Download Herieka et al online data supplement (Rev 1)

Data supplement
Herieka M. et al., “Reduced dietary intake of pro-inflammatory Toll-like receptor stimulants
favourably modifies markers of cardiometabolic risk in healthy men”
Table of contents
Page
Supplemental Methods
2
Supplemental Tables
5
Table S1: Baseline physical parameters of healthy volunteers
5
Table S2: Nutritional and TLR-stimulant content of processed food items and
pre-prepared meals provided to study participants
6
Table S3: Estimated nutritional content of diets based on typical daily intakes
during low and high PAMP arms of the chronic study
7
Table S4: Nutritional content of onion bhaji test meals
8
Table S5: Physiological parameters of volunteers before and after ingestion of
onion-based test meals
9
Supplemental Figures
Page
Figure S1: Graphical depiction of study protocols
10
Figure S2: Correlation of food-borne TLR4-stimulants with capacity of food
extracts to induce IL-6 production from human whole blood
11
Figure S3: Kinetics of bacterial growth in ready-prepared meals
12
Figure S4: Effect of storage time on TLR-stimulant content of pre-prepared
meals
13
Figure S5: Serum endotoxin and endotoxin neutralisation capacity before and
after chronic low and high PAMP diet interventions
14
Figure S6: Effects of low and high PAMP onion meals on PBMC cytokine
mRNA and serum CRP responses
15
Figure S7: Effects of low and high PAMP onion meals on post-prandial
leukocyte counts
16
Figure S8: Effects of low and high PAMP onion meals on post-prandial serum
triglyceride and cholesterol concentrations
17
Figure S9: Effects of low and high PAMP onion meals on glucose, insulin and
HOMA-IR
18
Figure S10: Change in plasma endotoxin after low and high PAMP onion test
meals
19
Supplemental methods
Power calculation and determination of sample size
To our knowledge, experimental dietary PAMP intake has not been examined in man
previously. However, Taudorf et al (Clin Vaccine Immunol 2007;14:250-5) reported the
results of intravenous administration of very low dose (0.3 ng/kg) bacterial endotoxin in
human volunteers by means of a 4 h infusion, which we considered may be more reflective of
the type of exposure arising from slow absorption of an orally administered dose over the
post-prandial phase than studies using bolus administration of endotoxin. The authors
reported that the circulating granulocyte count increased significantly from a baseline of 2.9
billion cells/L to 4.2 billion cells/L, with standard deviation of 1.3. Power calculations
suggested that the minimum sample size required to observe the same change in granulocyte
counts with alpha of 5% was n=10 with power of 0.8, and n=13 with power of 0.9. This is
comparable to group sizes reported in several previous studies in which circulating endotoxin
was observed to increase in the post-prandial phase (typically n=10-12; e.g. as reviewed in
Herieka et al. Mol Nutr Food Res 2013;58:136-46). For both studies, the primary outcome
measure was circulating inflammatory marker status (white blood cell count and CRP).
Chronic study design
All volunteers for the chronic study were asked to complete a food frequency
questionnaire (see below) to record dietary habits with respect to intake of high PAMP foods
prior to study entry. Then, for a run-in period of 7 days, volunteers were asked to avoid
specific types of food that we found in recent studies to be at relatively high risk of
containing high concentrations of PAMPs [13,14]. These items included pre-prepared meals
or sandwiches, cheese, chocolates and any other food containing minced meat or readychopped vegetables stored at refrigeration temperature for an extended period of time. During
this period, subjects were invited to consume any quantity of fresh produce, including any
form of meat, fish or vegetables that had not been minced or processed unless immediately
before consumption, and there were no restrictions on salt, sugar or non-alcoholic beverages.
Subjects were also encouraged to snack preferentially on bread, cereal or fried potato
products when hungry during this period, since we found these typically contain very low
concentrations of soluble TLR-stimulants. Anthropomorphic measurements were made, and
fasting blood samples were collected on days 0 and 7 of this run-in period.
Then, over the next 4 days, subjects consumed a set lunch and evening meal provided to
them, each chosen on the basis of likelihood of high PAMP content from prior screens
(Figure S2). The meals provided ~1,500 kCal per day and each was consumed on or just
before the ‘best before’ date advertised on the packaging. The specific items provided for
each meal and their nutritional and PAMP contents are summarised in Table S2. Subjects
provided a fasting blood sample in the morning after the final day of the high PAMP diet.
Throughout the study, subjects were asked to maintain a quantitative diet diary (see below
for details) and to avoid excessive alcohol consumption for the duration of the study. No
significant side effects were reported on either arm of the study.
Acute study design
The acute study was of single-blinded, crossover design. 13 healthy male university
workers fasted overnight before giving blood then ingesting either a low PAMP (control) or
high PAMP breakfast meal on separate occasions with at least two weeks washout between
visits. Notably, as the vast majority of probiotic and fermentative organisms are Grampositive, and therefore do not secrete large quantities of soluble pro-inflammatory PAMPs
[12], the hypothesis could not be tested using ingredients based on such organisms. We
therefore made use of the prior observations that while TLR-stimulants are undetectable in
freshly chopped onion, γ-proteobacteria (which are a major source of soluble TLR2/4stimulants [14,15]) grow rapidly in chopped onion stored at refrigeration temperature [15].
Repeated testing of ready-chopped onion from a particular vendor revealed reproducibly high
content of PAMPs at the ‘best before’ date, while the product also did not contain any
additives or preservatives (other than a protective atmosphere). The low PAMP meal
therefore comprised 200 g of freshly chopped onion, battered with a flour/water mix, and
deep fried in vegetable oil for 5-8 minutes (onion bhajis), supplemented with 30 g full-fat
mayonnaise. The high PAMP meal equivalent was identical, with the exception that the onion
was purchased as a ready chopped convenience item, stored at refrigeration temperature until
preparation and consumption on the advertised ‘best before’ date. The meals appeared
identical to study participants. TLR-transfectant based bioassays confirmed that the two
meals differed significantly in their content of TLR2- and TLR4-stimulants (21.0±5.5 vs
746±324 μg, P=0.0286 and 14.6±5.3 vs 379±153 μg, P=0.0286, respectively, measured after
cooking), while nutritional contents of the complete meals were otherwise identical (Table
S4). Subjects provided a postprandial blood sample at 3 h and a second fasted sample at 24 h,
following advice to consume only low PAMP food items for intervening meals on both arms
of the acute study. There were no significant reported side effects of treatment with either
type of meal.
Estimation of dietary intakes before and during the study
To estimate the frequency of intake of food items at high risk of PAMP contamination
prior to study entry, volunteers completed a food frequency recall questionnaire. Specifically,
volunteers were asked to indicate numerically how many servings of the following types of
food item they habitually consumed each week: (i) Pre-prepared meals (e.g. ready meals
designed for microwave heating or chilled pizza) (ii) Foods containing minced meats (e.g.
sausages, pies, burgers, etc) (iii) Pre-packaged ready chopped vegetables, sandwiches or
salads (iv) Cheese, (iv) Chocolate. The number of servings of each type of food item was
weighted equally with respect to likely PAMP content, assigning a value of 1 per serving (see
Table 1 in main text).
Volunteers for the chronic study were also asked to maintain a comprehensive diet diary,
recording quantities of all meals, drinks and snacks for the duration of both arms of the study.
The primary information required from these diaries was frequency of intake of foods at high
risk of PAMP contamination (see Results and Table 1 in main text). However, as some
diaries contained incomplete quantitative information with respect to portion sizes (i.e.
serving weights), we aimed to explore the likely daily intake of major nutrients on each arm
of the chronic study by modelling two average days, based on the information provided in the
diet diaries. For this analysis, it was assumed that daily caloric intake was roughly equivalent
on each arm (~2,500 kCal/day).
The nutritional content of foods consumed on a typical high PAMP day were estimated by
calculating the average of the daily total energy, fat, saturated fat, carbohydrate, sugar, fibre,
protein and salt contents of the foods which were provided to volunteers, based on the
nutritional information presented on the food packaging (see Table S2). To these values were
supplemented the nutritional content of items chosen to represent typical ingredients of
supplemental meals recorded in the diet diaries, which comprised: breakfast cereal (100 g),
semi-skimmed milk (125 ml), one apple (125 g), one serving of peas (100 g), one small
serving of chocolate (30 g) and bread with margarine and jam (80 g). Nutritional and caloric
content of these foodstuffs were identified from online databases of food nutrient content
(e.g. www.nutritiondata.self.com).
The nutritional content of foods consumed on a typical low PAMP day were estimated by
modelling an average day’s intake based on the types of food most frequently reported in the
diet diaries. The modelled foods were: breakfast cereal (100 g), semi-skimmed milk (125 ml),
bread (160 g), chicken breast (100 g), banana (200 g), apple (125 g), beef steak (250 g),
french fries (150 g), carrots (80 g), peas (80 g), chocolate (15 g), potato chips (32 g). These
quantities were chosen to approximate those reported by the more complete diaries.
Estimated energy intake on both modelled days amounted to ~2,500 kCal (see Table S3).
Biochemical measurements
Leukocyte counts were measured using a AcTdiff™ Analyzer (Beckman Coulter). mRNA
was extracted from peripheral blood mononuclear cells (PBMC) prepared immediately from
blood samples by density gradient centrifugation and converted to cDNA for real-time PCR
analysis of gene expression. For flow cytometry, a whole blood staining protocol was
employed, followed by erythrocyte lysis and flow cytometry for leukocyte CD11b, CD16 and
CD14 surface markers using a Coulter Epics XL-MCL™ flow cytometer (Beckman Coulter).
Plasma was prepared from blood samples by centrifugation (1,000 g, 15 mins), aliquoted and
stored at -20oC before batch analysis for CRP and insulin by ELISA (R&D systems,
Millipore). Triglycerides, cholesterol and glucose were measured by enzymatic assay (Wako
and Invitrogen). Plasma endotoxin was measured by kinetic limulus assay (Associates of
Cape Cod), following denaturation of masking proteins at 70oC for 10 minutes and using
pyrogen free reagents throughout.
For measurement of TLR-stimulant concentrations in foodstuffs, extracts of each food
were made by blending a representative sample in 10 volumes of distilled water and filtersterilising (0.2 μm) centrifugally cleared supernatants, as described previously [13]. Then,
HEK-293 cells were transfected in 96-well plates with NF-κB-sensitive reporter (pGL3ELAM), thymidine-kinase promoter-driven renilla reporter, human CD14 and TLR2 or
TLR4/MD2. After 48 h, cells were challenged with medium alone (negative control), serial
dilutions of standards (Pam3CSK4 or E. coli LPS, for TLR2- and TLR4-stimulants,
respectively), or 1:400 dilutions of each food extract in culture medium. Results are presented
as biological activities relative to defined quantities of Pam3CSK4 or E. coli LPS, as
described previously [13].
Table S1: Baseline physical parameters of healthy volunteers
Parameter
Age (years)
n (M:F)
Systolic BP (mm Hg)
Diastolic BP (mm Hg)
BMI (kg/m2)
Abdominal circumference (cm)
White blood cell count (109/L)
Total cholesterol (mM)
LDL-cholesterol (mM)
HDL-cholesterol (mM)
Triglycerides (mM)
Glucose (mM)
Insulin (μU/ml)
HOMA-IR
Chronic study
37.5 ± 10.0
11:0
124.8 ± 11.2
77.8 ± 9.5
24.8 ± 2.1
89.9 ± 7.3
5.2 ± 1.7
5.72 ± 1.25
3.79 ± 1.23
1.45 ± 0.53
1.05 ± 0.52
4.77 ± 0.39
3.65 ± 2.19
0.78 ± 0.48
Acute study
27.8 ± 10.7
13:0
127.9 ± 12.7
73.9 ± 11.6
24.2 ± 1.94
87.3 ± 5.2
5.7 ± 0.7
5.51 ± 1.41
3.47 ± 1.30
1.52 ± 0.38
1.21 ± 0.50
4.97 ± 0.45
4.20 ± 1.70
0.95 ± 0.34
Indicated measurements are fasting values measured at entry to each study. Only two
volunteers participated in both studies. Results are presented as mean ± SD. BP, blood
pressure; HOMA-IR, homeostatic model of insulin resistance.
Supplemental Table 2: Nutritional and TLR-stimulant content of processed food items
and pre-prepared meals provided to study participants
Item
Pork sausage rolls
Cheese and onion rolls
Lasagne
Cornish pasty
Fish pie
Cheese and onion pasty
Pork sausage rolls
Bolognase pasta
Cheese and onion pasty
Cornish pasty
Spaghetti bolognase
Item
Pork sausage rolls
Cheese and onion rolls
Lasagne
Cornish pasty
Fish pie
Cheese and onion pasty
Pork sausage rolls
Bolognase pasta
Cheese and onion pasty
Cornish pasty
Spaghetti bolognase
Day/
meal*
Serving
(g)
Energy
(kCal)
Fat
(g)
Sat
(g)
8L
8L
8E
9L
9E
10L
10L
10E
11L
11L
11E
180
80
400
300
800
150
120
600
150
150
800
501
204
562
761
653
410
334
798
397
381
886
21.6
10.9
12.9
33.9
17.6
23.9
14.4
34.2
20.9
17.0
33.6
9.4
5.8
6.6
15.3
9.6
11.9
6.2
18.6
10.2
7.7
14.4
Day/
meal*
8L
8L
8E
9L
9E
10L
10L
10E
11L
11L
11E
Sugar
(g)
2.7
1.8
8.4
6.0
4.8
2.4
1.8
17.4
2.7
3.0
13.6
Fibre
(g)
4.3
1.8
4.5
5.7
5.6
3.9
2.9
5.4
5.0
2.9
12.0
Protein
(g)
13.1
5.2
16.5
20.7
32.0
7.1
8.8
40.2
8.9
10.4
32.8
Salt
(g)
1.6
0.6
1.5
1.8
4.0
0.8
1.1
3.0
1.5
0.9
3.2
TLR2
(μg) **
30
6.9
146
8.1
12
10
20
18
10
4.1
29
Carbohydrate
(g)
61.0
20.6
23.4
90.0
88.0
40.1
40.7
79.2
41.1
45.0
106.4
TLR4
(μg) **
2,714
127
6,442
124
218
125
1,809
232
125
62
459
Values are presented per total serving as provided to volunteers. * Code indicates the day of
study each food item was provided to volunteers, followed by L or E to distinguish lunch or
evening meal, respectively. ** Soluble stimulants of TLR2 or TLR4 were measured in
aliquots of the same batch of each food type measured at the same time as provided to
volunteers (i.e. on the advertised ‘best before’ date), by TLR-transfectant-based bioassay (see
methods). TLR2- and TLR4-stimulant measurements are presented as μg per total serving
relative to Pam3CSK4 or E. coli lipopolysaccharide biological activity, respectively. Foods
were tested after heating using a microwave oven when recommended by the supplier.
Supplemental Table 3: Estimated nutritional content of diets based on typical daily
intakes during low and high PAMP arms of the chronic study
3.9
1.4
0.5
4.0
0.2
0.0
7.8
2.1
0.0
0.0
2.8
0.9
23.5
25.9
6.0
6.4
0.0
24.0
13.0
0.0
0.9
2.4
2.4
8.4
0.7
90.1
8.5
0.0
4.3
1.0
5.2
3.0
0.0
5.3
2.4
6.4
0.1
1.3
37.5
8.2
4.5
12.6
16.0
2.2
0.0
53.0
4.5
0.8
6.4
1.1
2.2
111.6
Salt (g)
69.5
6.0
75.0
18.0
46.0
17.0
0.0
55.5
6.4
16.8
8.5
17.4
336.1
Protein (g)
Fibre (g)
6.6
2.3
3.2
18.0
0.6
0.0
18.0
21.0
0.0
0.0
4.6
9.4
83.6
Saturated
(g)
Fat (g)
Energy
(kCal)
391
63
393
293
178
65
375
432
28
94
80
163
2555
Sugar (g)
100
125
160
100
200
125
250
150
80
80
15
32
Carbohydrate (g)
Cereal
Milk*
Bread **
Chicken breast
Banana
Apple
Beef steak
French fries
Carrots
Peas
Chocolate
Potato chips
Total
Serving (g)
Food type
(A) Estimated daily nutrient intake during the low PAMP week
0.7
0.0
1.6
1.1
0.0
0.0
0.8
0.8
0.1
0.0
0.0
0.4
5.5
Protein (g)
Salt (g)
3.9
1.4
0.0
0.0
5.6
1.3
29.0
41.1
Fibre (g)
6.6
2.3
0.0
0.0
9.2
5.8
60.0
83.8
Saturated
(g)
Fat (g)
Energy
(kCal)
391
63
65
118
159
259
1470
2525
Sugar (g)
100
125
125
100
30
80
-
Carbohydrate (g)
Cereal
Milk**
Apple
Peas
Chocolate
Bread***
Provided****
Total
Serving (g)
Food type
(B) Estimated daily nutrient intake during the high PAMP week
69.5
6.0
17.0
21.0
17.0
43.5
159.0
333.0
25.9
6.0
13.0
3.0
16.8
8.5
16.0
89.2
8.5
0.0
3.0
8.0
0.2
2.4
13.0
35.1
8.2
4.5
0.0
8.0
2.3
6.4
49.0
78.3
0.7
0.0
0.0
0.0
0.1
0.9
5.0
6.7
In order to estimate the nutritional content of the diets consumed during the low and high
PAMP arms of the study, two representative 24 h periods were modelled. The first (typical
low PAMP day) is modelled on average daily intakes of specific food types collated from the
diet diaries (see summary statistics in Table 1). The second is based on the same breakfast but
different intake of fruits, vegetables and chocolate in accordance with food frequencies
reported in the diet diaries, in combination with the meals provided to subjects. * Semiskimmed milk. ** 4 slices of white bread. *** 2 slices with jam and margarine. **** These
are the average daily intake values for all of the provided meals combined (see Table S2 for
breakdown by meal/day).
Supplemental Table 4: Nutritional content of onion bhaji test meals
Energy
Total fat
Saturated fat
Carbohydrate
Sugar
Fibre
Protein
Salt
TLR2-stimulants*
TLR4-stimulants*
Low PAMP
4,097 kJ
60 g
6g
92 g
24 g
10 g
14 g
0.5 g
21.0 ± 11.1 μg
14.6 ± 10.5 μg
High PAMP
4,097 kJ
60 g
6g
92 g
24 g
10 g
14 g
0.5 g
746 ± 648 μg **
379 ± 306 μg **
* TLR2- and TLR4-stimulant values are presented as μg per total serving relative to
Pam3CSK4 or E. coli lipopolysaccharide standards, as measured using TLR-transfectant
based bioassay of extracts of each food as supplied to the subjects immediately after cooking
(see methods section). ** P<0.05 vs low PAMP meals.
Supplemental Table 5: Physiological parameters of volunteers before and after
ingestion of onion-based test meals
Temperature
(oC)
Pulse rate
(bpm)
Systolic
(mmHg)
Diastolic
(mmHg)
Low PAMP meal
0h
3h
36.3
36.6
(0.4)
(0.4)
64.9
64.8
(11.1)
(11.7)
127.9
127.3
(12.7)
(9.8)
73.9
72.1
(11.6)
(13.8)
24 h
36.5
(0.3)
62.7
(10.6)
125.1
(9.2)
72.8
(10.3)
High PAMP meal
0h
3h
36.2
36.5
(0.2)
(0.3)
65.8
62.6
(10.6)
(9.6)
123.7
125.2
(8.1)
(8.8)
75.7
72.9
(11.6)
(10.6)
24 h
36.3
(0.4)
66.3
(8.4)
126.9
(11.2)
74.4
(10.7)
Values are presented as mean ± (SD). BPM, beats per minute; mmHg, millimetres of
mercury.
Supplemental Figure 1: Graphical depiction of study protocols.
The protocols for the chronic and acute dietary PAMP intervention studies are shown in
panels A and B, respectively. Details of the low PAMP dietary advice and the high PAMP
meals provided to volunteers are reported in the main text.
Supplemental Figure 2: Correlation of food-borne TLR4-stimulants with capacity of
food extracts to induce IL-6 production from human whole blood.
In order to identify high PAMP foods for use as test meals in the chronic study, filtersterilised extracts of 15 pre-prepared meals and 8 baked goods containing minced meat or
chopped onion, were added at 1:100 dilution to whole blood cultures (n=4 individual donors)
in microtitre plates and IL-6 was measured at 24 h (A). TLR4-stimulants were also measured
in each extract by TLR-transfectant bioassay as described in the methods (B). Spearman
correlation analyses revealed strong correlations between food content of TLR4-stimulants
and the capacity of each extract to induce IL-6, TNF-α and IL-1β production by cultured
blood (C). All foods were tested at the advertised ‘best before’ date.
Supplemental Figure 3: Kinetics of bacterial growth in ready-prepared meals.
Four identical examples of representative pre-prepared meals (2x lasagne and 2x spaghetti
bolognese), were purchased from local supermarkets and stored at refrigeration temperature
for the indicated periods relative to the advertised ‘best before’ date on the package (as
indicated by arrows on x-axes). Aerobic mesophile bacteria were measured as colony
forming units (CFU) per gramme by streaking on plate-count agar. Colonies were counted
after incubation of plates in air at room temperature for 72 h.
Supplemental Figure 4: Effect of storage time on TLR-stimulant content of preprepared meals.
Four identical examples of representative pre-prepared meals (2x lasagne and 2x spaghetti
bolognese), were purchased from local supermarkets and stored at refrigeration temperature
for the periods indicated in Supp Fig 3 relative to the advertised ‘best before’ date on the
package. TLR2- and TLR4-stimulants (panels A and B, respectively) were measured in each
food extract using HEK-293 transfectants as described in the methods section. Results are
presented as mean biological activities relative to Pam3CSK4 (TLR2-stimulants) or E. coli
LPS (TLR4-stimulants).
Supplemental Figure 5: Serum endotoxin and endotoxin neutralisation capacity before
and after chronic low and high PAMP diet interventions.
Serum endotoxin was measured in fasted samples from healthy male subjects (n=11) at study
entry (visit 1), after 7 days low pathogen-associated molecular pattern (PAMP) diet (visit 2)
and after 4 days high PAMP diet (visit 3), using the kinetic limulus amoebocyte lysate assay
after denaturation of limulus-activity masking proteins in plasma by heating at 70oC for 10
minutes (A). In order to measure endotoxin neutralisation capacity (ENC), this heating step
was omitted and plasma samples were spiked with 20 ng/ml LPS for 1 h prior to assay. ENC
is expressed as a percentage suppression of the signal induced by 20 ng/ml LPS in the
absence of plasma (B). Results are presented as means ± SE.
Supplemental Figure 6: Effects of low and high PAMP onion meals on PBMC cytokine
mRNA and serum CRP responses.
Normalised mean (±SE) mRNA expression of IL-6 (A) and CCL-2 (B) relative to 36B4
mRNA in PBMC collected before or 3 h after ingestion of a low or high PAMP meal is
shown. CRP was measured in plasma samples collected at 0 and 24 h by high-sensitivity
ELISA (C). Results were analysed using linear mixed models. * P<0.05 vs baseline by
Student’s T-test with Bonferroni correction for multiple testing.
Supplemental Figure 7: Effects of low and high PAMP onion meals on post-prandial
leukocyte counts
Mean (±SE) leukocyte counts measured in blood samples of healthy male volunteers (n= 13)
provided with either a low or high pathogen-associated molecular pattern (PAMP) onionbased test meal on two separate occasions. Blood samples were collected at 0 h (fasted
sample), 3 h (postprandial) and 24 h (fasted). Linear mixed models revealed no significant
effect of meal alone, but the meal x time interaction was significant for total leukocyte count
(P=0.039) and monocyte count (P=0.041). Post-hoc comparison of fasted sample means was
performed by Student’s T-test with Bonferroni correction for multiple testing. ** P=0.013 vs
baseline.
Supplemental Figure 8: Effects of low and high PAMP onion meals on post-prandial
serum triglyceride and cholesterol concentrations.
Plasma triglycerides (A), LDL-C (B) and HDL-C (C), were measured in blood samples of
healthy male volunteers (n=13) collected before ingestion of a low- or high PAMP test meal
(fasted, 0 h), postprandially (3 h) and the next day (fasted, 24 h) after the test meals. Results
were analysed using linear mixed models and are presented as means ± SE.
Supplemental Figure 9: Effects of low and high PAMP onion meals on glucose, insulin
and HOMA-IR.
Plasma glucose (A), insulin (B) and HOMA-IR (C), were measured in fasted blood samples
of healthy male volunteers (n=13) collected before ingestion of a low- or high-PAMP test
meal (0 h), or 24 h after the test meals. Results were analysed using linear mixed models and
are presented as means ± SE.
Supplemental Figure 10: Change in plasma endotoxin after low and high PAMP onion
test meals.
Endotoxin was measured in blood samples of healthy male volunteers (n=13) collected
before ingestion of a low- or high PAMP test meal (fasted, 0 h) and postprandially (3 h) after
the test meals using the limulus amoebocyte lysate assay. Results are presented as mean (±
SE) change in endotoxin concentrations relative to baseline. A paired sample T-test was used
to test significance. Post-prandial LPS concentrations were not significantly different from
baseline.