Introduction
Increases in core temperature, and heat stress along with intestinal hypoperfusion, and ischaemia–reperfusion (I–R), contribute to intestinal injury and changes in permeability during exercise [
1‐
7]. These physiological responses are likely exacerbated when exercising in hot and humid environments. Moreover, exercise in the heat is both common and necessary in many sport (for athletes) or work contexts (e.g. military, firefighters). However, under extreme circumstances exhaustive exercise in such environments can result in severe endotoxaemia (the translocation of bacterial lipopolysaccharide (LPS) into the central circulation) [
8,
9], through intestinal injury and changes in permeability, which is thought to be associated with acute inflammation, sepsis, shock and organ failure that in rare cases may be fatal.
Nutritional interventions to maintain the integrity of the intestinal barrier and, therefore, avoiding these complications during and following exercise in the heat are somewhat limited. However, bovine colostrum (Col) has shown to be both effective in blunting the heat-induced increase in permeability in vitro and in vivo in animals and humans [
1,
2,
10,
11]. Previously Col has blunted both the exercise-induced increase in intestinal permeability and circulating intestinal fatty acid-binding protein (I-FABP) (a marker of intestinal cellular injury) in the exercise stress model in humans where an increase of ~ 1.5 °C in core temperature was observed [
1,
2,
11].
It is important to note, however, that this increase (following 20 min of running at 80%
\({\dot {\text{V}}}{\text{O}_{2\text{peak}}}\)) may be relatively mild considering that strenuous athletic events in the heat can evoke much higher elevations in core temperatures [
12,
13]. The implications of changes to the intestinal barrier during exercise and heat is an issue of increased significance as there is a global increase in ambient temperatures during athletic competition [
14].
We have previously shown that changes in plasma I-FABP immediately following treadmill running occur concurrently (and correlate) with changes in intestinal permeability as measured by the 5-h urinary excretion ratio between lactulose and rhamnose (L/R) [
2], and also that both are blunted by Col supplementation. However, a recent study [
15] has indicated that when core temperature is elevated above 39 °C during exercise then Col may not be effective in blunting the increase in plasma I-FABP. Although, in this study [
15] there were variable running times between the two conditions [placebo (Plac) and Col], imposing different stresses that may have compromised the validity of these results.
Some previous investigations have quantified changes in circulating LPS following exercise [
16,
17] to indicate changes in intestinal permeability. However, there are issues pertaining to the collection and analysis of blood samples [through the limulus amoebocyte lysate assay (LAL)], in addition to the detection of LPS, suggesting that this marker may not be ideal [
18,
19]. Some clinical studies have measured intestinal-derived circulating bacterial DNA via 16S rDNA PCR assays to indicate changes in intestinal permeability [
19‐
22], which has been suggested to be more specific to targeted bacterial strains and can overcome some of the sensitivity and specificity issues that exist for the LPS LAL assays. However, circulating bacterial DNA (as a marker of bacterial translocation) has not previously been assessed within an exercise and heat stress model.
Therefore, the primary aim of this study is to determine whether 14 days of oral Col supplementation can blunt the heat- and exercise-induced increase in intestinal injury measured by plasma I-FABP. The secondary aim was to determine the effects on circulating bacterial DNA, as a marker of bacterial translocation. It is hypothesised that exercise and heat would present a larger challenge to intestinal integrity (greater increase in plasma I-FABP) and intestinal permeability than we observed in exercise alone in our previous work [
2], resulting in a large increase in this injury marker and circulating bacterial DNA. However, it is hypothesised that Col supplementation would blunt these increases.
Discussion
The main findings of this study are that (1) plasma I-FABP increased when running at 70%
\({\dot {\text{V}}}{\text{O}_{2\text{peak}}}\) in the heat for 60 min, (2) 14 days of oral Col supplementation was able to blunt the increase in plasma I-FABP, but did not influence the exercise response of plasma Bacteroides/total 16S rDNA (although there was a main effect of arm). This is the first study to demonstrate the efficacy of Col in vivo on intestinal injury in exercising humans within a heat and exercise stress model and expands on our previous work showing the benefit of Col on the intestines following exercise [
1,
2,
11].
The increase in plasma I-FABP would suggest that intestinal barrier integrity in the current study was compromised as changes in plasma I-FABP have previously been shown to be associated with increases in permeability [
2]. Furthermore, these increases in plasma I-FABP following exercise were to a greater extent than observed in our previous study during thermoneutral conditions [
2], and may be associated with changes in circulating Bacteroides/total 16S rDNA. The 407 ± 194% increase in plasma I-FABP was over double the increase we have observed previously [
2], where we reported a concurrent increase in intestinal permeability as measured by urinary L/R (5 h). Previous investigations reporting either significant increases in core temperature or an end core temperature > 39 °C (or both as seen in the present study) with treadmill running observed significant changes in permeability as measured by urinary L/R (5 h) [
2,
3,
11,
34]. A recent study has suggested that when heat stress induces increase in core temperature to greater than 39 °C then Col may not be efficacious [
15]. However this study [
15] had significant limitations: they only reported a 162% increase in plasma I-FABP despite exercising at higher ambient temperature than the current study (40 vs 30 °C), which was also reflected in a marginal greater rise in core temperature. Furthermore, the increase (162%) in I-FABP previously reported [
15] is one of the lowest reported in the literature, lower than reported increases following high intensity interval training (172%) [
32], following exercise in the heat (168%) [
35], and following cycling in a controlled thermoneutral condition (172%) [
31]. This relatively low post-exercise increase in plasma I-FABP may explain their lack of reported effect [
15] of Col supplementation. Moreover, McKenna et al. [
15] reported high pre-exercise levels of plasma I-FABP which may also offer an explanation, and finally but perhaps most significantly, there were variable running times between the two conditions, imposing different stresses between conditions (placebo and Col), which likely confound their results.
Running in the heat causes a greater redistribution of blood flow towards the cutaneous region to dissipate heat resulting in greater hypoperfusion, and ischaemia in the intestines than exercising in temperate conditions [
36]. The increase in plasma I-FABP during exercise appears to be as a result of increases in core temperature, hypoperfusion and I-R [
2,
5,
31]. Both comparable physiological temperatures (and lower) as reported in the present study, and hypoperfusion and subsequent ischaemia result in tight junction (TJ) breakdown [
37,
38]. Col has been previously shown to blunt the in vivo increase in intestinal permeability in animals heated to a core temperature of 41.5 °C [
10]. The mechanism for the efficacy of Col is through an up-regulation of TJ proteins (such as Claudin-1, Claudin-2 and zonula occluden-1 protein), and its ability to induce favourable changes in caspase-3, caspase-9, Baxα and Bcl-2 as has been demonstrated in vitro in human cell lines [
1,
11,
39]. Therefore, Col is able to upregulate TJ proteins, maintaining cell to cell contact (and thus cellular integrity under stress) preventing cellular damage, as indicated by a blunting of plasma I-FABP in the Col arm. Moreover, direct protective cellular effects (e.g. up-regulation of heat shock protein [HSP]) prompted by Col, which has previously been demonstrated [
1,
11], may also blunt the increase in plasma I-FABP.
Recent studies have shown that significant rises in core temperature [measured at the rectum and oesophagus (approx. 1.5–2 °C)] during exercise in the heat result in significant elevations in plasma I-FABP [
2,
15,
40]. The larger increase in core temperature (measured at the rectum) in the present study (~ 2.5 °C) in comparison with our previous work (1.5 °C increase) [
2], was also reflected by greater increases in plasma I-FABP. Not only does this highlight the stress that both exercise and heat place on the intestine (in accordance with changes in circulating I-FABP) but furthermore shows that this dose and timing (20 g day
−1 for 14 days) of Col supplementation are only able to partly blunt intestinal injury during exercise with combined heat stress. This finding is in agreement with our results from a series of previous investigations [
1,
2,
11] showing that this supplementation regime cannot fully blunt the increase in intestinal damage during exercise. This also supports the notion that combinations of supplements to confer further benefit against intestinal damage in the exercise (and heat) stress model may be more efficacious [
1].
Consistent with previous studies [
5,
41], plasma I-FABP levels approximated pre-exercise levels in the current study 60 min following the termination of exercise which likely underlines the ability of the intestines to withstand short periods of reduced perfusion (ischaemia) and to rapidly restore the intestinal barrier (restitution). Full restoration of the intestinal barrier through intestinal cell migration 60 min after a 30 min ischaemic period has previously been reported [
42]. However, even these comparatively short periods of intestinal barrier injury (where permeability is increased) are likely still of clinical relevance. For example, LPS translocation has previously been described following exercise of shorter duration than the present study [
43]. It is possible that Col could enhance the migration of intestinal cells in this post-ischaemic period which has been previously shown in vitro [
44].
In the present study, and previously we have shown that Col supplementation can blunt both the exercise-induced increase in intestinal permeability as measured by the urinary L/R (5 h) [
1,
2,
11] and cellular damage as indicated by plasma I-FABP [
2]. However, it may be considered more pertinent to investigate the effect of Col supplementation on the consequence of this increase in permeability (i.e. bacterial translocation) as this is associated with endotoxaemia and in some cases (although rare) severe outcomes. Although the aforementioned issues pertaining to the collection and analysis of blood samples, in addition to the detection of LPS (some components of human plasma can interfere with the LAL assay resulting in false positives), suggest that the use of this marker to assess the impact of intestinal permeability changes is less than ideal [
18,
19]. Therefore, in the present study we measured the ratio of Baceteroides to total bacterial DNA. Bacteroides account for around 25% of the anaerobes found in the gastrointestinal tract, and are the most prevalent bacteria in the gut [
45]. In the present study, Bacteroides/total 16S rDNA appeared to be lower overall following 14 days of Col supplementation which may indicate a direct effect of Col on intestinal permeability and subsequent bacterial translocation but it must be noted that there was no arm × time interaction showing the overall response to exercise was not influenced by Col. There was large inter-participant variation in this measure, and whilst this method appears advantageous in comparison with LPS assays (e.g. LAL) due to specificity for a particular bacterial strain, it may require a larger sample size to detect the magnitude of effect observed in this study. Alternatively, it may be valuable to also measure other abundant gut bacterial species at the same time to further explore the effects of exercise and Col. This is the first study to assess the effect of Col supplementation in an exercise and heat stress model with measures of bacterial translocation using the nucleic acid testing (NAT) model for bacterial DNA. It provides further evidence that 14 days of Col supplementation can reduce the increase in exercise-induced intestinal permeability, and preliminary evidence that this may reduce bacterial translocation, but the latter requires further research (i.e. with a larger sample size and/or adding other bacterial strains to the detection panel). This study and others [
2,
30‐
32] have observed a large inter-participant variability (Plasma I-FABP was highly variable in both arms) in plasma I-FABP; therefore, future investigations should endeavour to understand how plasma I-FABP is influenced by factors such as diurnal variation and how it changes in the post-prandial period.
A limitation of the present study is that we did not attain training history or current training status from participants, it has been speculated that regular training may be protective against exercise-induced changes in intestinal permeability through an up-regulation in HSP [
22,
46]. However, this has yet to be reliably demonstrated [
47], and furthermore in the current study there was little variation for
\({\dot {\text{V}}}{\text{O}_{2\text{peak}}}\) values (SD < 5 mL kg
−1 min
−1) indicating a similar level of aerobic fitness for participants in the study (whom were all regular exercisers). Finally, the protocol employed in this study required participants to exercise at the same relative intensity, therefore, imposing a similar physiological stress between participants despite small variances in fitness through training status.