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Rapid EPA and DHA incorporation and reduced PGE2 levels after one week intervention with a medical food in cancer patients receiving radiotherapy, a randomized trial

Clinical Nutrition, 3, 32, pages 338 - 345

Summary

Background & aims

In cancer patients, metabolic alterations, reduced immune competence and anti-cancer treatment can increase the risk of infections. A rapid-acting nutritional intervention might reduce this risk and support overall treatment. The present study investigated whether one week of intervention with a specific medical food led to fatty acid incorporation and functional immunological changes.

Methods

In a randomized, double-blind study, 38 cancer patients receiving radiotherapy consumed daily for one week 400 ml of specific medical food, which is high in protein and leucine, and enriched with fish oil and specific oligosaccharides (Active group), or iso-caloric/iso-nitrogenous product (Control group). Blood samples were taken at day 0 (baseline) and day 7.

Results

After one week of intervention, the incorporation of EPA and DHA in white blood cells was significantly higher in the Active group (2.6% and 2.6% of total fatty acids) compared to the Control group (1.0% and 2.2% of total fatty acids) (p < 0.001 and p < 0.05). Serum PGE2 levels decreased in the Active group and increased in the Control group (p < 0.01). No differences were observed on cytokine production in LPS-stimulated whole blood cultures.

Conclusions

In cancer patients receiving radiotherapy, nutritional intervention with a specific medical food rapidly increased the percentage EPA and DHA in white blood cell phospholipids and reduced serum levels of the inflammatory mediator PGE2 within one week.

Clinical registration number: NTR2121 .

Keywords: Cancer, Nutrition, EPA, Immunity, PGE2, Radiotherapy.

Abbreviations: COX - cyclooxygenase, CRP - C-reactive protein, DHA - docosahexaenoic acid, ECOG - Eastern Cooperative Oncology Group, EPA - eicosapentaenoic acid, FCShi - heat-inactivated fetal calf serum, IFN - interferon, IL - interleukin, LPS - lipopolysaccharide, MDSC - myeloid derived suppressor cells, PGE2 - prostaglandin E2, PUFA - poly-unsaturated fatty acids, RBC - red blood cells, TNF - tumor necrosis factor, WBC - white blood cells.

1. Introduction

In many cancer patients, metabolic alterations lead to a chronic condition of catabolism, including involuntary weight loss, wasting, anorexia, asthenia and fatigue, resulting in a poor performance status and reduced quality of life. 1 Other important features include the presence of a chronic inflammatory state and, paradoxically, a state of impaired immune responsiveness.2 and 3 Several mediators that are either tumor- or host-derived (e.g. pro-inflammatory cytokines, chemokines and prostaglandins) induce a cascade of events leading to a suppressed immune function, thereby reducing the acute response to infectious triggers and facilitating the escape of tumor cells from immune surveillance.4, 5, and 6 The risk of immune deficiency is even higher after anti-cancer treatment. Surgery, radiotherapy and chemotherapy are associated with suppression of the cellular immune system and lead, in combination with malnutrition, to a reduced treatment efficacy and a higher frequency and severity of infectious and other complications.3, 7, 8, 9, and 10 In addition, anti-cancer treatment can lead to damage of the gastrointestinal (GI) mucosa, a diminished barrier function and a change in the intestinal microbiota contributing to local and systemic inflammation. 11

To reduce the risk of (infectious) complications and to support the performance status (daily living abilities) of cancer patients, a multi-targeted approach should be applied of which nutritional support is an integral part. Every effort should be made to prevent involuntary weight loss and delayed treatment schedules. In malnourished patients, pre-operative nutritional support is associated with a 50% reduction of post-operative complications. 9 In patients receiving radiotherapy, nutritional supplementation resulted in reduced weight loss and fewer treatment interruptions due to a reduction in acute mucositis and/or maintenance of performance status (daily living abilities).9 and 12 Nutritional interventions have been recommended as an integral part of anti-cancer therapy to improve clinical outcomes and quality of life.3, 9, 13, and 14 Cancer patients often start with an anti-cancer treatment soon after diagnosis and it is therefore of obvious clinical relevance to provide the optimal nutritional support as early as possible.

Immune modulatory effects of long-chain PUFA are well described. Both (n-6) and (n-3) PUFA play a major role in immune regulation and the balance between them may affect the development and severity of inflammatory diseases. 15 However, to modify cell function and obtain beneficial immune modulatory effects, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) must be effectively incorporated into cell membrane phospholipids. In the literature, the majority of studies providing EPA and DHA examine fatty acid incorporation and immune modulatory activities after 4, 8 or 12 weeks of supplementation.16 and 17 In a previous study in healthy volunteers, however, nutritional intervention with a recently developed specific medical food* significantly increased the percentage of EPA into white blood cell (WBC) phospholipids within one week. Additionally, ex vivo immune responsiveness to LPS was increased significantly. 18 The aim of the present study was, to investigate the rapid-acting effects of the medical food on the fatty acid incorporation into WBC phospholipids and the subsequent changes on parameters of immune function in cancer patients receiving radiotherapy. This medical food is high in protein and leucine and is enriched with emulsified fish oil (containing EPA and DHA) and a specific oligosaccharide mixture, to reduce the inflammatory state and support immune function in cancer patients, aiming to reduce complications and to provide optimal treatment support.

*A medical food is in the USA defined in 21 U.S.C. § 360ee(b) 3 as “a food which is formulated to be consumed or administered enterally under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognizable scientific principles, are established by medical evaluation”. A comparable definition exists in the harmonized legislation of the European Union (cf. Article 1, 2(b) of Commission Directive 1999/21/EC of 25 March 1999 on dietary foods for special medical purposes).

2. Materials & methods

A randomized, controlled, double-blind study was conducted in order to investigate the incorporation of EPA and DHA into WBC phospholipids and to determine the effects on functional immune parameters after one week of nutritional intervention with a medical food in cancer patients receiving radiotherapy, compared with the effects of an iso-caloric and iso-nitrogenous control product. Secondary study objectives were to investigate the incorporation of EPA and DHA into red blood cell (RBC) phospholipids and plasma and to assess the effects of the medical food on inflammatory status. Moreover, nutritional status, safety and compliance were determined in these patients as well.

2.1. Subjects

In the period between October 2009 and January 2010, 39 patients with histologically confirmed solid tumor(s), receiving radiotherapy, were recruited from the Department of Radiology, Division of Radiooncology, Freiburg University Hospital, Freiburg, Germany. Patients had an age of 18 years and above, a pathologically confirmed diagnosis of a solid tumor, a body mass index (BMI) between 18.5 and 30 kg/m2, were willing and able to abstain from alcohol use, smoking, fish or fish oil-, vitamin-, herbal- or other oil-containing supplements and were included in the study after written informed consent was obtained. Exclusion criteria were a life expectancy less than 3 months, surgery, chemotherapy and/or hormone therapy in the previous 6 weeks, radiotherapy (before the current treatment cycles) in the previous 6 weeks, an Eastern Cooperative Oncology Group (ECOG) performance status of higher than 2, an altered immune function (e.g. caused by a major active infection, autoimmune disease, active allergy, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, or by use of medication such as immunosuppressive drugs, immunomodulators or systemic corticosteroids), a dependency on tube feed or parenteral nutrition during the previous 4 weeks, use of fish oil-, herbal-, or other oil-containing supplements during the previous 4 weeks, currently smoking or having smoked in the past 6 months, intolerance of or allergy to dairy products, fish or other ingredients of the study products, pregnancy or lactation, dementia or an altered mental status that would prohibit the understanding and giving of informed consent, any other medical condition that might interfere with the safety of the patient or the outcome parameters or uncertainty about the willingness or ability of the patient to comply with the protocol requirements, according to the investigator's judgment. Patients were advised to use paracetamol if analgesics were required during the study period and not to use NSAIDs (including aspirin) in the 48 h before their visits.

2.2. Study design

The study was conducted in compliance with the principles of the ‘Declaration of Helsinki’ (59th WMA General Assembly, Seoul, October 2008) according to the ICH-GCP guidelines and was approved by the Ethics Committee of the Albert Ludwigs University, Freiburg, Germany. After an initial pre-screening of patients undergoing out-patient radiotherapy by reviewing their electronic records, the remaining group was interviewed and patients fulfilling the selection criteria were screened and asked to participate in the study by the study coordinator. Subject characteristics, relevant medical history and anthropometrics were determined at visit 1 (baseline). Patients were randomized to the Active group receiving the medical food or to the Control group receiving an iso-caloric Control product using a computerized randomization program with a block size of 4. All subjects were asked to consume the study products for seven successive days in addition to their normal food, starting with one dose in the afternoon of day 0, 2 doses on days 2–6 and one dose in the morning of day 7.

The morning of day 0, before visit 1, all patients consumed one unit of iso-caloric Control product to minimize differences at baseline. At visit 1, before the first dose of the Active medical food or Control product and at visit 2 (day 7), after the morning dose of the Active medical food or Control product, body weight was measured and blood was drawn for the measurement of several immune, nutritional and safety parameters. The amount of study product taken was recorded daily in a diary by the patient. Additionally, compliance, the use of concomitant medication or nutritional supplements, the occurrence of adverse events and diary completion were monitored at day 7. Patients with an intake of <85% (less than 12 doses) of the minimum amount of 2 × 200 ml Active or Control product per day, or an intake of <2 of the 3 products on days 6 and 7, were considered as noncompliant and therefore as a protocol violation.

2.3. Nutritional intervention

The prescribed product intake during the study was 2 doses (2 × 200 ml sip feed) of either the Active medical food or the Control product daily. The Active medical food is an energy dense (163 kcal/100 ml), nutritionally complete oral supplement that is high in protein and leucine and is enriched with emulsified fish oil (providing 2.4 g EPA and 1.2 g DHA daily), specific oligosaccharides and a balanced mix of vitamins, minerals and trace elements ( Table 1 , Nutricia N.V., Zoetermeer, The Netherlands). The Control product is an energy dense (160 kcal/100 ml), iso-caloric and iso-nitrogenous, commercially available, standard nutritional product ( Table 1 , Fortisip Extra, Nutricia N.V., Zoetermeer, the Netherlands). All products were provided in white cartons (in two different flavors) with letters A–D to ensure blinding.

Table 1 Nutritional composition of the Active medical food and Iso-caloric control product (Fortisip Extra) in grams per 100 ml.

Ingredients Active medical food Iso-caloric control product
Macronutrients
 Energy (kJ/kcal) 683/163 675/160
 Carbohydrates (g) 17.4 18.1
 Protein (g) 9.9 10.0
 Whey (g) 3.2 2.0
 Casein (g) 5.6 8.0
 Added amino acids: free leucine (g) 1.1 0
 Total fat (g) 5.3 5.3
 EPA (g) 0.6 0
 DHA (g) 0.3 0
 Oligosaccharides (g) 1.4 0
 GOS (g) 1.2 0
 FOS (g) 0.2 0
Minerals & trace elements
 Sodium (mg) 110 50.0
 Potassium (mg) 215 220
 Chloride (mg) 140 81.0
 Calcium (mg) 147 280
 Phosphorus (mg) 115 197
 Magnesium (mg) 28.2 40.0
 Iron (mg) 1.9 2.2
 Zinc (mg) 2.1 2.4
 Copper (μg) 288 338
 Manganese (mg) 0.7 0.6
 Fluoride (mg) 0.2 0.2
 Molybdenum (μg) 16.0 19.0
 Selenium (μg) 13.5 19.0
 Chromium (μg) 11.0 13.0
 Iodine (μg) 21.0 35.0
Vitamins
 Vitamin A (μg-RE) 130 188
 Vitamin D3 (μg) 1.1 2.5
 Vitamin E (mg-α-TE) 3.2 2.3
 Vitamin K (μg) 8.5 10.0
 Thiamin (B1) (mg) 0.2 0.3
 Riboflavin (B2) (mg) 0.3 0.3
 Niacin (B3) (mg-NE) 2.9 4.0
 Pantothenic acid (B5) (mg) 0.9 1.0
 Vitamin B6 (mg) 0.6 0.4
 Folic acid (μg) 53.0 50.0
 Vitamin B12 (μg) 0.6 0.7
 Biotin (μg) 6.4 7.5
 Vitamin C (mg) 21.0 19.0
 Carotenoids (mg) 0.3 0
Other
l-Carnitine (mg) 10.9 0
 Choline (mg) 59.0 69.0
 Taurine (mg) 13.3 0

Values represent the amount of ingredients of the medical food in grams per 100 ml. Bold values indicate total amounts.

Abbreviations: EPA, eicosapentaenoic acid; DHA, docosahexaenoid acid; GOS, galactooligosaccharides; FOS, fructooligosaccharides.

2.4. Study outcome

The primary outcome parameters of the study were the percentages of EPA and DHA of total phospholipid fatty acids of WBC and the ex vivo LPS-stimulated cytokine and prostaglandin E2 (PGE2) production in whole blood after one week of intervention, as markers for immune function. Secondary parameters were the percentages of other n-3 and n-6 poly-unsaturated fatty acids (PUFA) of total phospholipid fatty acids of WBC and RBC and plasma and serum levels of pro-inflammatory cytokines, C-reactive protein (CRP) and PGE2 as markers for the inflammatory state of the patients.

WBC and RBC were isolated from heparinized blood using a density gradient and stored at −80 °C until use. In short, 30 ml heparin blood was diluted with 10 ml PBS (Gibco BRL, Life Technologies, Merelbeke, Belgium) + 2% heat-inactivated fetal calf serum (FCShi) (Hyclone, Perbio Science, Etten-Leur, The Netherlands) and divided over two Leucosep tubes pre-filled with Ficoll-Isopaque (Greiner Bio-One B.V., Alphen aan den Rijn, the Netherlands). Tubes were centrifuged for 10 min at 1000 g (RT, no brake), WBC were collected and washed with PBS + 2% FCShi. WBC were resuspended in cold culture medium (RPMI-1640 containing 25 mM HEPES and 2 mM l-glutamine; Life-Technologies, enriched with 100 kU/l penicillin/streptomycin) with 10% FCShi, counted and centrifuged for 10 min at 17,000 g (RT). WBC pellets were stored at −80 °C until analysis. RBC were collected after gradient centrifugation and stored at −80 °C until analysis. Plasma was obtained by centrifugation of 5 ml heparinized blood for 5 min at 1300 g (RT) and stored at −80 °C until analysis. Phospholipid fatty acids of WBC, RBC and plasma were analyzed by gas chromatography as described before. 19

The whole blood assay was performed by adding 100 μl/well blood to 50 μl/well culture medium in a 96-well plate (flat-bottom, polystyrene, BD Falcon Erembodegem Aalst, Belgium). Blood was subsequently incubated with 50 μl/well LPS (final concentration 100 μg/L, Escherichia coli, B55:O55, Sigma–Aldrich Chemie, Steinheim, Germany) or culture medium (control) for 20 h at 37 °C in a humidified environment containing 5% CO2. Afterward, plates were centrifuged for 5 min at 250 g (RT) and supernatants were harvested and stored at −80 °C until analysis. Serum was obtained from blood collected in serum tubes (clotting tubes), which were incubated for 2 h at RT. Afterward, blood was centrifuged for 10 min at 1300 g (RT) and serum was stored at −80 °C until analysis. Cytokine production (interleukin (IL)-8, IL-1β, IL-6, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10) was measured using a Bio-Plex Cytokine bead immunoassay (Bio-Rad, Veenendaal, The Netherlands) according to the manufacturer's protocol. PGE2 was measured using a commercial enzyme immunoassay (Biotrak Amersham, Buckinghamshire, UK) according to the manufacturer's protocol.

In addition, the following parameters were determined: white and red blood cell count and differential, hemoglobin, pre-albumin, albumin, and calcium and also safety parameters for liver function (ALAT and ASAT), kidney function (creatinine) and partial thromboplastin time were measured at the Tumor Biology Center, Freiburg, Germany.

2.5. Statistical analysis

The primary parameters have not been reported in cancer patients receiving radiotherapy before. Therefore, the expected difference in the percentage of EPA in plasma phospholipids between the Active and Control group and its variance was estimated based on three studies in healthy volunteers.18, 20, and 21 Group size was based on the expected difference of the LPS-stimulated cytokine production in whole blood cultures between the Active and Control group. Its variance was estimated based on one study in healthy volunteers. 18 With a significance level (α) of 0.05, a standard deviation of 24% and a power of 80%, a sample size of 13 per group would allow for a statistically significant difference after one week of nutritional intervention. Taking into account a drop-out rate of 20% and the possible effects of the Control sip feed, a sample size of 18 for each of the two groups would be sufficient to detect a statistically significant result between the groups.

All subjects who received the study products were included in the intention-to-treat (ITT) analysis. For baseline comparisons and efficacy analysis, the differences between the Active and Control group were determined. The LPS-stimulated data were corrected for the non-stimulated measurements by subtraction of the latter (resulting negative values were cut off at zero). ANOVA with treatment as covariate was used to analyze the measurement of the study parameters. When the data were not normally distributed, the Mann Whitney U test was used. For the nominal variables a Fisher's exact test was used and for the ordinal variables the difference between the Active and Control group were compared with the Mann Whitney U test. All adverse events were assessed and for affected patients medical history and medication use were checked. The statistical analyses were performed using SAS version 9.1.3.

3. Results

3.1. Study population and compliance

In total 534 subjects, scheduled to undergo out-patient radiotherapy, were pre-screened in the study by reviewing their electronic records. Afterward, 349 patients were interviewed and of the 45 subjects that were screened in the study, 39 subjects were randomized and 38 subjects received the study products ( Fig. 1 ), since 1 subject withdrew shortly after signing the informed consent form. Patients failed to be included in the study due to disinterest (n = 108), appointment time for radiotherapy unsuitable to process blood samples (n = 81), smoking (n = 57), cancer diagnosis within last 3 months (n = 54), NSAID use (n = 46), concomitant chemotherapy (n = 29), obesity (n = 21), surgery within past 6 weeks (n = 20), no solid tumor (n = 20), inability to swallow (n = 19), use of oral nutritional supplements (n = 17), language difficulties (n = 16), radiotherapy completed within less than 5 days (n = 16), and other causes (n = 37).

gr1

Fig. 1 Trial profile; screening, randomization and study completion. *Included in ITT analysis.

Of the 38 subjects that received the study products, 20 were allocated to the Active product and 18 to the Control product. These subjects were all included in the ITT analysis. A total of 2 subjects (1 subject in the Active group and 1 subject in the Control group) terminated the study early due to the development of nausea, resulting in no follow-up data. Of the remaining 36 subjects, 3 subjects violated the protocol (2 subjects in the Active group and 1 subject in the Control group) by non-compliance or the use of corticosteroids. The average compliance with consumption of the products was similar for both groups (92% for the Active product and 93% for the Control product).

3.2. Baseline characteristics

The baseline characteristics of the patients are presented in Table 2 . At baseline, the mean age of the total patient group was 62.7 ± 11.0 years with a body weight of 70.8 ± 12.6 kg. The Active and Control group matched well, with the only baseline difference being that patients in the Active group had lost weight in the past three months (−1.8% ± 4.8%) while patients in the Control group had gained in weight (1.5% ± 6.3%, p < 0.01). In 50% of the patients the tumor was located in the breast and in 26% of the patients the tumor was located in the prostate. Other locations of the tumor included the gynecologic area (5.3%), urinary tract (5.3%), lungs (2.6%), head and neck (2.6%), esophagus (2.6%) and others (5.3%). TNM stage ranged from I–IV, and was equally distributed among the study groups. Before radiotherapy, most patients had already received one or more previous treatments, including surgery (n = 29), radiotherapy (n = 1), chemotherapy (n = 12) and hormone therapy (n = 11), but no differences between the Active and the Control group were detected. The planned radiotherapy schedule of the patients was 6.3 ± 1.4 weeks with an average planned dose of 53.4 ± 16.2 Gy divided over a total of 28.7 ± 9.8 fractions, which was not different between the groups.

Table 2 Baseline characteristics of the study groups.

Variable Presented as Active (n = 20) Control (n = 18) Total (n = 38)
Sex n (%)      
Female   6 (30%) 8 (44%) 14 (37%)
Male   14 (70%) 10 (56%) 24 (63%)
Age (years) mean ± SD 61.4 ± 13.1 64.3 ± 8.1 62.7 ± 11.0
BMI (kg/m 2 ) mean ± SD 25.1 ± 3.3 24.6 ± 3.7 24.8 ± 3.5
Bodyweight (kg) mean ± SD 70.8 ± 13.2 70.8 ± 12.3 70.8 ± 12.6
Body weight change in past 3 months (%) mean ± SD −1.8 ± 4.8 a 1.5 ± 6.3 −0.2 ± 5.7
Time since diagnosis (months) mean ± SD 8.0 ± 11.5 15.6 ± 40.9 11.6 ± 29.2
Tumor location (primary) n (%)      
Lung   0 (0%) 1 (5.6%) 1 (2.6%)
Head and neck   1 (5.0%) 0 (0%) 1 (2.6%)
Gynecologic   1 (5.0%) 1 (5.6%) 2 (5.3%)
Breast   11 (55%) 8 (44%) 19 (50%)
Prostate   5 (25%) 5 (28%) 10 (26%)
Urinary tract   1 (5.0%) 1 (5.6%) 2 (5.3%)
Esophagus   0 (0%) 1 (5.6%) 1 (2.6%)
Other   1 (5.0%) 1 (5.6%) 2 (5.3%)
Staging (TNM stage) n (%)      
I   8 (40%) 4 (22%) 12 (32%)
II   3 (15%) 5 (28%) 8 (21%)
III   4 (20%) 3 (17%) 7 (18%)
IV   4 (20%) 5 (28%) 9 (24%)
Other   1 (5.0%) 1 (5.6%) 2 (5.3%)
Previous treatment n (%)      
Surgery   17 (85%) 12 (67%) 29 (76%)
Radiotherapy   0 (0%) 1 (5.6%) 1 (2.6%)
Chemotherapy   5 (25%) 7 (39%) 12 (32%)
Hormone therapy   7 (35%) 4 (22%) 11 (29%)
No treatment   1 (5.0%) 3 (17%) 4 (11%)
Curative   19 (100%) 12 (80%) 31 (91%)
Palliative   0 (0%) 3 (20.0%) 3 (8.8%)
Radiotherapy schedule mean ± SD      
Number of weeks   6.4 ± 1.0 6.3 ± 1.7 6.3 ± 1.4
Planned dose (Gy)   54.0 ± 16.9 52.7 ± 15.9 53.4 ± 16.2
Number of fractions   31.0 ± 9.0 26.1 ± 10.3 28.7 ± 9.8
Dose per fraction (Gy)   1.9 ± 0.1 2.1 ± 0.4 2.0 ± 0.3
Curative n (%) 20 (100%) b 14 (78%) 34 (89%)
Palliative   0 (0%) 4 (22%) 4 (11%)

a Significantly different from the Control group, p < 0.01 (Mann Whitney).

b The distribution of patients over Curative and Palliative treatment is significantly different from the Control group, p < 0.05 (Fisher's exact).

Data represent the baseline characteristics as the number of subjects (n) and percentages or means ± SD of the Active group (n = 20), the Control group (n = 18) and the total patient group (n = 38).

Abbreviations: BMI, body mass index; Gy, gray (unit of radiotherapy); TNM, tumor, node, metastasis.

3.3. Efficacy

After one week of intervention, the incorporation of EPA, DPA and DHA into WBC phospholipids was significantly higher in the Active group compared to the Control group (p < 0.001, p < 0.01 and p < 0.05 for EPA, DPA and DHA, respectively, Table 3 ). By contrast, the incorporation of arachidonic acid (AA) was significantly decreased in the Active group compared to an increase in the Control group (p < 0.001). The percentage of total n-3 PUFA showed a significantly higher increase in the Active group compared to the Control group, whereas the percentage of total n-6 PUFA in WBC phospholipids was decreased in the Active group compared to the Control group ( Table 3 , p < 0.001), as was also observed for the ratio n-6/n-3 PUFA (p < 0.001). In RBC phospholipids, the EPA and DHA incorporation were increased in the Active group compared to a decrease in the Control group after the intervention (p < 0.001 and p < 0.05 for EPA and DHA, respectively, Table 3 ) but no effect of the intervention on DPA and AA was observed. In plasma, the percentages of EPA, DPA and DHA were significantly increased in the Active group compared to the Control group (p < 0.001), but no effect on AA was observed.

Table 3 Incorporation of EPA, DPA, DHA, AA, total n-3 fatty acids, total n-6 fatty acids and ratio total n-6/n-3 fatty acids of total phospholipid fatty acids in WBC, RBC and in plasma of patients in the Active group (n = 20) and the Control group (n = 18) presented as percentages at day 0 and day 7 or as the Δd7-d0.

  Group EPA DPA DHA AA Total n-3 Total n-6 n-6/n-3
WBC
Day 0 Active 0.4 ± 0.3 2.3 ± 0.4 2.2 ± 0.5 20.4 ± 1.0 5.2 ± 1.0 30.8 ± 1.9 6.1 ± 1.1
Control 0.6 ± 0.4 2.3 ± 0.6 2.2 ± 0.7 19.3 ± 2.0 5.3 ± 1.1 29.4 ± 2.4 5.8 ± 1.0
Day 7 Active 2.6 ± 1.0 a 3.1 ± 0.7 b 2.6 ± 0.5 c 18.0 ± 1.9 b 8.4 ± 1.7 a 27.1 ± 2.2 a 3.4 ± 0.9 a
Control 1.0 ± 0.5 2.4 ± 0.4 2.2 ± 0.6 19.8 ± 1.9 5.7 ± 1.0 30.3 ± 1.7 5.4 ± 0.9
Δd7-d0 Active 2.1 ± 1.0 a 0.7 ± 0.6 b 0.4 ± 0.4 c −2.5 ± 2.2 a 3.1 ± 1.5 a −3.8 ± 2.5 a −2.7 ± 1.0 a
Control 0.5 ± 0.7 0.0 ± 0.5 0.0 ± 0.5 0.3 ± 1.6 0.5 ± 1.2 0.8 ± 2.5 −0.4 ± 1.2
RBC
Day 0 Active 1.0 ± 0.3 2.3 ± 0.3 3.8 ± 0.7 11.8 ± 0.8 7.3 ± 1.1 28.9 ± 1.5 d 4.1 ± 0.7
Control 1.1 ± 0.4 2.3 ± 0.4 3.7 ± 1.1 11.4 ± 1.0 7.3 ± 1.5 27.8 ± 1.4 4.0 ± 1.0
Day 7 Active 2.3 ± 0.5 a 2.3 ± 0.5 3.8 ± 1.0 11.0 ± 1.1 8.6 ± 1.7 e 26.6 ± 1.8 3.2 ± 0.6 e
Control 0.9 ± 0.4 2.2 ± 0.6 3.5 ± 1.2 10.8 ± 2.5 6.7 ± 2.0 26.9 ± 5.5 4.4 ± 1.3
Δd7-d0 Active 1.3 ± 0.3 a 0.0 ± 0.4 0.1 ± 0.6 d −0.8 ± 1.0 1.3 ± 1.2 e −2.4 ± 1.9 e −0.9 ± 0.4 e
Control −0.2 ± 0.2 −0.1 ± 0.5 −0.2 ± 0.6 −0.6 ± 2.5 −0.6 ± 1.3 −0.9 ± 5.7 0.4 ± 0.7
Plasma
Day 0 Active 1.1 ± 0.4 1.0 ± 0.2 3.3 ± 0.9 9.7 ± 1.0 5.6 ± 1.2 33.7 ± 2.2 6.3 ± 1.5
Control 1.0 ± 0.4 1.0 ± 0.2 3.1 ± 0.9 9.6 ± 1.6 5.5 ± 1.2 32.7 ± 1.5 6.3 ± 1.8
Day 7 Active 5.6 ± 0.9 a 1.7 ± 0.3 a 4.9 ± 0.7 a 9.2 ± 1.2 12.5 ± 1.4 a 28.0 ± 1.6 a 2.3 ± 0.3 a
Control 1.0 ± 0.4 1.0 ± 0.2 3.2 ± 1.0 9.4 ± 1.7 5.5 ± 1.3 33.8 ± 2.3 6.6 ± 2.2
Δd7-d0 Active 4.6 ± 0.8 a 0.7 ± 0.2 a 1.7 ± 0.5 a −0.4 ± 0.8 6.9 ± 1.1 a −5.9 ± 2.0 a −4.1 ± 1.4 a
Control 0.0 ± 0.4 −0.0 ± 0.1 0.0 ± 0.7 −0.3 ± 0.9 0.0 ± 1.0 1.0 ± 2.0 0.3 ± 1.1

a Significantly different from Control p < 0.001 (ANOVA).

b Significantly different from Control p < 0.01 (ANOVA).

c Significantly different from Control p < 0.05 (ANOVA).

d Significantly different from Control p < 0.05 (Mann–Whitney).

e Significantly different from Control p < 0.001 (Mann–Whitney).

Data represent means (%) ± SD.

Abbreviations: EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid; AA, arachidonic acid.

The production of pro-inflammatory cytokines (IL-8, IL-1β, IL-6, TNF-α, IFN-γ and IL-10) and PGE2 was measured in LPS-stimulated whole blood ( Table 4 ). A good stimulation response to LPS was observed for all the mediators at the different time points. However, levels of IL-6 were above the detection limit of the assay and therefore not measurable. After one week of nutritional intervention no significant differences between the Active group and Control group were observed for any of the pro-inflammatory mediators.

Table 4 Production of cytokines and PGE2 in LPS-stimulated whole blood of patients in the Active group (n = 19) and the Control group (n = 17) at day 0 and day 7 and calculated as the Δd7-d0.

  Group IL-8 IL-1β TNF-α IFN-γ IL-10 PGE2
Day 0 Active 2121 (1378–2578) 1256 (943–2120) 3166 (2479–5940) 822 (636–965) 531 (334–700) 1422 (815–4915)
Control 2271 (1692–2964) 1277 (821–1759) 3550 (2637–4909) 816 (689–1049) 395 (303–630) 761 (388–1186)
Day 7 Active 1640 (1179–2341) 1408 (957–1906) 3996 (2644–5084) 679 (533–831) 638 (435–689) 1107 (608–3482)
Control 2324 (1800–3065) 1263 (672–1784) 3616 (1850–6100) 696 (335–1187) 545 (289–655) 742 (395–1575)
Δd7-d0 Active −196 (−719–264) 83.8 (−162–292) 339 (−749–921) −45.5 (−284–48.2) 75.3 (−43.0–164) −206 (−2000–636)
Control 68.5 (−505–487) 88.2 (−277–354) −282 (−892–380) 0.2 (−191–213) 35.1 (−54.0–214) −43.6 (−350–606)

Data represent medians (pg/ml) and interquartile ranges (25th–75th percentiles).

Abbreviations: IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; PGE2, prostaglandin E2.

Serum concentrations of pro-inflammatory cytokines were relatively low, with most levels below or just above the detection limit of the assay. Serum levels of only IL-6 and IL-8 could be measured properly, with average levels of 0.99 pg/ml and 4.23 pg/ml in the Active group and 0.75 pg/ml and 3.47 pg/ml in the Control group at day 0 and levels of 0.90 pg/ml and 4.33 pg/ml in the Active group and 1.01 pg/ml and 4.77 pg/ml in the Control group at day 7. No significant differences were observed between the Active and Control group. By contrast, average serum PGE2 levels were decreased from 627 pg/ml at day 0–463 pg/ml at day 7 in the Active group and increased from 433 pg/ml at day 0–736 pg/ml at day 7 in the Control group. The change (delta d7-d0) was significantly different in the Active group (−78 pg/ml) and the Control group (202 pg/ml, p < 0.01, Fig. 2 ). By contrast, serum CRP levels were relatively low and no differences were observed between the Active and Control group.

gr2

Fig. 2 Change in serum PGE2 levels (pg/ml) of patients in the Active group (n = 19) and Control group (n = 16). Data are presented as the delta of d7-d0 in means ± SEM. **Significant difference between the Active and the Control group, p < 0.01 (ANOVA). Abbreviations: PGE2, prostaglandin E2.

No differences were observed between the Active group and Control group in total and differential blood cell count (leukocytes, neutrophils, monocytes, lymphocytes, basophils and eosinophils) at baseline and after one week (data not shown).

As already mentioned, the body weight change in the previous 3 months was significantly different between the Active and Control group, however body weight at baseline was not different between the groups. After one week of nutritional intervention, body weight increased from 73.4 kg at day 0 to 73.9 kg at day 7 in the Active group and decreased from 72.8 kg at day 0 to 71.7 kg at day 7 in the Control group, but there was no significant difference between groups. Furthermore, no significant differences between the Active and Control group were observed in serum concentrations of pre-albumin, albumin and total calcium.

3.4. Safety and tolerability

A total of 32 adverse events (AEs) were reported, 19 in the Active group (in 13 patients) and 13 in the Control group (in 10 patients). No serious adverse events were reported. The number of patients with at least one AE was not different between the Active and Control group. Most adverse events were gastrointestinal with flatulence, constipation and eructation most frequently observed in the Active group and diarrhea, flatulence, nausea and gastroesophageal reflux most frequently observed in the Control group. Blood safety parameters all remained within the respective reference ranges and no clinically relevant changes in liver and kidney function or in partial thromboplastin time were observed (data not shown).

4. Discussion

In the literature, incorporation of EPA into WBC is generally described after a 4-, 8-, or even 12-week intervention period, reaching levels of 2.5% of total lipids after 4 weeks of supplementation with fish oil providing 2.1 g EPA and 1.1 g DHA per day. 22 Interestingly, in a previous study in healthy volunteers, incorporation of EPA into WBC phospholipids was demonstrated within one week of nutritional intervention with the medical food. 18 Additionally, immune modulatory effects were observed by the increased production of pro-inflammatory cytokines in LPS-stimulated whole blood cultures within one week of nutritional intervention. Comparably, in this double-blind, randomized, controlled study in cancer patients receiving radiotherapy, a rapid incorporation of EPA and DHA into phospholipids of WBC, RBC and plasma was observed after one week of intervention with the fish oil-enriched medical food compared to an iso-caloric and iso-nitrogenous Control product. Moreover, serum levels of the inflammatory mediator PGE2 were significantly decreased in the Active medical food group compared to an increase in the Control group, whereas no effects were observed on the production of pro-inflammatory cytokines in LPS-stimulated whole blood cultures.

Cancer patients often suffer from a severe systemic inflammatory state that accounts for the production of chemokines, pro-inflammatory cytokines, prostaglandins and reactive oxygen/nitrogen species, inducing a cascade of events leading to a suppressed immune function.3, 6, and 23 Prostaglandin E2 (PGE2) is a major inflammatory and immune suppressive mediator and is described to play a role in various human malignancies, including colon, lung, breast and head and neck cancer, and is often associated with a poor prognosis. 4 It is produced by different types of cancer cells and their surrounding cells during the course of inflammation in response to growth factors, hormones and inflammatory cytokines.4 and 24 PGE2 can act by regulating cell proliferation, apoptosis, migration and invasion of tumor cells, by secretion of growth factors, pro-inflammatory mediators and angiogenic factors or it can act as a chemotactic factor for myeloid derived suppressor cells (MDSCs), which have been demonstrated to inhibit immune surveillance and to be potent suppressors of anti-tumor immunity.4, 5, and 24

In cancer patients receiving anti-cancer treatment, inflammatory and immune suppressive effects were even more pronounced. Surgery, radiotherapy and chemotherapy are associated with suppression of the cellular immune system and lead, in combination with malnutrition, to a reduced treatment efficacy and a higher frequency and severity of (infectious) complications.3, 7, 8, and 9 Moreover, in patients receiving radiotherapy, the radiation exposure is associated with an increase in eicosanoid production, in which PGE2 is predominantly detected. This is confirmed by a positive Pearson correlation between serum PGE2 and AA in white blood cells of patients in the Control group (ρ = 0.54 with p = 0.03). Moreover, inhibition of these prostaglandins can potentiate the anti-tumor effects of radiotherapy, with a main role for COX-2 being described. 25 On that account a reduction in PGE2 levels, as observed in the present study, might be beneficial for patients receiving radiotherapy, to reduce the inflammatory state, improve immune responsiveness and reduce infections, and possibly potentiate the anti-tumor effects of the radiation. Each of the product features (high protein, leucine, fish oil and specific oligosaccharides) may have played a role in this process, but based on preclinical studies, overlapping biological activities and synergistic interactions between these ingredients are hypothesized to lead to the overall effect.19, 26, and 27

In contrast to serum levels of PGE2, levels of other inflammatory markers were very low and around the detection limit of the assay, whilst before the start of the study a more severe inflammatory state of these patients was expected. 23 It appears that the condition of the patients in this study is better than expected, which might also be expressed by a good immune responsiveness. A possible explanation for the low inflammatory state of the patients might be the fact that in total 76% of the patients had had a previous operation, which might indicate that the tumor is dissected and less inflammatory cytokines become systemically available. Moreover, the low levels of inflammatory mediators suggest the absence of a true catabolic and cachectic state of the patients, which is confirmed by the low amount of weight loss in the Active group (−1.8 ± 4.8%) and even weight gain in the Control group (1.5 ± 6.3%) in the previous 3 months. The patients in the active group gained 0.5 kg body weight, whereas patients in the control group on average lost 1.1 kg. However, in this short term intervention with the Active medical food weight changes did not reach statistical significance when compared to an iso-caloric, iso-nitrogenous Control product, while in a previous study in esophageal cancer patients body weight was significantly increased after a period of 4 weeks of nutritional intervention with the Active medical food, compared to a Control product (submitted for publication).

In conclusion, the present study demonstrates a rapid incorporation of EPA and DHA into WBC and a significant reduction of serum PGE2 levels in cancer patients receiving radiotherapy after a one week of nutritional intervention with a medical food, which is high in protein and leucine and enriched with emulsified fish oil (containing EPA and DHA) and a specific oligosaccharide mixture compared with the effects of an iso-caloric and iso-nitrogenous control product. Moreover, the medical food is well appreciated. Patients like the two different taste variations resulting in a high compliance rate of study product intake. No clinically relevant safety concerns were reported and no changes in blood safety parameters were measured. Consequently, these results show that nutritional intervention with the specific medical food may represent a new opportunity for applications in cancer patients being an integral part of disease management to provide optimal treatment support. Additional research is recommended to elucidate the potential immunological effects in different types and stages of cancer.

Role of the funding source

The study was funded by Nutricia Advanced Medical Nutrition, Danone Research, Centre for Specialised Nutrition, including a role in the study design, analysis, interpretation of the data and writing the manuscript, as indicated in the affiliations.

Statement of authorship

JF, MB, BJW, APV, AvH, MHO, MH and JA designed the research; JF and UF conducted the research under supervision of APV, MH, JG, AvH and JA; MB and MA conducted the study management tasks; JF wrote the manuscript; BJW, AvH, MHO, JG and JA had primary responsibility for the interpretation and final content; all authors read and approved the final manuscript.

Conflict of interest

As indicated in the affiliations, JF, MB, APV and AvH are employed within Nutricia Advanced Medical Nutrition, Danone Research, Wageningen, The Netherlands and MHO and JA serve on the Nutricia Scientific Advisory Board on Oncology.

Acknowledgments

The authors would like to thank M. Balvers and N. Buurman for their technical assistance, Y.C.W. van Oossanen and M.M. van Keulen from the Research office of the Meander Medical Center and L. Homans from the Research office of the Gelderse Vallei Hospital and W. de Graaf for their study management tasks, Dr. M. Azémar for his input on the laboratory analysis and Dr. R. Hobo and Dr. S. Swinkels for data management and statistics.

References

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Footnotes

a Nutricia Advanced Medical Nutrition, Danone Research, Centre for Specialised Nutrition, Wageningen, The Netherlands

b Department of Pharmacology & Pathophysiology, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, The Netherlands

c ProQinase GmbH, Tumor Biology Center, Freiburg, Germany

d Tumor Biology Center, Freiburg University, Freiburg, Germany

e Department of Gastroenterology and Hepatology, Gelderse Vallei Hospital, Ede, The Netherlands

f Clinical Study Section, Clinic for Radiooncology, University Clinic, Freiburg, Germany

g Department of Internal Medicine and Gastroenterology, Meander Medical Centre, Amersfoort, The Netherlands

Corresponding author. J. Faber. Nutricia Advanced Medical Nutrition, Danone Research, Centre for Specialized Nutrition, P.O. Box 7005, 6700 CA Wageningen, The Netherlands. Tel.: +31 317 467922; fax: +31 317 466500.


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