight 6 months prior (5.1 8.8%) (p < 0.01). Compared to the control group and their usual intake, patients ingested less energy (p < 0.01). Patients also ingested less protein (p < 0.01) and 18:3n-3 FA (p < 0.05), compared to the control group. Patients plasma concentrations of 18:0, 24:0 FA and the 18:0/18:1 FA ratio were higher (p < 0.001), and 16:0 (p Z 0.001) and 18:2n-6 (p Z 0.02) concentrations were lower than that of the control group. Conclusions: The weight loss and low nutrient intake among patients suggest the importance of nutrition intervention at an early stage of the diagnosis and plasma 18:0/18:1 FA ratio may be an indirect marker of FA metabolism disregulation in these patients. ª 2008 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
* Corresponding author. Programa de Po ¸a ¸a ´s-graduac ˜o em Nutric ˜o Humana, Faculdade de Cie ˆncias da Sau ´de, Universidade de Brası´lia, Brası´lia, DF, 70910-900, Brazil. Tel.: þ55 61 3307 2548; fax: þ55 61 3273 3676. E-mail address: [email protected] (M.K. Ito). 0261-5614/$ - see front matter ª 2008 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2008.03.006
372
Introduction Cancer of the cervix is the second leading cancer affecting women worldwide, most of them in developing countries. In Brazil, estimates indicate that about 20,000 women were diagnosed with uterine cervical neoplasm in 2006.1 Human papillomavirus infection is the single most important risk factor for this type of cancer, but together with other relevant cofactors, nutrients and foods may play a role in cervical carcinogenesis,2 protecting against or stimulating its development. Among food factors, dietary polyunsaturated fatty acids of the n-3 series (n-3 PUFA) have shown a protective effect against many cancers.3e5 Tumor initiation, i.e. DNA adduct formation and promotion of colon cancer were prevented when rats were fed n-3 PUFA from fish oil.6 and in mice bearing human breast cancer xenograft and fed on a 10% n-3 PUFA diet, the tumor growth rate was significantly slower than the n-6 PUFA diet.7 In cancer patients, the supplementation of diets with n-3 PUFA inhibited weight loss8 and increased life span.9 Parallel to these PUFA protective effects on tumor cells, elevated fatty acid synthase (FAS) activity has been identified in human breast cancer cells,10 particularly in aggressive types, and many other malignancies.11 The enzyme FAS is responsible for de novo synthesis of medium and long chain saturated fatty acids and its overexpression was shown to increase the ratio of saturated to unsaturated fatty acids in tumor tissues.11 Under normal physiological circumstances, FAS activity is inhibited by long chain fatty acids.12 Therefore, this alteration has been implicated with cancer proliferative properties as it reflects the activity of several genes involved in lipid metabolism.13 The increased expression of the FAS gene and its activity have been hypothesized to play a central role in the neoplastic transformation of cells in the early stages of cancer development.14 Additionally, Menendez et al.15 demonstrated the existence of a strong inhibitory effect of 18:3n-6 (gamma-linolenic acid) and 18:3n-3 (alpha linolenic acid, ALA) fatty acids on human breast cancer associated tumor cell FAS, in vitro. Thus, the understanding of the mechanism involved in the inhibition of tumor cell FAS activity is of great interest due to its potential cancer treatment target.11 However, studies reporting the fatty acid status of cancer patients are rare. Women with higher 18:0/18:1 fatty acid ratio in the blood were found to be at higher risk for breast cancer16 and Leeuwen et al.,17 studying patients with lung, esophageal or pancreatic cancer, found reduced plasma concentration of n-3 PUFA in pancreatic patients compared to the control group and suggested that plasma fatty acids may vary among different cancers. Thus, considering the lack of information on fatty acid status in uterine cervical cancer patients and given the potential therapeutic possibilities of these essential nutrients, the aim of this study was to investigate the plasma phospholipid fatty acid profile, along with nutrition status and intake parameters, of untreated uterine cervical cancer patients, in order to examine their relationship with the established disease.
Materials and methods Study outline A cross-sectional controlled study was conducted with 29 women with uterine cervical cancer during initial diagnosis
A.Q. Lisboa et al. and before the initiation of treatment and a control group of 25 healthy women. During the period from January 2002 to March 2003 all patients at the radiotherapy service of the Brası´lia Districtal Hospital, in Brasilia, who fulfilled the inclusion criteria, were invited to participate in the study. The inclusion criteria were: histopathologically confirmed invasive epidermoid carcinoma of the cervix with indication for radiotherapy with or without sensitizing chemotherapy as initial treatment, without co-morbidities, except for nutritional and psychological ones. Patients with malignant neoplasia of a histological type differing from uterine cervix epidermoid carcinoma or a second neoplasia or a distant metastasis were excluded. Other criteria of exclusion were: local recidivation after surgery and patients under 18 or above 65 years of age. Patients were classified according to their disease stage, following the International Federation of Gynecology and Obstetrics (FIGO) staging classification.18 This research was approved by the Research Ethics Committees of the Federal District State Health Secretariat and of the Faculty of Medicine of the University of Brası´lia and all participants signed a written consent before entering the study.
Anthropometry The nutritional status of the participants was determined by the body mass index (BMI) calculated from body weight (Soehnle Alpha electronic scale, 0.1 kg) divided by the square of the height (Dexter height scale, 0.1 cm), in kg/ m2. The usual weight (UW), consisting of weight 6 months prior to the interview and the weight change (WC) during that period were informed by the patient. The percentage of weight change (%WC) was calculated based on the UW.
Food intake A semi-quantitative food frequency questionnaire (FFQ) based on foods consumed by the Brazilian population19 was used to determine usual food intake. To assess recent consumption, the 24-h recall method was used. Illustrative material was used to help people identify the portions consumed. Daily ingestion of calories and the energy percentages from macronutrients were calculated. Additionally, total PUFAs and the fatty acids: linoleic (LA) (18:2 n-6), alpha-linolenic (ALA) (18:3 n-3), arachidonic (AA) (20:4 n-6), eicosapentaenoic (EPA) (20:5 n-3), and docosahexaenoic (DHA) (22:6 n-3) in the diet were calculated. The nutritional composition of food questionnaires was calculated using the NutriSurvey for Windows software program. The participants were questioned about the presence of symptoms related to dietary intake, such as constipation, appetite, nausea and/or vomiting and diarrhea.
Plasma phospholipid fatty acids Blood samples were collected from the arm vein into a heparinized test tube. The plasma was separated by centrifugation and stored at 80 C until analysis. Total lipids were extracted from plasma (1 ml) by the Folch et al.,20 method. The extracted lipid was evaporated under N2 and resuspended in 20 ml of chloroformemethanol 2:1
FA and nutritional status of uterine cervical neoplasm patients
373
(v/v) for phospholipids separation using silica gel high performance thin-layer chromatography (HPTLC) plates (Merck). The solvent system for HPTLC separation was hexane:diethyl ether:glacial acetic acid (80:20:2, v/v/ v).21 The phospholipid band was removed from the plate and methylated with KOH at 0.5 M in methanol, in the presence of 12% BF3 in methanol.22 Fatty acid methyl ester samples were analyzed by gas chromatography (Shimadzu, model 17A) equipped with a flame ionization detector and DB-Wax capillary column (J&W Scientific), 30 m 0.25 mm 0.25 mm. Injector and detector were kept at 250 C. The oven temperature was a gradient of 150 C initial temperature for 1 min, increments of 5 C/min up to 220 C which was maintained for 12 min. The sample was injected in split mode (1:50) and hydrogen was used as carrier gas. Fatty acids were identified by comparing the individual retention times to external standards of fatty acids (Sigma). Results were expressed as percentage of fatty acid in relation to the total peak area of fatty acids identified in chromatograms.
patients had a significant weight loss (3.6 5.6 kg) in relation to their weight 6 months prior to the interview (p < 0.01) and in relation to the weight change of the control group (þ0.2 2.2 kg) during the same period (p < 0.01).
Statistical analysis
Food intake and related symptoms
Student’s t-test was used to compare independent unpaired data from patients and control group. For correlation analyses, Pearson or Spearman coefficients were used, according to the sample normality distribution. The statistical software package used was the SAS version 8.2. Differences with a two-tailed value of p < 0.05 were considered statistically significant.
Among the gastrointestinal symptoms, 58.6% of patients reported intestinal constipation, compared to 11.8% of the control group. Furthermore, 44.8% of patients reported anorexia, 34.5% nausea and/or vomiting and 3.4% diarrhea, even before initiating the treatment. In terms of usual intake (Table 3), the FFQ showed no statistical difference in the energy intake between the groups. Patients had lower usual protein intake (p < 0.01), of 59.4 23.4 g/day, compared to the control group (78.9 22.0 g/day). Patients’ lower protein intake was counterbalanced by a higher intake of carbohydrates (p < 0.05). Patients also had higher than usual intake of 20:4n-6 compared to the control group (p < 0.01). However, the recent calorie intake (1087 496 kcal/ day) of the patients was significantly reduced compared to their usual intake assessed by the FFQ (p < 0.01) and to the control group’s recent intake (1493 471 kcal/day) (p < 0.01). Consequently, a shift from a normocaloric diet of 24 8 kcal/kg BW, to a hypocaloric one of 19 9 kcal/ kg BW (p < 0.01), was observed among patients. Recent protein intake was also lower among patients (53 31 g/ day) than the control group (72 33 g/day) (p < 0.05). In terms of the fatty acids ingested, the most striking difference was observed in the ALA intake of the patients, which decreased from their usual intake of 1.04 0.48 g/day to 0.71 0.36 g/day (p < 0.01). This recent intake was lower than that of the control group (p < 0.05). In addition, compared to their usual ingestion, patients had reduced their recent carbohydrate (p < 0.05), PUFA (p < 0.01) and LA (p < 0.05) intake. In the control group, habitual and recent intakes were similar (Table 3).
Results The patients’ and control groups’ mean SD ages were 46 8.7 years and 44 8.7 years, respectively, with no significant difference between them (Table 1). As for the clinical staging, 48% of patients were in stage IIa or IIb and 52% of them in stage IIIb. The cancer patients showed a significantly higher mean number of parity than the control group (p < 0.05).
Nutrition status Patients and control groups were similar in weight, with mean BMI ranging from 24.3 to 24.7 kg/m2, respectively (Table 2). However, compared to their usual weight,
Table 1 Clinical characteristics of the cervical cancer patients and control group Variables
Control
Patients
n Age (years) Parity (no.) Clinical staging IIa IIb IIIb
25 44 (28e63) 2 (0e12)
29 46 (29e60) 4 (1e12)a
e e e
4 10 15
a
p < 0.05.
Table 2 Anthropometric data (mean SD) of untreated cervical cancer patients and the control group
Usual weight (kg) Weight (kg) BMI (kg/m2) WC (kg) WC (%)
Control (n Z 24)
Patients (n Z 29)
59.8 10.7 59.8 10.4 24.7 4.9 0.2 2.2 0.4 3.2
61.8 14.8 58.1 13.2 24.3 4.8 3.6 5.6a 5.1 8.8a
BMI, body mass index; WC, weight change. a p < 0.01.
Plasma phospholipid fatty acids In both groups, the fatty acids with highest concentrations were, in decreasing order: LA, AA, stearic acid (18:0), palmitic acid (16:0), oleic acid (18:1n-9) and DHA. These fatty acids represented more than 80% of the total fatty acids identified (Table 4).
374 Table 3
A.Q. Lisboa et al. Nutrient composition of habitual (FFQ) and recent (24 h recall) diets of cancer patients and control group Control (n Z 23)
Calories (kcal/day) kcal/kg Protein (g/day) Carbohydrates (%) Lipid (%) Polyunsaturated (g) Fatty acids (g) 18:2 n-6 20:4 n-6 18:3 n-3 20:5 n-3 22:6 n-3
Patients (n Z 29)
FFQ
24 h recall
FFQ
24 h recall
1641 563 28 9 78.9 22.0 50.4 8.6 27.4 6.9 8.1 4.1
1493 471 25 10 72.5 33.0 49.6 13.4 29.7 10.4 7.8 5.9
1446 470 24 8 59.4 23.4d 56.9 6.6d 26.0 5.8 8.1 4.0
1087 496b,d 19 9b,c 53.7 31.5c 52.0 8.0a 27.5 7.6 5.5 2.9b
6.26 3.60 0.08 0.04 1.13 0.49 0.03 0.04 0.10 0.11
6.14 5.36 0.14 0.20 1.10 0.67 0.06 0.10 0.14 0.16
6.13 3.42 0.23 0.22d 1.04 0.48 0.03 0.06 0.15 0.17
4.21 2.29a 0.16 0.15 0.71 0.36b,c 0.03 0.05 0.09 0.12
FFQ, food frequency questionnaire; kcal/kg, kilocalories ingested per kilogram of body weight; protein (g/kg), grams of ingested protein per kilogram of body weight. a Significantly different from food frequency questionnaire (p < 0.05). b Significantly different from food frequency questionnaire (p < 0.01). c Significantly different from control group (p < 0.05). d Significantly different from control group (p < 0.01).
Patients showed a striking higher 18:0/18:1 ratio (2.27) than the control group (1.84) (p < 0.001) (Table 4), as a result of changes in the saturated and in the monounsaturated fatty acid concentrations. Patients had higher percentages of 18:0 (16.39%) (p < 0.001) and tetracosanoic acid (24:0) (2.92%) (p < 0.001) and lower percentages of 16:0 (12.94) (p Z 0.001) than the control group. Of the monounsaturated fatty acids, a non-significant reduction (p < 0.06) of 18:1 and increases in 20:1 and 22:1 were observed. Among n-3 PUFA, patients had a tendency toward reduced EPA (p Z 0.06) and no difference was observed in other n-3 PUFA or in their total value. Among the n-6 PUFA, patients had lower concentration of LA compared to the control group (p Z 0.02). A positive correlation between the LA intake and its plasma concentration was seen (r Z 0.46; p Z 0.04). The EPA intake was positively correlated to its plasma phospholipids concentration (r Z 0.50; p Z 0.02) as well as with plasma ALA concentration (r Z 0.54; p Z 0.01).
Discussion Weight loss and altered 18:0/18:1 ratio in the plasma phospholipid of uterine cervical cancer patients were the two most relevant results observed in the present study. The significant weight loss before the initiation of the treatment (5.1 8.8%) seen in the patients of this study (Table 2) was similar to that observed by Leeuwen et al.17 in patient with lung cancer (4.8 2.0%) and esophageal cancer (5.7 0.9%). Similar weight loss was also reported by Orr et al.23 for patients with Stage III and IV uterine cervical neoplasm and the observed weight loss was not correlated to age or race. Tunca24 found a slight weight loss (0.5 5.6 kg) among patients with cancer of the cervix (n Z 21) but considered it not nutritionally significant. In our study, the side effects, such as anorexia, reported by
almost 50% of patients, and nausea and/or vomiting seen in 34.5% of them, were unexpected and may partly explain the reduction in calorie intake (p < 0.01) of the patients (Table 3) which, in turn, might be related to the observed weight loss. Although not commonly seen in uterine cervical neoplasms, decreased body weight of oncologic patients is a multi-causal phenomenon. In advanced stages of the disease, cancer cachexia may develop due to a series of interactions between the advanced disease, host metabolic alterations and the prescribed anti-tumor treatment.25 In the early stages of the disease, however, cancer of gastrointestinal origin and surrounding organs is known to lead to early weight loss as a consequence of reduced food intake.26 Psychological causes of weight loss may also be present, reflecting the patients’ anxiety about cancer, its possible progression, depression and anticipatory phenomena.27 In fact, Rezende28 had observed psychological deterioration in the patients of the present study. According to Wilson,25 these factors may lead to reduction in food intake and, therefore, may partly explain the weight loss observed in our patients. This finding underscores the impact of the cancer diagnosis on the patients’ psychological status and calls attention to the importance for healthcare providers to deliver appropriate psychological and dietary interventions in the early stages of treatment to help patients cope with this common symptom.25,26 The higher plasma ratio of 18:0/18:1 fatty acids in cancer patients of this study (Table 4) is in accordance with similar changes observed in surgically removed colonic adenocarcinoma cells.11 To our knowledge, this is the first report of such a change observed in uterine cervical cancer patients. In their study, compared to normal colonic mucosa, Rakheja et al.11 observed that 18:0 in malignant cell biopsies was the only fatty acid that was significantly increased, resulting in a significantly higher 18:0/18:1 ratio of 6.44, in comparison to a ratio of 3.92 for normal colonic mucosa. Similar to our results in the plasma, the ratio of
FA and nutritional status of uterine cervical neoplasm patients Table 4 Mean SD of the composition of plasma phospholipid fatty acids of the control group and of untreated cervical cancer patients, as the percentage of total area Fatty acids Saturated 16:0 17:0 18:0 19:0 20:0 21:0 22:0 24:0 Total Monounsaturated 16:1n-7 18:1n-9 20:1 22:1n-9 Total Polyunsaturated n-6 18:2n-6 20:2n-6 20:3n-6 20:4n-6 22:2n-6 Total n-6 Polyunsaturated n-3 18:3n-3 20:5n-3 22:3n-3 22:6n-3 Total n-3 Total polyunsaturated Ratio n6/n3 18:0/18:1
Control (n Z 24)
Patients (n Z 20)
p-value
15.15 1.72 0.38 0.07 14.56 1.33 0.06 0.07 0.17 0.09 4.97 1.13 0.12 0.06 2.28 0.58 38.00 2.01
12.94 2.24 0.37 0.06 16.39 1.41 0.10 0.09 0.17 1.10 5.37 1.83 0.16 0.12 2.92 0.44 38.76 3.01
0.001 0.63 <0.001 0.15 0.88 0.40 0.15 <0.001 0.34
0.34 0.10 8.15 1.50 0.23 0.06 0.12 0.08 8.85 1.63
0.28 0.15 7.36 1.25 0.30 0.14 0.23 0.23 8.17 1.27
0.13 0.06 0.06 0.06 0.13
22.97 3.10 0.74 0.19 0.36 0.22 16.97 2.92 2.35 2.35 43.39 1.94
20.46 4.05 0.71 0.29 0.25 0.19 18.45 3.21 4.00 4.82 43.88 4.49
0.02 0.69 0.08 0.11 0.17 0.65
0.25 (0.14) 1.20 (0.73) 1.15 (0.37) 7.16 (1.84) 9.76 (2.17) 53.15 (2.44)
0.19 (0.12) 0.88 (0.34) 1.14 (0.39) 7.26 (2.12) 9.47 (2.17) 53.35 (3.34)
0.19 0.06 0.89 0.86 0.65 0.82
4.95 (1.19) 1.84 (0.39)
4.96 (1.15) 2.27 (0.36)
0.50 <0.001
16:0/16:1 did not change between adenocarcinoma and normal cells.11 Nevertheless, the increased ratio of 18:0/ 18:1 observed here suggests the possibility of the plasma fatty acid ratio being a surrogate marker of the overexpressed FAS and possibly of the altered fatty acid metabolism, as seen in other cancer cells. Except for lactating breast and cyclic endometrium, in well-nourished humans the FAS enzyme activity is low.12 Therefore, the increased FAS activity seen in mammary, prostatic, endometrial, ovarian, gastric, esophageal, pulmonary, colonic, among other common human cancers,11 might be occurring in cervix cancer cells as well. FAS expression was seen particularly in aggressive varieties of some of these cancers.13 Although the mechanism involved in the overexpression of this enzyme in tumor cells has not been fully elucidated, it is thought to play an important role in the growth of malignant cells since FAS inhibition was cytotoxic to cancer cells in vitro.10,13,15 The demonstration of the prominent
375 inhibitory effect of the fatty acids ALA and DHA on breast cancer-associated FAS, as opposed to AA, by Menendez and co-workers,15 helps explain some of the mechanisms involved in the protective effect of n-3 PUFA against tumor cells.3e9 Furthermore, because the FAS enzyme synthesizes de novo medium and long chain saturated fatty acids, it has been hypothesized that saturated fatty acids might be more abundant than PUFA in cancer cells, being a potential therapeutic target, due to, for example, the different freezing temperature of these FA.11 Results on the 18:0/ 18:1 FA ratio from Leeuwen et al.’s17 study of pancreatic, lung and esophageal cancer patients and Mosconi et al.’s29 patients with untreated esophageal (n Z 7), stomach (n Z 3) and pancreatic (n Z 2) cancer do not match the present study. Aside from the fact that no fatty acid or food intake information was provided in their studies, which could have influenced their results, part of the explanation may come from the fact that their patients’ tumor histology varied and the results of 18:1 FA in plasma were the sum of 18:1n-7 and 18:1n-9 FA.17 Nevertheless, the increased 18:0/ 18:1 ratio seen in the present study calls for further investigation of its possible relationship with changes in FAS activity in tumor cells and of the mechanisms involved in such changes in uterine cervical neoplasms. In terms of the fatty acid intake as a whole, our plasma phospholipid results are in line with other studies that correlate intake with plasma and/or red blood cell fatty acids. Habitual fat intake of control and patient groups (Table 3) were in accordance with the usual intake of Brazilian women.19,30 In general, cooking oil based on soybean is the main source of fat in the Brazilian diet.30 In comparison to their usual intake and that of the control group, patients had reduced ingestion of 18:3n-3 fatty acid (p < 0.01), which is the precursor of EPA. Plasma EPA concentration was positively correlated to its intake, as well as to 18:3n-3 intake. Patients were also ingesting less 18:2n-6 (p < 0.05) and their plasma18:2n-6 was lower (p < 0.05), compared to the control group. Some recent publications have established plasma phospholipid fatty acids as a marker for dietary fat intake.31,32 In one study, the decreased plasma 18:2n-6 was considered the strongest predictor of a low fat intake.32 Thus, if we consider plasma phospholipid fatty acids as biochemical markers of intake, our results on the patients’ diet indicating no change between habitual and recent intake (26.0% from FFQ and 27.5% from R24) may be overestimating the patients’ recent fat intake. The concept of a biochemical marker of fat intake needs further studies. Likewise, patients’ lower plasma 18:2n-6 concentration strengthens the results of Mosconi et al.29 in malnourished cancer patients, as well as those from pancreatic17 and lung33 cancer patients. These authors found decreased 18:2n-6 fatty acids in plasma and/ or red blood cell membrane phospholipids of patients in comparison to healthy subjects. Although a study that analyzed the composition of fatty acids of lysophosphatidic acid and lysophosphatidylinositol in plasma of ovarian, endometrial and uterine cervical cancer patients detected higher unsaturated fatty acids, such as 18:2n-6, 18:1 and 20:4 fatty acids, in most advanced cases34 Qi et al.,35 who investigated the influence of dietary 18:2n-6 fatty acid on cancer development using human papillomavirus 16 transgenic mice fed on diet rich
376 in corn oil, concluded that in contrast to skin cancer, high dietary fat had no effect on the development of uterine cervical cancer. It is interesting to note that while skin cancer growth was enhanced with the diet, estrogen was the cofactor needed for cervical cancer development,35 indicating the involvement of different cellular and tissue specific factors mediating tumor cell proliferation. Therefore, the low level of plasma 18:2n-6 in patients of the present study is suggestive of a lowered intake rather than disease related metabolic alterations. There are several limitations in this study. Results from cross sectional studies can only infer about the association between results. The food intake assessment methods used in the present study are known for their intrinsic limitations. The FFQ and R24 intake methods must rely on patients’ memory with its corresponding intra- and inter-subject and observer measurement errors, therefore the differences observed need to be evaluated with caution. However, assuming that the intra-observer errors were uniform throughout the study because the same person carried out all the interviews, we were able to observe important differences between patients and healthy volunteers. In conclusion, in our study we observed weight loss in uterine cervical cancer patients even before the initiation of the treatment, associated with a reduction in calorie intake and presence of symptoms such as anorexia, nausea and vomiting. The weight loss and low nutrient intake of cervical cancer patients present at the time of disease diagnosis suggest the importance of nutrition intervention at an early stage of the disease treatment. The increased ratio of plasma 18:0/18:1 fatty acids may be an indirect marker of FA metabolism dysregulation in these patients.
Conflict of interest statement There are no financial conflicts of interest for any of the authors of this work.
Acknowledgements Contributors: A.L. and M.R. carried out the sample and data analyses. A.L. drafted the manuscript. M.M. and M.I. conceived the study, participated in the design and the coordination of the study, assisted in the statistical analyses and were responsible for the final writing of the manuscript.
References 1. Estimativa 2006, Incide ˆncia de Ca ˆncer no Brasil. Brasil. Ministe ¸˜ ao a ´rio da Sau ` Sau ´de. Secretaria de Atenc ´de, Instituto Nacional de Caˆncer. Rio de Janeiro: INCA, 2005 [accessed November 1, 2007, at, http://www.inca.gov.br/estimativa/2006/ versaofinal.pdf]. 2. Garcia-Closas R, Castellsaque X, Bosch X, Gonzalez CA. The role of diet and nutrition in cervical carcinogenesis: a review of recent evidence. Int J Cancer 2005;117:629e37. 3. Robinson LE, Clandinin T, Field CJ. R3230AC rat mammary tumor and dietary long-chain (n-3) fatty acids change immune cell composition and function during mitogen activation. J Nutr 2001;131:2021e7.
A.Q. Lisboa et al. 4. Boudreau MD, Sohn KH, Rhee SH, Lee SW, Hunt JD, Hwang DH. Suppression of tumor cell growth in nude mice and in culture by n-3 polyunsaturated fatty acidy: mediation through cycooxygenase-independent pathways. Cancer Res 2001;61:1386e91. 5. Kato T, Hancock RL, Mohammadpour H, McGregor B, Manalo P, Khaiboullina S, et al. Influence of omega-3 fatty acids on the growth of human colon carcinoma in nude mice. Cancer Lett 2002;187:169e77. 6. Davidson LA, Nguyen DV, Hokanson RM, Callaway ES, Isett RB, Turner ND, et al. Chemopreventive n-3 polyunsaturated fatty acids reprogram genetic signatures during colon cancer initiation and progression in the rat. Cancer Res 2004;64:6797e804. 7. Hardman WE. (n-3) Fatty acids and cancer therapy. J Nutr 2004;134:S3427e30. 8. Barber MD, Ross JA, Preston T, Shenkin A, Fearon KCH. Fish oil-enriched nutritional supplement attenuates progression of the acute-phase response in weight-losing patients with advanced pancreatic cancer. J Nutr 1999;129:1120e5. 9. Gogos CA, Ginopoulos P, Salsa B, Apostolidou E, Zoumbos N, Kalfarentzos F. Dietary omega-3 polyunsaturated fatty acids plus vitamin E restore immunodeficiency and prolong survival for severely III patients with generalized malignancy. A randomized control trial. Cancer 1998;82:395e402. 10. Kuhajda FP, Jenner K, Wood FD, Henningar RA, Jacobs LB, Dick JD, et al. Fatty acid synthesis: a potential selective target for antineoplastic therapy. Proc Natl Acad Sci USA 1994;91: 6379e83. 11. Rakheja D, Kapur P, Hoang MP, Roy LC, Bennett MJ. Increased ratio of saturated to unsaturated C18 fatty acids in colonic adenocarcinoma: implications for cryotherapy and lipid raft function. Med Hypotheses 2005;65:1120e3. 12. Volpe JJ, Vagelos PR. Saturated fatty acid biosynthesis and its regulation. Annu Rev Biochem 1973;42:21e60. 13. Kuhajda FP, Pizer ES, Li JN, Mani NS, Frehywot GL, Townsend CA. Synthesis and anti-tumor activity of a novel inhibitor of fatty acid synthase. Proc Natl Acad Sci USA 2000; 97:3450e4. 14. Menendez JA, Colomer R, Lupu R. Why does tumor-associated fatty acid synthase (oncogenic antigen-519) ignore dietary fatty acids? Med Hypotheses 2005;64:342e9. 15. Menendez JA, Ropero S, Mehmi I, Atlas E, Colomer R, Lupu R. Overexpression and hyperactivity of breast cancer-associated fatty acid synthase (oncogenic antigem-519) is insensitive to normal arachidonic fatty acid-induced suppression in lipogenic tissues but it is selectively inhibited by tumoricidal alpha-linolenic and gamma-linolenic fatty acids: a novel mechanism by which dietary fat can alter mammary tumorigenesis. Int J Oncol 2004;24:1369e83. 16. Chaje `s V, Hulte ´n K, Kappel ALV, Winkvist A, Kaaks R, Hallmans G, et al. Fatty-acids composition in serum phospholipids and risk of breast cancer: an incident case-control study in Sweden. Int J Cancer 1999;83:585e90. 17. Leeuwen SDZ, Heijden MSVD, Rietveld T, Berg JWOVD, Tilanus HW, Burgers JA, et al. Fatty acids composition of plasma lipids in patients with pancreatic, lung and oesophageal cancer in comparison with healthy subjects. Clin Nutr 2002;21: 225e30. 18. Percorelli S, Benedet JL, Creasman WT, Sheperd JH. FIGO staging of gynecologic cancer. 1994e1997 FIGO Committee on Gynecologic Oncology. International Federation of Gynecology and Obstetrics. Int J Gynecol Obstetr 1999;65:5e10. 19. Sichieri R, Everhart JE. Validity of a Brazilian food frequency questionnaire against dietary recalls and estimated energy intake. Nutr Res 1998;18:1649e59. 20. Folch J, Lees M, Stanley GHS. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226:497e509. 21. Christie WW. HPTLC and lipids. A practical guide. 2nd ed. 1987.
FA and nutritional status of uterine cervical neoplasm patients 22. Ito MK, Simpson KL. The Biosynthesis of u3 fatty acids from 18: 2 n-6 in Artemia ssp. Comp Biochem Physiol 1996;115B:69e76. 23. Orr JW, Wilson K, Bodiford C, Cornwell A, Soong SJ, Honea KL, et al. Nutritional status of patients with untreated cervical cancer. Am J Obstet Gynecol 1985;151:625e31. 24. Tunca JC. Nutritional evaluation of gynecologic cancer patients during initial diagnosis of their disease. Am J Obstet Gynecol 1983;147:893e6. 25. Wilson RL. Optimizing nutrition for patients with cancer. Clin J Oncol Nurs 2000;4:23e8. 26. Nitenberg G, Raynard B. Nutritional support of the cancer patient: issues and dilemmas. Crit Rev Oncol Hematol 2000; 34:137e68. 27. Lesko LM. Psychosocial issues in the diagnosis and management of cancer cachexia and anorexia. Nutrition 1989;5:114e6. 28. Rezende, M. Influe ˆncia da radioterapia sobre a capacidade fagocita ´ria e o estado psicolo ´gico em mulheres com carcinoma epidermo ´ide de colo uterino. Master’s dissertation, Faculdade de Cie ˆncias da Sau ´de, Universidade de Brası´lia; 2003. 29. Mosconi C, Agradi E, Gambetta A, Bozzetti F, Galli C. Decrease of polyunsaturated fatty acids and elevation of the oleic/stearic acid ratio in plasma and red blood cell lipids of malnourished cancer patients. J Parenter Enteral Nutr 1989;13:501e4.
377 30. Cunha J, Costa THM, Ito MK. Influences of maternal dietary intake and suckling on breast milk lipid and fatty acid composition in low-income women from Brasilia, Brazil. Early Hum Dev 2005;81:303e11. 31. Raatz SK, Bibus D, Thomas W, Kris-Etherton P. Total Fat intake modifies plasma fatty acid composition in humans. J Nutr 2001; 131:231e4. 32. King IB, Lemaitre RN, Kestin M. Effect of a low-fat diet on fatty acid composition in red cells, plasma phospholipids and cholesterol esters: investigation of a biomarker of total fat intake. Am J Clin Nutr 2006;83:227e36. 33. Prisco D, Paniccia R, Coppo M, Filippini M, Francalanci I, Brunelli T, et al. Platelet activation and platelet lipid composition in pulmonary cancer. Prostaglandins Leukot Essent Fatty Acids 1995;53:65e8. 34. Shen Z, Wu M, Elson P, Kennedy AW, Belinson J, Casey G, et al. Fatty acid composition of lysophosphatidic acid and lysophosphatidynositol in plasma from patients with ovarian cancer and other gynecological diseases. Gynecol Oncol 2001;83: 25e30. 35. Qi M, Chen DZ, Liu K, Auborn KJ. N-6 polyunsaturated fatty acids increase skin but not cervical cancer in human papillomavirus 16 transgenic mice. Cancer Res 2002;62:433e6.