Key words
gastrointestinal - epithelium - endotoxin - inflammatory cytokine - immunoglobulin
- exercise
Introduction
It is now well established that endurance exercise promotes integrity disturbances
to
the gastrointestinal epithelium due to primary pathways (i. e., circulatory-
and neuroendocrine-gastrointestinal) associated with exercise-induced
gastrointestinal syndrome (EIGS) [1]
[2]
[3]
[4]. The secondary outcomes of these disturbances may
include intestinal epithelium cell injury, with or without epithelial rupture, and
hyperpermeability. Such perturbations to intestinal epithelial integrity may result
in the translocation of pathogenic content from the intestinal lumen (i. e.,
bacterial endotoxins, such as lipopolysaccharide (LPS)) into circulation,
heightening the systemic inflammatory response that is commonly observed with
prolonged and strenuous exercise stress [5]
[6]. These EIGS biomarkers appear to be exacerbated when
exercise is performed in the heat (i. e.,≥35°C ambient
temperature (Tamb)) resulting in≥39.0°C core body
temperature [7]
[8]
[9]
[10], and of longer
exercise duration (i. e.,≥3 h) [11].
Exacerbation of these biomarkers suggests that exertional-heat stress and exertional
stress duration may play a key role in the magnitude of exercise-associated
disturbance to gastrointestinal integrity, and subsequently the magnitude of
translocation of intestinal lumen originating bacterial pathogenic agents. Such
translocation, in response to compromised epithelial integrity, may have clinical
(e. g., sepsis) or sub-clinical (e. g., gastrointestinal symptoms
(GIS)) implications [4]
[12]
[13]
[14]
[15]
[16],
which may be associated with impairment to exercise [17]
[18]
[19].
Systemic endotoxemia has previously been classified as a pre- to post-exercise change
of≥5 pg/ml in bacterial endotoxins, with a concomitant reduction in
anti-endotoxin antibody immunoglobulins (Ig), namely IgM and IgG, as defined by
earlier field studies; however, the clinical significance of these exercise-induced
changes is yet to be clarified due to potential exposure and immune tolerance [12]
[13]
[20]
[21]
[22]
[23]
[24]. Moreover, it is common for both field and
laboratory-controlled studies to measure plasma endotoxin concentration without the
supportive analysis of anti-endotoxin antibodies. Nevertheless, recent
laboratory-controlled studies have incorporated endogenous endotoxin core antibodies
(EndoCAb) within the suite of EIGS biomarkers and suggest that measuring
anti-endotoxin antibodies may provide a more comprehensive and reliable
interpretation of endotoxin translocating activities in response to endurance
exercise [9]
[10]
[25], compared with endotoxin assessment (e. g.,
LAL chromogenic endpoint assay) alone. This proposal is due to: 1) sample
collection, processing, and analysis procedural issues [26]
[27]
[28];
2) liability of circulating bacterial endotoxins to consistent immune and hepatic
clearance [29]
[30]
[31]; and 3) lack of assessment procedures to globally
detect a vast array of bacterial endotoxins with pathogenic properties [32]
[33]
[34]
[35]. These potential
limitations may mask the full magnitude of exercise-associated endotoxemia and its
contribution as a confounding factor to accurately determine exercise-associated
endotoxemia.
Produced primarily by lymphocyte B cells and/or plasma cells, EndoCAb react
to a number of gram-negative bacterial species antigens [22]. As such, plasma EndoCAb concentration is suggested to be a marker
for systemic endotoxin exposure [36]. Within a healthy
human population, resting plasma concentrations range between 35–250
MU/ml. These values are in accordance with median rangers observed in
healthy blood donors with high antibody titre [37]
[38]. In response to a modest acute
transient endotoxin exposure (e. g.,≤0.3 EU/ml), there is a
transient increase in circulating EndoCAb (e. g., ~100–250
MU/ml), associated with immune activation of lymphocyte-B and/or
plasma cells [36]
[39]
[40]. With a substantial systemic
endotoxin load (e. g., 0.5–1.0 EU/L), as per the case of
sepsis, a depression in EndoCAb is observed (e. g.,≤35
MU/ml), likely attributed to an increase in antibody utilization that
overrides baseline and in-situ production levels. This functional aspect
suggests EndoCAb may play an important part in the overall and correct
interpretation of exercise-associated systemic endotoxin, and subsequent systemic
inflammatory response profile, as part of EIGS assessment [9]
[10]
[25]
[41].
With this in mind, the current study therefore aimed to investigate the impact of
exertional and exertional-heat stress on systemic EndoCAb (i. e., IgG, IgM,
and IgA, collectively) concentration. Based on previous exercise gastroenterology
research outcomes and clinical manifestation of changes to EndoCAb in response to
systemic endotoxin exposure, it was hypothesised that the proposed exertional stress
would result in increased concentrations of EndoCAb, but exertional-heat stress
would result in a depressed response.
Materials and Methods
Participants
Forty-four (n=26 males and n=18 females) individuals trained in
endurance running volunteered to participate in the study and experimental
procedures ([Table 1]). All participants provided
written informed consent, which received approval from the local ethics
committee, and conformed to the 2008 Helsinki Declaration for Human Research
Ethics and meet the ethical standards of the International Journal of Sports
Medicine [42]. The laboratory’s standard
exclusion criteria has been previously reported [17]. For female participants, the experimental trial was scheduled
during the early-mid follicular phase of their menstrual cycle (n=14),
when taking oral contraceptive pill (n=1), or postmenopause
(n=3). Resting estrogen levels (DKO003/RUO; DiaMetra, Italy)
were measured for verification (<50 pg/ml) and did not differ
between trials [43].
Table 1 Descriptive participant characteristics of
volunteers undertaking one of three different experimental running
exercise protocols (P1-HIIT: 2 h high intensity interval training
running with plyometric drop jumps in temperate ambient conditions,
P2-EHS: 2 h steady state running in hot ambient conditions, and
P3-SS: 3 h steady state running in temperate ambient
conditions).
|
All n=44
|
P1-HIIT n=17
|
P2-EHS n=14
|
P3-SS n=13
|
|
Male=26 Female=18
|
Male=10 Female=7
|
Male=9 Female=5
|
Male=7 Female=6
|
|
Age (yr)
|
36 (34 to 39)
|
32 (28 to 36)
|
35 (31 to 39)
|
45 (29 to 50)
|
|
Height (m)
|
1.73 (1.71 to 1.76)
|
1.74 (1.70 to 1.78)
|
1.75 (1.70 to 1.79)
|
1.72 (1.67 to 1.77)
|
|
Body mass (kg)
|
68.5 (65.6 to 71.5)
|
68.7 (63.4 to 73.9)
|
67.3 (61.4 to 73.1)
|
69.7 (64.2 to 75.2)
|
|
Body fat mass (%)
|
18.8 (17.1 to 20.6)
|
18.1 (14.9 to 21.3)
|
18.0 (14.8 to 21.2)
|
20.7 (17.3 to 24.1)
|
|
V̇
O
2max
(min/kg/min)
|
56.8 (54.1 to 59.4)
|
54.5 (51.5 to 57.5)
|
63.2 (57.6 to 68.8)
|
52.8 (48.7 to 56.9)
|
|
Training load (min/week)
|
504 (440 to 568)
|
410 (310 to 509)
|
623 (483 to 762)
|
499 (421 to 577)
|
|
Modality
|
|
Endurance running n=5 Ultra-endurance running
n=7 Triathlon n=5
|
Endurance running n=8 Ultra-endurance running
n=6
|
Endurance running n=13
|
Mean (95% CI)
Preliminary measures
Prior to the first experimental trial, baseline measurements for height
(Stadiometer, Holtain Limited, Crosswell, Crymych, United Kingdom), body mass
and body composition by multi-frequency bioelectrical impedance analysis (MBIA,
Seca 515 MBCA, Seca Group, Hamburg, Germany), and V̇ O2max
(Vmax Encore Metabolic Cart, Carefusion, San Diego, CA, United States) were
recorded; and familiarisation of the running exercise procedures was undertaken.
V̇ O2max was estimated by means of a continuous incremental
exercise test to volitional exhaustion on a motorized treadmill (Technogym,
Cesena, Italy), as previously reported [44]. To
determine running speeds for the respective exercise trials, the speed at
approximately 60% of V̇ O2max at 1% gradient
(10.1±1.6 km/h) was determined and verified from the V̇
O2-work rate relationship. In addition, for the high intensity
interval training simulation (HIIT), the speed at approximately 50
(7.3±1.0) km/h), 55–60 (8.6±1.4 km/h),
70–75 (10.5±1.6 km/h), and 80–85
(12.3±2.0) km/h)% V̇ O2max and
1% gradient was extrapolated and verified.
Experimental procedure
On a separate occasion,≥1 week after the incremental exercise test,
participants were provided with a low FODMAP diet (9.8±2.1
MJ/day (143±31 kJ/kg/day) energy, 351±64
g/day (5.1±0.9 g/kg/day; 62% of total
energy intake contribution) carbohydrate, 92±33 g/day protein
(1.3±0.5 g/kg/day; 16% of total energy intake
contribution), 56±21 g/day fat (0.8±0.3
g/kg/day; 22% of total energy intake contribution),
44±8 g/day fibre, and 2±0 g/day total FODMAP)
the day before the experimental trial to reduce GIS confounded from the lead-in
diet [7]
[45]. Food
and fluid provisions were designed in accordance with current nutrition
guidelines for endurance athletes, with estimated total daily energy expenditure
determined via the Cunningham equation, and adjusted for a resting non-training
day [46]. In addition, dietary provisions were
aimed to provide<2 g FODMAP per meal using a FODMAP specific database
(Monash University, FoodWorks Professional 7, Xyris, Brisbane, Australia) [7]
[45]. Participants
reported to the laboratory at 0800h after consuming the standardised pre-trial
low FODMAP meal (2.9±0.7 MJ, 99±28 g carbohydrate, 25±6
g protein, 20±5 g fat, 11±4 g fibre, 1±1 g total
FODMAP), consumed at 0700h. The meal was consumed 2 h prior to the start of
exercise, simulating real-life translational practice in the target population
[47]
[48]. A
dietary log containing the prescriptive diet determined ingestion compliance and
food waste. Participants refrained from strenuous exercise 48 h before the
respective trial.
Prior to the commencement of exercise at ~0900h, participants were asked
to void before nude body mass measurement, and MBIA to determine total body
water (TBW). Blood samples were then collected by venepuncture from an
antecubital vein into lithium heparin (6 ml, 1.5 IU/ml heparin) and
K3EDTA (4 ml, 1.6 mg/ml EDTA) vacutainers. Pre-exercise
resting rectal temperature (Tre) was then recorded, with participants
inserting a thermocouple 12 cm beyond the external anal sphincter (Alpha
Technics Precision Temperature 4600 Thermometer, Oceanside, CA). As part of
exercise gastroenterology intervention studies reported elsewhere [7]
[11]
[49]
[50]
[51], participants undertook one of three endurance
exercise protocols ([Fig 1]).
Fig 1 Schematic illustration of the three experimental exercise
protocols. P1-HIIT: 2 h high intensity interval training running with
plyometric drop jumps in temperate ambient conditions, P2-EHS: 2 h
steady state running in hot ambient conditions, and P3-SS: 3 h steady
state running in temperate ambient conditions. Abbreviations: FODMAP,
fermentable oligo-, di-, monosaccharides and polyols; HR, heart rate;
RPE, rating of perceived exertion; TCR, thermal comfort rating; GIS,
gastrointestinal symptoms; Tamb, ambient temperature;
VO2max, maximal oxygen uptake; BM, body mass; w/v, water
volume equivalent.
Protocol 1 (P1-HIIT): Starting in a euhydrated state (plasma osmolality:
291±8 mOsmol/kg and TBW: 60.7±3.4%) participants
(n=17) undertook 2 h high intensity interval running exercise (HIIT)
session in temperate ambient conditions (ambient temperature (Tamb)
23.4±0.9°C and 43±7% relative humidity (RH)),
with dual fan wind speed set at 10.6 km/h. The protocol involved 3 rounds of
running for 3.5 min at 55–60% V̇ O2max, 1 min
of running at 65–70% V̇ O2max, and 30-s
running at 75–80% V̇ O2max, followed by 20
plyometric drop (50 cm) jumps of alternating legs. Participants then returned to
the treadmill to walk until the 20-min cycle was completed. This was repeated 6
times. During exercise, participants were provided with room temperature water
equivalent to 3 ml/kgBM/h (208±32 ml/h). Total distance over the 2-h
protocol was 16.2±2.5 km with 120 plyometric drop jumps.
Exercise-associated body mass loss and post-exercise plasma osmolality were
1.8±1.0% and 293±10 mOsmol/kg, respectively.
Protocol 2 (P2-EHS): Starting in a euhydrated state (plasma osmolality:
296±4 mOsmol/kg and TBW: 59.4±3.5%) participants
(n=14) undertook 2 h of exertional-heat stress (EHS), comprising running
exercise on a motorised treadmill at the previously determined speed
representing 60% V̇ O2max in hot ambient conditions
(Tamb 35.7±0.9°C and
23.3%±3.2% RH), with dual fan wind speed 10.6 km/h. Room
temperature water was provided ad libitum (662±279 ml) for autonomy over
drinking patterns to minimise programmed drinking induced occurrence of GIS.
Total distance over the 2-h protocol was 21.5±3.3 km.
Exercise-associated body mass loss and post-exercise plasma osmolality were
2.1±0.9% and 297±6 mOsmol/kg, respectively.
Protocol 3 (P3-SS): Starting in a euhydrated state (plasma osmolality:
293±9 mOsmol/kg and TBW: 57.5±3.5%) participants
(n=13) undertook 3-h steady state (SS) running exercise on a motorised
treadmill at the previously determined speed representing 60% V̇
O2max in temperate ambient conditions (Tamb
23.1±1.2°C and 43.6±5.5% RH), with dual fan wind
speed set at 10.6 km/h. During exercise, participants were provided with a room
temperature carbohydrate (dextrose-fructose solution) beverage containing
64±15 g/h, 10% w/v, 509 mOsmol/kg) at 0 min and every 20 min
thereafter for the first 2 h, and allowed to consume water ad libitum. Total
water intake during the first 2h equated to 650±147 ml/h. In the
3rd hour, room temperature water was provided ad libitum,
equating to 276±15 ml. Total distance over the 3 h protocol was
27.3±2.1 km. Exercise-associated BM loss and post-exercise plasma
osmolality were 1.1±0.4% and 292±9 mOsmol/kg,
respectively.
The exercise protocols were employed in accordance with previous exercise
experimental models known to disturb gastrointestinal epithelial integrity to
levels of clinical and performance significance. Standard physiological strain
variables (i. e., Tre, heart rate (HR), rating of perceived
exertion (RPE), McGinnis 13-point thermal comfort rating (TCR), and body mass)
were measured at regular intervals during running, as previously reported [7]
[11]
[49]
[50]
[51]. Immediately after exercise, a blood sample was
collected and nude body mass measured, as previously described.
Sample analysis
Whole blood hemoglobin was determined by a HemoCue system (Hb201; HemoCue,
Ängelholm, Sweden), and hematocrit was determined by the capillary
method with a microhematocrit reader (ThermoFisher Scientific), both from
heparin whole blood samples. Haemoglobin and haematocrit values were used to
estimate changes in plasma volume (PV) relative to baseline and used
to correct plasma variables. The remaining heparin and K3EDTA whole
blood samples were centrifuged at 4000 rpm (1500 g) for 10 min within 15
min of sample collection. Plasma was aliquoted into 1.5 ml micro-storage tubes
and frozen at –80°C until analysis, except for 2 x 50 µl
heparin plasma that was used to determine plasma osmolality (POsmol)
in duplicate (CV: 1.7%) by freezepoint osmometry (Osmomat 030, Gonotec,
Berlin, Germany). Plasma concentration of endogenous endotoxin core antibodies
(EndoCAb) IgM, IgA, and IgG were determined by ELISA (EndoCAb, HK504, Hycult
Biotech, Uden, Netherlands). All variables were analysed in duplicate as per
manufacturer’s instructions, with standards and controls on each plate,
and each participant assayed on the same plate. The CV for plasma IgM, IgA, and
IgG were 7.9%, 7.7%, and 13.2%, respectively.
Data analysis
Confirmation of adequate statistical power for the primary research are
previously described [7]
[11]
[49]
[50]
[51]. Participants and researchers
at the time of data collection were unaware that the data would be used for
analysis of pre- and post-exercise plasma anti-endotoxin antibody concentration
in response to various exertional and exertional-heat stress protocols. Based on
the statistical test, mean, standard deviation, and effect size; and applying a
standard alpha (0.05) and beta value (0.80) the current participant sample size
is estimated to provide adequate statistical power
(power* 0.80–0.99) for detecting significant exercise
magnitude and sub-group differences (G*Power 3.1, Kiel, Germany), and is
in accordance with participant sample sizes previously used to explore
gastrointestinal epithelial integrity biomarkers for EIGS [9]
[10]
[25]
[52]
[53]. Descriptive data in text are presented as
mean±standard deviation (SD). Primary and secondary variable data in
text and tables are presented as mean and 95% confidence interval,
unless otherwise indicated. For clarity, data in figures are presented as
individual responses and mean. All data were checked for normal distribution by
Shapiro-Wilks test of normality, prior to applying appropriate parametric or
non-parametric statistical tests. Paired sample t-tests or non-parametric
equivalents (Wilcoxon signed-rank) were used to assess pre- to post-exercise
anti-endotoxin antibody concentration within exercise bouts. One-way ANOVA or
non-parametric equivalents (Wilcoxon signed-rank) with post hoc analysis, were
used to assess pre-exercise anti-endotoxin antibody concentration and ∆
pre- to post-exercise response magnitudes between exercise bouts. Statistics
were analysed using SPSS statistical software (v.27.0, IBM SPSS Statistics, IBM
Corp., Armonk, NY, USA) with significance accepted at p<0.05.
Results
Dietary and exercise control
No significant difference was observed between foods and fluids provided to
participants and actual consumption of these food and fluids in P1-HIIT, P2-EHS,
and P3-SS. Overall,>95% of the foods and fluids provided to
participant were consumed within the three experimental designs. All
participants confirmed they refrained from strenuous exercise for 48 h before
each trial.
Physiological and thermoregulatory strain
A significant change was observed during P1-HIIT for RPE (p<0.001), but
not HR (160 (157 to 163) bpm) or TCR (8 (8 to 9)), with RPE increasing from the
first 20-min HIIT cycle (11 (10 to 11)) to cycle 4 (80-min; 13 (12 to 14)) and
onwards (120-min; 14 (12 to 15)). Tre was measured pre- and
immediately post-exercise, with a significant increase (p<0.001)
observed from pre- (36.5 (36.1 to 36.8)°C] to post-exercise (37.9 (37.6
to 38.1)°C).
A significant change was observed for RPE (p=0.02) during P2-EHS with a
significant increase at the end of exercise (120 min) (13 (11 to 14)) compared
to 15 min into exercise (10 (9 to 11)). From pre-exercise resting values (37.0
(36.8 to 37.3)°C), Tre significantly increased from 30-min
exercise until the end of exercise (peak Tre: 38.9 (38.5 to
39.4)°C) (p<0.001). HR increased from 144 (134 to 153) bpm
(15-min) to 158 (145 to 172) bpm (120-min), but the increase was not
significant. TCR remained constant throughout P2-EHS (9 (9 to 9)).
A significant change was observed for RPE (p=0.002) and Tre
(p=0.036) on P3-SS. RPE increased from 90 min into exercise until
completion (13 (12 to 15)) compared with 30 min (11 (10 to 11). Tre
increased in the last 30 min of exercise until completion (peak Tre:
38.6 (38.3 to 38.8)°C) compared with pre-exercise (36.9 (36.7 to
37.1)°C). HR increased from 133 (128 to 138) bpm (15 min) to 141 (134 to
148) bpm (180 min), but the increase was not significant. TCR remained constant
throughout P3-SS (8 (7 to 8)).
Effect of exercise protocols on plasma EndoCAb concentration
Pre- and post-exercise plasma concentrations for each respective protocol are
described in [Table 2]. Overall resting
pre-exercise levels for plasma IgM, IgA, and IgG were (mean and 95% CI),
173 (132 to 214) MMU/ml, 37 (29 to 44) AMU/ml, and 79 (48 to
109) GMU/ml, respectively. Resting pre-exercise plasma IgM concentration
significantly differed between protocols (p=0.035); whereby, P2-EHS
presented the lowest mean baseline values ([Table
2]). No significant differences were observed between protocols for
pre-exercise plasma IgA (p=0.742) and IgG (p=0.308)
concentrations. In P1-HIIT and P3-SS, plasma concentrations of all EndoCAb did
not significantly differ from pre- to post-exercise. In P2-EHS, plasma
concentrations of anti-endotoxin antibodies of IgA (p=0.017) and IgG
(p=0.016), but not IgM (p=0.158), significantly increased from
pre- to post-exercise.
Table 2 Pre- and post-exercise anti-endotoxin antibody
plasma concentrations across the three different experimental
running exercise protocols (P1-HIIT: 2 h high intensity interval
training running with plyometric drop jumps in temperate ambient
conditions, P2-EHS: 2 h steady state running in hot ambient
conditions, and P3-SS: 3 h steady state running in temperate ambient
conditions).
|
P1-HIIT
|
P2-EHS
|
P3-SS
|
|
n=17
|
n=14
|
n=13
|
|
IgM (MMU/ml)
|
IgA (AMU/ml)
|
IgG (GMU/ml)
|
IgM (MMU/ml)
|
IgA (AMU/ml)
|
IgG (GMU/ml)
|
IgM (MMU/ml)
|
IgA (AMU/ml)
|
IgG (GMU/ml)
|
|
Pre-exercise
|
229 (150 to 308)
|
32 (22 to 42)
|
90 (21 to 160)
|
106aa (87 to 125)
|
40 (22 to 57)
|
54 (27 to 80)
|
173 (82 to 263)
|
39 (26 to 52)
|
90 (31 to 149)
|
|
Post-exercise
|
215 (135 to 294)
|
32 (21 to 43)
|
169 (-70 to 408)
|
127 (92 to 162)
|
49* (28 to 69)
|
80* (40 to 121)
|
133 (87 to 178)
|
39 (28 to 50)
|
91 (9 to 172)
|
Mean (95% CI): * p<0.05 vs. pre-exercise;
aa
p<0.01 vs. P1-EHS.
Plasma EndoCAb concentration between protocols
[Fig 2] illustrates the mean and individual
participant magnitude of pre- to post-exercise change for plasma concentrations
of anti-endotoxin antibody between protocols. There was no significant
difference in the magnitude of pre- to post-exercise change for plasma IgM
concentration between protocols (p=0.135). There was no significant
difference, but a trend in the magnitude of pre- to post-exercise change for
plasma IgA concentration between protocols (p=0.058); whereby a greater
change was seen in P2-EHS, compared with P1-HIIT and P3-SS. A significant
difference between protocols for the magnitude of pre- to post-exercise change
for plasma IgG concentration was observed (p=0.037). However, when
significance was adjusted by the Bonferroni correction for multiple tests, no
significant difference existed between the magnitude of pre- to post-exercise
change for anti-endotoxin antibody IgG across the protocols
(p>0.05).
Fig 2 Magnitude of pre- to post-exercise change in plasma
concentrations of endogenous endotoxin core antibody (EndoCAb) IgM (A),
IgA (B), and IgG (C) across the three different experimental running
exercise protocols (P1-HIIT: 2 h high intensity interval training
running with plyometric drop jumps in temperate ambient conditions,
P2-EHS: 2 h steady state running in hot ambient conditions, and P3-SS: 3
h steady state running in temperate ambient conditions). Mean and
individual responses (n=44). Outlier removed from [Fig 2C] (n=1): P1-HIIT
IgG=1741 GMU/ml.
Plasma EndoCAb by biological sex
Pre-exercise anti-endotoxin antibody concentrations of IgM, IgA and IgG were the
same in both male and female participants across the three exercise protocols.
There was a significant difference between biological sex in plasma
concentration of anti-endotoxin antibody IgA (p<0.05), but not IgM or
IgG, in response to exercise, with a mean increase in female participants (8.61
(2.48 to 14.70) AMU/ml) and a decrease in male participants
(–1.34 (–5.57 to 2.90) AMU/ml) from pre- to
post-exercise ([Fig 3]).
Fig 3 Magnitude of pre- to post-exercise change in plasma
concentrations of endogenous endotoxin core antibody (EndoCAb) IgM (A),
IgA (B), and IgG (C) by biological sex. Mean and individual responses
(n=44): p<0.05 vs biological sex.
Outlier removed from [Fig 3C]
(n=1): male IgG=1741 GMU/ml.
Discussion
The current study aimed to investigate the impact of exertional and exertional-heat
stress on systemic EndoCAb concentration. To our knowledge this is the first to
comprehensively assess EndoCAb responses to a variety of exercise stress models and
using rigorous control to avoid artificial impact of confounders know to perturb the
epithelial integrity of the gastrointestinal tract in response to exercise. Contrary
to our hypothesis, all systemic anti-endotoxin classed antibodies measured in this
current study were not substantially impacted with exertional stress, characterised
by 3-h steady state and 2-h high intensity interval running exercise. Instead of the
expected reduction in all EndoCAb Ig in response to exertional-heat stress, a
significant increase was observed for plasma IgA (Δ pre- to post-exercise (9
(2 to 16) AMU/ml)) and IgG (27 (3 to 50) GMU/ml) concentrations, but
not for plasma IgM concentration (21 (-6 to 47) MMU/ml). However, the
magnitude of change for IgA and IgG was modest and within normative values for a
resting healthy human population [37]
[38]. The current findings suggest such indirect
biomarkers to describe the magnitude of translocation of intestinal lumen pathogenic
endotoxin into systemic circulation are not particularly sensitive to exercise
stress. This is in the context of: 1) the rigorous confounder control within the
current study (i. e., pre and during exercise food and fluid intake,
hydration status, circadian variation, ambient conditions, and female menstruation
cycle, measurement of physiological and thermal strain known as EIGS exacerbation
factors) [3], 2) the diversity in exercise
experimental models used, and 3) the overall physical strain of the experimental
models being synonymous with promoting EIGS [1]
[2]. Moreover, although significant differences in
EndoCAb responses were observed between males and females (i. e., biological
sex differences), the magnitude of difference appears modest, within normative
reference values for resting healthy human populations, and of no clinical
relevance.
Within a healthy individual, plasma concentrations of EndoCAb at rest are sufficient
for immunocompetence and range from an arbitrary value of 35 to 250 median-units
(MU)/ml, based on healthy blood donors with high Ig titres [37]
[38]. EndoCAb
responses in systemic circulation primarily act to tag microbial endotoxins
(e. g., rough and smooth LPS and/or lipid-A from various pathogenic
bacteria such as E.coli) for immune cell detection, neutralisation
and/or clearance by innate and/or adaptive immune responses [22]
[54]
[55]. For example, the mechanistic explanation for
EndoCAb activity may include Fc receptor antibody-dependant immune activation of
phagocytes (i. e., IgA), complement-dependant cytotoxicity (i. e.,
IgM and IgG), and/or antibody-dependant cell-based cytotoxicity
(i. e., IgA and IgG) [56]
[57]
[58]. It has
previously been documented that in response to a modest acute transient endotoxin
exposure (e. g.,≤0.3 EU/ml, equivalent to 30 pg/ml),
there is a transient increase in circulating EndoCAb (e. g., IgM≥250
MU/ml and IgG ~100–250 MU/ml), due to the
circulating endotoxin exposure activation, generally irrespective of the bacterial
species origins of the endotoxin [36]
[39]
[40]. Whilst, a
substantial systemic endotoxin load (e. g., 0.5–1.0 EU/L,
equivalent to 50–100 pg/ml), as per the case of sepsis, a depression
in EndoCAb is observed (e. g.,≤35 MU/ml), likely attributed
to the increased antibody utilization that overrides baseline starting and
in-situ production levels [36]
[39]
[40]
[59]. Therefore, clinical relevance is indicated
at<35 MU/ml, suggesting a state of immunosuppression as a result of
Ig consumption>production rate in response to illness and/or
pathogenic infection [36]. In the current study,
overall resting pre-exercise levels for plasma EndoCAb IgM, IgA, and IgG were 173
(132 to 214) MMU/ml, 37 (29 to 44) AMU/ml, and 79 (48 to 109)
GMU/ml, respectively. Therefore, values appear to be within normative health
ranges, although it is recognised and accepted that plasma IgA concentrations are
generally lower that those presented for IgM and IgG, due to IgA’s
predominant role within the gastrointestinal tract lumen (e. g., secretion
of IgA into lumen through epithelial cells), and not necessarily within internal
circulation [60]. The inclusion of IgA and IgG in the
current study are a novel contribution to scientific literature, while pre-exercise
resting values for IgM are similar to previous studies; whereby, mean values of 90
to 127 MMU/ml have been reported [9]
[10]
[25]. Although the
pre-exercise resting levels of EndoCAb differed substantially between the protocols
within the current study and previous studies, they were within resting normative
ranges.
Circulating levels of EndoCAb are reported to increase in response to mild endotoxin
presence in systemic circulation, but depress in response to severe and exaggerated
endotoxin load [36]
[59].
These clinical characteristics between endotoxemia and EndoCAb responses have also
been observed in exercise research. Whereby, prolonged duration endurance events
(e. g., triathlon and marathon) and ultramarathon events have resulted in a
detectable systemic endotoxin load with a concomitant reduction in EndoCAb IgM
and/or IgG [20]
[21]
[22]
[23],
in adjunct with altered systemic inflammatory cytokines, mimicking SIRS [13]. It is therefore expected that exertional
and/or exertional-heat stress of sufficient magnitude would show similar
outcomes. Laboratory control studies that used 2-h exertional-heat stress
(60% V̇ O2max, 35°C Tamb), and
reported a modest plasma endotoxin load post-exercise (Δ 9.6 pg/ml),
also observed a modest reduction in anti-endotoxin antibody IgM (12%),
concomitant with a greater systemic inflammatory response, compared with exercise
at
20°C and 30°C Tamb
[9]
[10]
[25]. Apart from the
exertional-heat stress model with water consumption to maintain euhydration [61], all other exercise models, with and without
nutrient feeding during, showed pre- to post-exercise increases in plasma IgM
concentration. Other studies have used more subtle stress models, and subsequently
have reported none to very modest disturbances to pre- to post-exercise
anti-endotoxin antibody values (e. g., 30-min 60–65% heart
rate reserve, up to 90 min walking in temperate and hot ambient conditions [62]
[63]
[64]. These observations suggest exertional-heat stress
and duration of exertional stress may play a key role in the magnitude of
exercise-associated disturbance to gastrointestinal integrity, and subsequently the
translocation magnitude of intestinal lumen originating microbial pathogenic
agents.
In the current study, 2 h of high intensity interval running with plyometric jumps
did not promote substantial changes to circulating EndoCAb values. This is not
surprising considering this exercise stress load has been reported to result in no
substantial change to sCD14, LBP, and SIR-profile [49]
[50]
[51].
Similarly, 3 h steady state running did not result in any substantial changes to
circulating EndoCAb values, even though IgM reduced by 23% pre- to
post-exercise, but was not to a significant extent and showed large individual
variation. These outcomes were surprising considering this exercise stress load has
previously been reported to result in substantial increases in pre- to post-exercise
sCD14, LBP, and SIR-profile [11], suggesting some
evidence of exercise-associated endotoxemia, but a lack of EndoCAb responding
accordingly. Finally, 2 h of exertional-heat stress increased all EndoCAb to a
modest degree, with IgM failing to reach significance. These outcomes were also
surprising considering previous research using that same exercise and heat load has
previously reported depressed IgM responses [9]
[25]. It is however important to note that the peak
Tre and TCR in previous research was greater (39.6°C and
11-very hot, respectively), compared with the current study (i. e.,
38.9°C and 10-hot) that did not reach the target threshold established to
promoted substantial gastrointestinal integrity perturbations
(circulatory-gastrointestinal pathway) synonymous with EIGS
(i. e.,≥39.0°C); thus, potentially providing some insight
into the differences in EndoCAb outcomes, namely IgM. Nevertheless, exertional-heat
stress of the current study was accompanied by modest increases in pre- to
post-exercise sCD14, LBP, and SIR-profile [7],
suggesting a mild endotoxin exposure and not synonymous with sepsis associated
EndoCAb systemic release and consumption [36].
Collectively and from a research and practice perspective, considering pre- and
post-exercise plasma EndoCAb Ig concentrations were within the normative values, it
appears even strenuous prolonged exercise experimental protocol, with or without
additional heat strain, do not substantially push EndoCAb to clinical relevance
(e. g., activation at>250 MU/ml or suppression at<35
MU/ml), which have only been observed in ultra-endurance field events.
In conclusion, exertional and exertional-heat stress, synonymous with EIGS and
perturbations to intestinal epithelial integrity leading to pathogenic bacterial
endotoxin translocation into systemic circulation and subsequent systemic
inflammatory responses, resulted in modest disturbances to circulating EndoCAb
concentration. Both pre- and post-exercise values were within normative ranges for
a
healthy population, suggesting the exercise-associated magnitude of change of
EndoCAb biomarkers (i. e., IgM, IgA, and IgG) presents a low response
sensitivity to a variety of exertional and exertional-heat stress loads. Within the
EIGS, the previous suggestion to use EndoCAb Ig as a marker to detect endotoxin
exposure in systemic circulation should be used with caution within an exercise
model and as a supportive biomarker instead of a primary biomarker.
