Oncology

ONC: Dietary Supplements and Medical Food Supplements Containing Fish Oil (2013)

Citation:

Pratt VC, Watanabe S, Bruera E, Mackey J, Clandinin MT, Baracos VE, Field CJ. Plasma and neutrophil fatty acid composition in advanced cancer patients and response to fish oil supplementation. Br J Cancer. 2002 Dec 2;87(12):1370-8.  

PubMed ID: 12454764
 
Study Design:
Randomized Controlled Trial
Class:
A - Click here for explanation of classification scheme.
Quality Rating:
Negative NEGATIVE: See Quality Criteria Checklist below.
Research Purpose:

1. To assess the fatty acid composition of plasma and neutrophil phospholipids (PL)of advanced cancer and to compare this with other patient groups of healthy subjects.

2. To assess n-6 and n-3 fatty acid content and extent of incorporation of supplemented oils into plasma and neutrophil phospholipids in pateints with advanced cancer.

3.To identify relationships among n-6 and n-3 fatty acids in plasma and cell phospholipids and some selected parameters of nutritional status.

Inclusion Criteria:
  • Presence of anorexia,defined as >3 on a visual analog scale.
  • Loss of 50% or more of their pre-illness body weight.
  • The ability to maintain oral intake.
  • Normal cognition,defined as a normal Mini-Mental State Exam(Periera et al,1998) for age and level of education.
  • Ability and willingness to give written ,informed consent
Exclusion Criteria:
Exlusion critria was not delineated.
Description of Study Protocol:

Recruitment : In the absence of a large base of reference data,the  patients groups,which represent a wide range of patient groups from critically ill to healthy subjects were used to obtain a context for the range of plasma phospholipids(PL) fatty acids observed in advanced cancer patients in this study. This study collected data in healthy subjects (n=6), advanced cancer (n=23), burn injury (n=18), and high dose chemotherapy with SCT (n=6) on a total of 53 subjects.

1. Advanced cancer patients were a cohort of subjects participating in a larger study. A random sub-sample of 14 subjects per treatment arm were assigned to receive more detailed measures of fatty acid as described in the present study.

2. Burn injury patients- recruitment methods were not detailed.

3. High dose chemotherapy patients-recruitment methods were not detailed.

4. Healthy subjects-recruitment methods were not identified.

 

Design

Eligible participants were randomised using a computer generated sequence to receive identical gelatin capsules containing fish oil or placebo(olive oil) for 14 days. The two treatment arms did not differ in age ,weight,presence of metastasis or mean number of capsules per day.

Due to the limited reference data for fatty acid composition of plasma in advanced cancer patients,multiple subject groups were selected for comparison. Each group of subjects had plasma and cellular level PL compostion assessed using the same laboratory as as the advanced cancer group.

 Participants were provided one of the following:

  • 1 gram fish oil - 18 capsules/day x 14 days
  • 1 olive oil - 18 capsules/day x 14 days (PLACEBO)

The following measures were taken:

  • Blood for plasma and Neutrophil phospholipid: Day 0 & 14
  • Weight: Day 1 & 14
  • 3 Day Food Record: Beginning & End of 14d period (days not specified)

Blinding used -all investigators and health care workers were blinded to the treatment received throughout the trial.

 

Intervention : Advanced cancer patients

  • Eligible participants were allocated to receive identical 1 gram gelatin capsules containing fish oil(180 mg EPA and 120 mg DHA Banner Pharmacaps,Olds,AB,Canada) or placebo (olive oil).
  • Olive oil was was selected as the placebo ,as it was not expected to alter plasma lipids or essential fatty acid metabolism.
  • Subjects were instructed to take 18 capsules orally each day for 14 days.

Table 1 Fatty Acid Composition of the fish oil and olive oil
 

                                Percent  Fatty   Acids                

Fatty acid         

Fish Oil

Olive Oil(placebo)

C 14:0

               6.9                

 0.1

C 16:0

14.8

 19.6

C 16:1(7)

7.8

 1.8

C 18:0

3.1

 2.4

C 18:1(9)

8.9

 60.3

C 18.1(7)

2.8

 2.5

C 18:2(6)

1.3

 12.3

C 18:3(3)

0.8

 0.6

C 18:4(3)

2.8

 nil

C 20:0

0.5

 0.1

C 20:1a

2.0

 0.2

C 20:2(6)

0.2

 nil

C 20:3(3)

1.0

 nil

C 20:4(6)

0.8

 nil

C 20:5(3)

17.8

 0.1

C 22:0

0.1

 0.1

C 22: 1b

1.7

 nil

C 22:4(6)

0.4

 nil

C 22:5(3)

2.4

 nil

C 22:6(3)

12.8

 0.1

C 24:1

0.6

 nil

a Includes 20:1(11),20:1(9),20:1(7) bIncludes 22.1(11),20:1(9),20:1(7). Fatty acid composition of fish oil and olive oil capsules was determined by gas liquid chromatography.

 Statistical Analysis:

  • Power Calculation: N based on the ability to detect a change at 5% significance level in n-3 fatty acids in plasma after 2 weeks of supplementation with  fish oil.
  • Chemotherapy and burn patients were not compared statistically to the advanced cancer patients because they were not controlled cohorts for sex,age or body composition.     
  • T-test (differences between the high BMI and low BMI groups)
  • Correltation analysis (BMI,kcal kg-1 BW intake against plasma lipids)
  • Paired t-test (changes in fatty acids composition within a supplemental group)
  • Repeat measures ANOVA (Differences between groups and timepoints)
  • Least squared means (differences between treatments and day 0 and 14)
  • Data are reported as mean±s.e.m
  • P<0.05 
Data Collection Summary:

Timing of Measurements:

1. Advanced cancer patients:

  • Patients were instructed by a research assistant to keep a three day food record during three consecutive days both at the beginning and end of the 14 day supplemntation period .
  • On day 0 and day 14 of supplementation,a non-fasting sample of peripheral blood(16 ml)was drawn by a registered nurse during in-home visits or in the clinic.

2. Burn injury patients:

  • A non-fasting wenous blood sample (10 mL) was collected within the first 12 days(n=10) and after 50 days(n=8) following the burn injury. Timepoints were compared using a repeated measures ANOVA.

 3. High dose chemotherapy patients:

  • Blood (16 ML0 was collected into heparinsed tubes at stem cell harvest(~ 2 weeks post induction chemotherapy) and on the day of disharge( ~ 2 weeks post-HDCT). A paired t-test was used to compare pre-and post chemotherapy values.    

4. Healthy subjects: information not detailed.

Dependent Variables

  • Plasma phospholipids (PL)

  • Constituent fatty acids

  • Neutrophil phospholipids

  • Selected parameters  of nutritional status :

               -caloric intake was estimated using 3 day food records and analyzed

                 using the Food Processor II Nutrient Analysis  Program  with the                                       Canadian Nutrient Data Files.

               -fat intake estaminated using same analysis as caloric intake.

               -Body Mass Index calculations:

  • Weight(without shoes and wearing light clothes)were  obtained on day 1 and day 14.

  • Height and pre-illness weight were documented on day 1.     

     

    Independent Variable:
    • Quantity of EPA consumed daily

     

  • Description of Actual Data Sample:

     

    Initial N:

    • Advanced Cancer: n=23 [Fish Oil: n=13 (8 male, 5 female); Control: n=10 (10 male, 0 female)]
      • The patients with advanced cancer were a cohort of subjects participating in a larger study. A random sub-sample of 14 subjects per treatment arm were assigned to receive more detail measures of fatty acid as described in this study. Characteristics are in Table 2.
    • Burn Injury: n=18 (details not described)
    • High Dose Chemotherapy w/ SCT: n=6 (0 male, 6 female) (details not described
    •  Healthy subjects: n=6, details not described   

    Table 2 Characteristics of the advanced cancer patients
    A. Subjects randomly allocated to fish oil supplements

    Patient ID

    Age(y)

    Gender

    Weight(kg)

    Cancer Site

    Metastasis

    Mean # capsules/d

    EPA/d             (mg kg bw)

    DHA/d(mg kg bw)

    01

     48

     M

     73

     pancreas

     no

     16

     38.5

     25.7

    02

     65

     M

     87

     prostate

     yes

     17

     35.4

     23.6

    03

     61

     F

     N/A

     lung

     no

     8

     33.6

     22.4

    04

     73

     M

     54

     rectum

     yes

     8

     25.0

     16.7

    05

     58

     M

     68

     rectum

     yes

     17

     45.4

     30.3

    06

     67

     M

     92

     prostate

     yes

     12

     23.5

     15.7

    07

     82

     M

     63

     jejunum

     yes

     11

     32.1

     21.4

    08

     69

     F

     54

     rectum

     yes

     16

     52.9

     35.2

    09

     78

     M

     59

     lung

     no

     6

     17.9

     11.9

    10

     61

     F

     49

     breast

     yes

     6

     30.3

     20.2

    11

     77

     F

     62

     adenocarcinoma

     yes

     12

     37.2

     24.8

    12

     46

     F

     44

     unknown

     yes

     9

     36.8

     24.6

    13

     61

     M

     70

     Cecum

     yes

     16

     41.4

     27.6

    mean±s.e.m.

    65±3

     

    65±3

     

     

    12±1

    34.6±2.6

    23.1±1.7

    Table 2 Characteristics of the advanced cancer patients
    B. Subjects randomly allocated to olive oil supplementation

    Patient ID Age(y) Gender Weight(kg) Cancer Site Metastasis Mean # capsules/d
    01  69

     M

     73

     lung

    yes

     10

    02  63

     M

     73

     kidney

     yes

     18

    03  77

     M

     45

     parotid

     yes

     8

    04  81

     M

     67

     prostate

     yes

     14

    05  59

     M

     54

     leiomyosarcoma

     yes

     6

    06  73

     M

     58

     lung

     no

     9

    07  66

     M

     50

     stomach

     yes

     15

    08  62

     M

     55

     rectum

     yes

     6

    09  76

     M

     60

     colon

     yes

     7

    10  65

     M

     63

     lung

     yes

     7

    mean±s.e.m.

    69±3

     

    60±3

       

    10±1

    Attrition (final N): n=23 [Fish Oil: n=13 (8 male, 5 female); Control: n=10 (10 male, 0 female)]

    • Reasons for not completing the study including failure to maintain intake of the oil capsules,complications due to cancer progression , and death(n=4).

    Ethnicity: Not detailed.

    Other relevant demographics: Groups did not differ significantly in age,or the presence of metastasis. The primary site of the cancer varied widely among subjects in both groups(See Table 2).

    Anthropometrics :Groups did not differ significantly in body weight

    Location: University of Alberta, Canada 

     

    Summary of Results:
    • Advanced Cancer Patients
      • Total plasma PL and most individual fatty acids in the PL lowest of all groups studied. 
      • Measure of EFAD elevated (0.3±0.04)
    • Chemotherapy patients
      • Decreased level of long chain FA
      • All PUFA (except EPA & DHA) below limit of detection
      • Post high dose chemotherapy:
        • DHA undetectable
        • EPA fell to 7% of control values (0.1 µg/ml)

    Table 3 Plasma phospholipids by patient group
       

    Burn Injury

    Burn Injury

    Breast Cancer

    Breast Cancer

    Advanced cancer Pre-supplementation(n=23

    Fatty      Acid  

     

    Healthy subjects(n=6)

     

    < 12 days post burn injury(n=10)

    ug mL-1 plasma 

      >50 days post burn injury(n=10)

    ug mL-1 plasma 

    After Induction(n=3)

    ug mL-1 plasma 

    After high dose(n=3)

    ug mL-1 plasma 

     

    C16:0

     207±31

     135±7

     160±14

     74±5

     53±8

     64±6

    C18:0

     100±11

     73±3

     91±12

     52±16

     32±6

     39±6

    C18:(9)

     70±10

     62±3

     50±8

     41±9

     29±4

     24±3

    C18:2(6)

     175±30

     79±4

     98±14

     73±23

     38±7

     51±8

    C18:3(3)

     2±1

     1±0.2

     2±1

     1±0.2

     0.1±0.1

     0.4±0.1

    C20:2(6)

     2±0.4

     1±0.3

     2±0.2

     3±1

     1±0.2

     1±0.1

    C20:3(6)

     17±3

     15±3

     16±3

     14±6

     7±3

     1±0.2

    C20:4(6)

     68±11

     31±4

     46±6

     43±14

     35±9

     25±4

    C20:5(3)

     2±0.4

     2±0.3

     2±1

     1±1

     0.1±0.1

     1±0.2

    C22:4(6)

     2±0.3

     1±0.2

     1±0.2

      nila

     nila

     0.4±0.1

    C22:5(6)

     2±0.4

     1±0.1

     1±0.4

      nila

     nila

     1±0.2

    C22:5(3)

     4±1

     3±1

     3±1

     nila

     nila

     1±0.2

    C22:6(3)

     21±3

     10±1

     13±3

     1±1

     nila

     9±2

    Sum n-6

     266±46

     122±14

     165±22

    133±42

     80±16

     77±10

     Sum n-3

     29±4

     15±2

     25±3

     3±2

     0.3±0.3

     16±3

     n-6/n-3 ratio

     9±1

     8±1

     9±1

     103±24

     90±23

     6±0.4

    Sum PUFA

     298±50

     142±15

     191±24

     136±40

     80±16

     115±18

    Sum SFA

     321±42

     212±19

     281±30

     126±31

     85±13

     117±13

    Sum MUFA

     101±17

     78±8

     81±12

     49±10

     33±5

     33±4

     Total

     720±99

     432±39

     552±62

     447±121

     278±50

     244±27

    a nil=<0.04; Abbreviations:PUFA polyunsaturated fatty acids, MUFA monounsaturated fatty acids,SFA saturated fatty acids.

     

    • Advanced Cancer Patients
      • Baseline PL FA coincentrations different
      • Higher  levels of 20:4 n-6 in all PL classes of neutrophils led to higher n-6/n-3 FA ratio
        • +80% in PI,+ 80% in PC,+22% in PE and 100% in PS
      • Average levels EPA & DHA in neutrophil PL highly variable
        • EPA levels range: undetectable to 4%
      • Highest levels of n-6 FA in neutrophil PL
    • Chemotherapy Patients:
      • Undectable levels of n-3 FA in neutrophil PL post induction

    Table 4 Fatty acid composition of phospholipid fractions of neutrophils compared to other patient groups
        Burn    Injurya                    Burn    Injurya                    Breast Cancer Breast Cancer

    Advanced cancer pre-supplementation(n=23

     Fatty      Acid  

     

    Healthy subjects(n=6)

    % ww1

     

    < 12 days post burn injury(n=10)

    % ww1

      >50 days post burn injury(n=10)

    % ww1

    After Induction(n=3)

    % ww1      

    After high dose(n=3)

    % ww1        

     

    % ww1

           

    Phosphatidylcholine

         
    16:0  33±2

     31±1

     31±1  33±1  31±0.2  24±2
    18:0  12±1  12±1  12±1  12±1  13±2  11±1
    18:1(9)  25±0.4  31±1  24±2  28±1  30±1  28±2
    18:2(6)  10±1  9±1  10±2  15±1  15±1  14±1
    20:4(6)  8±1  3±0.4  7±1  4±1  4±0.4  12±1
    20:5(3)  2±1  1±0.2  1±1  0.2±0.2  1±0.2  1±0.3
    22:6(3)  0.3±0.2  0.4±0.1  1±0.1  nilb   0.1±0.1  1±0.2
    Sum  n-6  21±1  14±1  15±2  22 ± 0.4  21±1  29±2
    Sum n-3 3±1 2±0.3 3±0.4 0.4±0.2 1±1 2±1
    n-6/n-3  10.7±2.6  4.4±1.5  5±2  32±7  19±4  26±4
    Sum PUFA  24±1  17±1  22±1  22±0.2  22±1  31±2
    Sum SFA  46±1  48±1  48±2  50±0.4  47±1  37±2
    Sum MUFA  30±1  35±1  28±1  28±1  32±1  31±1
           
    Phosphatidylinositol
         
     16:0  18±4  24±3  17±5  36±3  26±2  9±2
     18:0  25±5  28±2  29±2  16±2  29±0.3  26±5
     18:1(9)  7±1  11±1  7±1  27±3  25±2  8±2
    18:2(6)  3±1  5±1  4±1  13±1  8±1  14±3
     20:4(6)  12±4  8±1  18±4  3±1  12±1  22±4
    20:5(3)  1±0.3  3±1  1±1  nilb  nilb  2±1
    22:6(3)  1±0.2  0.2±0.1  1±0.3  nilb  nilb  2±1
    Sum  n-6  25±5  17±2  25±3  17±2  21±1  40±5
    Sum n-3  3±0.2  3±1  4±1  nilb  nilb  5±1
    n-6/n-3 11±3 5.8±1.4 11±3 N/A N/A 15±4
    Sum PUFA  28±5  22±2  29±3  17±2  21±1  45±5
    Sum SFA  57±3  61±1  59±2  56±4  54±2  37±6
    Sum MUFA  13±1  17±1  16±2  27±3  25±2  14±2

    bnil=<0.04%. N/A=Not applicable. Data are expressed as means±s.e.m.  Abbreviations:PUFA polyunsaturated fatty acids, MUFA monounsaturated fatty acids,SFA saturated fatty acids.

     

    • Advanced Cancer Patients:
      • Significant reduction in 20:4 n-6 content in the PI fraction to levels similar to control subjects.
      • Total n-3 FA content of neutrophil PC directly related to plasma PL n-3 FAcontent (r 0.78, p0.001)
      • n-6/n-3 ration of plasma PL directly relatd to n-6/n-3 ratio of neutrophil PC (r 0.67, p 0.007)
      • No significant increase in plasma PL concentration

    Table 5 Effect of fish oil supplementation before and after 14 days in plasma and neutrophil PL
      Plasma PL Plasma PL Neutrophil PC Neutrophil PC Neutrophil PC Neutrophil PC
      PRE-FO Post FO Pre- FO Post FO Pre-FO Post FO
    Fatty acid (% fatty acid ) (% fatty acid ) (% fatty acid ) (% fatty acid ) (% fatty acid ) (% fatty acid )
    16:0  27.6±1.0  27.7±1.1  26.0±2.5  24.0±1.4  9.7±3.3  14.2±2.9
    18:0  13.9±0.5  14.9±0.8  11.9±0.7  13.2±1.1  28.0±7.2  25.0±4.0
    18:1n-9  10.9±1.0  10.3±0.5  25.8±1.9  30.5±2.0  7.3±2.6  14.3±3.0
    18:2n-6  19.9±0.9a  16.6±1.1b  15.2±2.5  11.1±1.1  12.1±4.6  7.1±1.4
    20:4n-6  10.2±0.7  9.2±0.8  14.1±2.3  11.9±2.6  26.8±5.9a  12.5±2.6b
    20:5n-3  0.7±0.1a  4.4±0.7b  0.2±0.1  0.1±0.1  2.1±1.5  1.6±0.8
    22:6n-3  4.1±0.6a  5.8±0.8b  0.7±0.3  0.8±0.4  1.4±0.7  4.1±1.9
    sum n-6  31.5±1.4  28.3±1.7  31.9±2.8  24.8±2.7  39.8±3.8  24.6±3.9
    sum n-3  6.2±0.6a  11.8±1.4b  1.6±1.0 1.6±0.8  3.5±0.5  8.3±3.4
     sum PuFA  40.5±1.9  41.2±2.1  33.6±3.2  26.4±3.1  44.5±3.8a  32.9±4.6b
    sum SFA  45.4±2.0  47.0±1.2  39.7±3.1  39.1±1.9  37.1±2.0  46.4±4.7
    sum MUFA  14.2±2.0  14.2±0.5  28.7±1.6  33.6±2.2  13.7±1.6a  20.8±2.5b

     Data are expressed as means±s.e.m.(n=13).ab Within a column grouping(i.e pre vs post FO) are significantly different.

    Other Findings:

    Advanced cancer patients

    • Caloric intake 488-2152 Kcal/day
      • 1/3 patient with intake < 1,000 Kcal/d
      • Mean percentage of calories derived from fat was 31±7%.
    • Caloric intake and total fat were highly correlated (r=0.97,P=0.0001).
    • Average BMI ± 20.7 kg/m2 (range 15-28)
      • Seperated into 2 groups (low BMI & high BMI)
      • No difference in caloric intake by BMI
    • Both BMI and caloric intake varied over a broad range and were not significantly correlated(P=0.14).

     

    Author Conclusion:
    • At baseline,patients with advanced cancer exhibited low levels(<30% of normal values of plasma phospholipids and constituent fatty acids and elevated 20:4 n-6 content in neutrophil phospholipids.
    • High n-6 /n-3 fatty acid ratios in neutrophil and plasma phospholipids were inversely related to body mass index.
    • Fish oil supplementation raised eicosapentaenoic acid and docosahexaenoic acid content in plasma but not neutrophil phospholipids.
    • 20:4 n-6 content was related directly to increases in eicosapentaenoic acid in plasma 
    • Limitations: The results presented here represent an initial approach and it is necessary to employ caution in comparing the data obtained from cancer patients with other patient groups which serve mainly to place the advanced cancer patiens in a context for discussion.  

        

    Funding Source:
    Government: Natural Science Engineering and Research Council of Canada
    Reviewer Comments:
    • "Double -blinding"  procedure was not utilized.
    • Advanced cancer group consisted of both hospital and outpatient subjects. Because the study also included outpatients, problems with patient compliance may have impacted the results of the study.
    • Limitations noted by the authors are very relevant in evaluating the study design employed. True  experiments assume that individuals in the experimental and control groups are equivalent,so that the only reason they might differ on the outcome of interest is the program or intervention itself. 
    • Power analysis goals were met for advanced cancer patients. "The number of patients entered into the study was based on the ability to detect a change at 5% significance level in n-3 fatty acids in plasma after 2 weeks of supplementation with  fish oil." 
    • Article confusing, data included on non-cancer patients included
    Quality Criteria Checklist: Primary Research
    Relevance Questions
      1. Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies) Yes
      2. Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about? Yes
      3. Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice? Yes
      4. Is the intervention or procedure feasible? (NA for some epidemiological studies) Yes
     
    Validity Questions
    1. Was the research question clearly stated? Yes
      1.1. Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified? Yes
      1.2. Was (were) the outcome(s) [dependent variable(s)] clearly indicated? Yes
      1.3. Were the target population and setting specified? No
    2. Was the selection of study subjects/patients free from bias? No
      2.1. Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study? No
      2.2. Were criteria applied equally to all study groups? No
      2.3. Were health, demographics, and other characteristics of subjects described? Yes
      2.4. Were the subjects/patients a representative sample of the relevant population? No
    3. Were study groups comparable? No
      3.1. Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT) Yes
      3.2. Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline? No
      3.3. Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.) Yes
      3.4. If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis? N/A
      3.5. If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.) N/A
      3.6. If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")? N/A
    4. Was method of handling withdrawals described? No
      4.1. Were follow-up methods described and the same for all groups? Yes
      4.2. Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.) No
      4.3. Were all enrolled subjects/patients (in the original sample) accounted for? Yes
      4.4. Were reasons for withdrawals similar across groups? No
      4.5. If diagnostic test, was decision to perform reference test not dependent on results of test under study? N/A
    5. Was blinding used to prevent introduction of bias? Yes
      5.1. In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate? Yes
      5.2. Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.) Yes
      5.3. In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded? N/A
      5.4. In case control study, was case definition explicit and case ascertainment not influenced by exposure status? N/A
      5.5. In diagnostic study, were test results blinded to patient history and other test results? N/A
    6. Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described? No
      6.1. In RCT or other intervention trial, were protocols described for all regimens studied? Yes
      6.2. In observational study, were interventions, study settings, and clinicians/provider described? N/A
      6.3. Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect? Yes
      6.4. Was the amount of exposure and, if relevant, subject/patient compliance measured? Yes
      6.5. Were co-interventions (e.g., ancillary treatments, other therapies) described? No
      6.6. Were extra or unplanned treatments described? No
      6.7. Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? No
      6.8. In diagnostic study, were details of test administration and replication sufficient? N/A
    7. Were outcomes clearly defined and the measurements valid and reliable? No
      7.1. Were primary and secondary endpoints described and relevant to the question? Yes
      7.2. Were nutrition measures appropriate to question and outcomes of concern? Yes
      7.3. Was the period of follow-up long enough for important outcome(s) to occur? ???
      7.4. Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures? Yes
      7.5. Was the measurement of effect at an appropriate level of precision? Yes
      7.6. Were other factors accounted for (measured) that could affect outcomes? No
      7.7. Were the measurements conducted consistently across groups? Yes
    8. Was the statistical analysis appropriate for the study design and type of outcome indicators? Yes
      8.1. Were statistical analyses adequately described and the results reported appropriately? Yes
      8.2. Were correct statistical tests used and assumptions of test not violated? Yes
      8.3. Were statistics reported with levels of significance and/or confidence intervals? Yes
      8.4. Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)? No
      8.5. Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)? No
      8.6. Was clinical significance as well as statistical significance reported? No
      8.7. If negative findings, was a power calculation reported to address type 2 error? Yes
    9. Are conclusions supported by results with biases and limitations taken into consideration? Yes
      9.1. Is there a discussion of findings? Yes
      9.2. Are biases and study limitations identified and discussed? Yes
    10. Is bias due to study's funding or sponsorship unlikely? Yes
      10.1. Were sources of funding and investigators' affiliations described? Yes
      10.2. Was the study free from apparent conflict of interest? Yes