Key words
exercise - immunoglobulin - nutrition - firefighters - training
Introduction
Intensive physical training alters body immune/inflammatory responses [1], predisposing athletes to infections and injuries with a deleterious impact on training
and performance [2]. The control of changes in the immune system is necessary to avoid pathological
adaptations. Immunoglobulins protect against antigens (i. e. viruses and bacteria,
or deleterious substances produced by the body itself) [2]. Five classes of immunoglobulins are formed by plasma cells: IgG, IgM, IgA, IgD,
and IgE, all having different properties and functions [3]. Most serum immunoglobulins are IgG (75%), followed by IgA (15%) and IgM (10%) [4]
[5], which are the main focus for this study. IgM is the first antibody to be expressed
and produced (main functions: activating complement and agglutination, and has isohaemagglutination
activity). IgG is the most abundant antibody in the blood and does the most in terms
of protection (main functions: opsonization, agglutination, placental transfer, antibody-dependent
cell-mediated cytotoxicity. and neutralization of toxins). IgA is the most abundant
immunoglobulin in the mucosal system (main functions: agglutination and antiviral
capacity). They are crucial components of humoral immunity, with pathogen neutralization
and opsonisation functions occurring during primary and/or secondary antibody responses
[2]
[3]
[4]
[5]
[6]. They also regulate cellular cytotoxic activity via sensitisation of natural killer
cells, phagocytes, and mast cells, and promote inflammatory responses and clearance
of immune complexes via activation of the complement system [2]
[6].
The literature review on immunoglobulin responses makes it possible to distinguish
between acute effects and chronic adaptations induced by different types of exercises
[2]
[7]
[8]. There seems to be a well-established pattern: the more intense and prolonged the
exercise, the more marked the acute response and the longer the recovery of baseline
levels of the immune system [2]. Endurance training increases IgA and IgM basal levels [1]. Moderate exercise tends to stimulate IgG production. However, master athletes engaged
in high-intensity and long-duration training, recreational athletes training at moderate
intensity and duration, and sedentary individuals presented similar IgG, IgA, and
IgM serum concentrations [1]. It was also evidenced that low- to moderate-intensity exercises increase IgA basal
levels [9]
[10] and that IgG and IgM are maintained at a high level across a training period [9]. Conversely, high-intensity training promoted an IgG and IgM decrease whereas IgA
remained stable [11], and a bout of 60 min downhill running (14 days after the first one) induced low
IgG and IgE serum concentrations (chronic effect) [2]. In addition, it was observed that some acute alterations induced by intensive physical
exercise are no longer visible after a few days of training [12]. Thus, even if systematic training seems to dampen the autoimmune inflammatory response,
there is an evident discrepancy among studies, probably due to exercise mode and training
specificity.
Micronutrient supplementation is widely used as a usual practice in disease and sport.
In patients with diabetes, it improved cellular immunity without significant effects
on IgA, IgM, and IgG serum concentrations [13] and, in healthy elderly sedentary persons, a milk-based wolfberry formulation increased
the serum IgG level [14]. Data are conflictual in sport-related studies, depending substantially on the type
of supplement used. In fact, whereas supplementation with the flavonoid Quercetin
did not damp the IgA salivary activity decrease after 160 km of running [15], melatonin ingestion increased serum IgA concentration 60 min after acute exercise
[16] and probiotics consumption attenuated the salivary IgA decrease post intensive military
training [17]. In addition, bovine colostrum supplementation prevented a post-exercise decrease
in serum IgG levels in highly trained cyclists [18] and increased serum IgA in distance runners [19]. Poor nutritional status acts as a deleterious co-factor on the immune system response
to exercise. In fact, there is an association between insufficiency of protein and
micronutrients in diet and immune dysfunction [1]. Studies focusing on the effect of single or multi-micronutrient supplementation
in serum immunoglobulin response to physical training are very scarce.
Firefighters are exposed to several environmental stressors such as smoke, chemical
exposure, heat, and intense physical exertion [20]
[21]. Thus, it is reasonable to seek nutritional resources, trying to mitigate the metabolic
effects of the imposed training load. Typically, firefighter recruits are selected
from an initial population of around several candidates and selected through demanding
physical tests and careful medical checks. After these procedures, the top recruits
join the recruiting process. During the recruit training period, firefighter recruits
are exposed to greater physical stress than professional firefighters in their regular
training. This is because during the training process of recruits, stressful situations
are experienced systematically and with greater incidence. Thus, we analysed the influence
of multi-micronutrient supplementation on resting serum immunoglobulins in subjects
undergoing an intensive physical training program. It was hypothesised that increasing
micronutrient intake would enhance the immunoglobulin response to five weeks of strenuous
training.
Materials and Methods
The participants in this study were chosen from an initial population of around 200
candidates who were selected through physical fitness tests. The top 25 joined the
recruiting process. Subjects with any incapacitating physical or organic pathology
were excluded. One firefighter recruit was excluded due to a shoulder injury prior
to the experiment.
Sample
Twenty-four male firefighters were randomly divided into supplemented and placebo
groups (concealed allocation was implemented). Regarding inclusion criteria, subjects
should be professional firefighter recruits, healthy (assessed through medical tests),
with no muscular bone or articular pathologies, without visual or hearing deficits,
and be classified in physical conditioning tests. Age, anthropometrics, and physical
conditioning characteristics were similar between groups ([Table 1]), with no dropouts during the study. All participants were informed about the benefits
and risks of participating in the current study prior to signing an informed consent
form, which was approved by the ethics board of the local university. Experimental
procedures were in accordance with the Helsinki Declaration and ethical principles
for medical research involving human subjects [22].
Table 1 Means and standard deviation (±SD) values of age, anthropometrics, and physical conditioning
characteristics of supplemented and placebo group.
|
Variables
|
Supplemented (n=12)
|
Placebo (n=12)
|
Paired t-test
|
|
Age (years)
|
22.1±1.9
|
23.9±0.3
|
ns
|
|
Height (cm)
|
174.5±3.8
|
174.3±3.5
|
ns
|
|
Body mass (kg)
|
68.9±7.4
|
69.3±12.3
|
ns
|
|
Fat mass (% body mass)
|
9.6±2.0
|
10.2±2.0
|
ns
|
|
Bench press with 50 kg (reps)
|
12.0±7.1
|
13.7±5.7
|
ns
|
|
Chin-ups (reps)
|
15.8±0.7
|
15.7±2.1
|
ns
|
|
Sprint 50 m (s)
|
7.00±0.0
|
6.97±0.05
|
ns
|
|
Cooper test (m)
|
3007±127
|
3120±106
|
ns
|
|
Relative peak power output (watt/kg)*
|
10.5±0.5
|
10.8±0.1
|
ns
|
|
Relative mean power output (watt/kg)*
|
7.8±0.5
|
8.1±0.3
|
ns
|
|
Relative minimum power output (watt/kg)*
|
5.7±0.8
|
5.8±0.6
|
ns
|
|
Fatigue index (%)*
|
45.9±6.4
|
45.2±6.8
|
ns
|
|
Squat jump (cm)
|
37.3±5.7
|
37.8±3.5
|
ns
|
|
Countermovement jump (cm)
|
38.3±6.2
|
39.4±5.8
|
ns
|
ns: t-test non-significant (p>0.05); *Wingate test.
Testing protocol
The present study was randomized, double-blinded, and placebo-controlled with the
supplemented group receiving, over 35 consecutive days, a proprietary supplement (Prisfar
Ever-Fit Plus with 15 mg of beta carotene, 200 mg of vitamin C, 136 mg of vitamin
E, 200 μg of selenium, 15 mg of zinc and 100 mg of magnesium). The other group received
a placebo powder (maltodextrin with artificial flavour and colour). The training period
included 5 microcycles of 5 training units (including 30 min of military drills plus
90 min of technical skills with and without fire protective clothing) and 2 resting
days. This and the contents of the weekly physical training program are displayed
in [Fig. 1]. Participants avoided physical exertion over the weekends during the study period.
Anthropometrical evaluation included stature; body mass; tricipital, bicipital, subscapular,
and suprailiac skinfold thickness; and fat mass [23].
Fig. 1 The physical training program.
All participants received the same physical training and professional skills program
during 3 months prior to the experiment. Physical fitness in-between groups was similar
at the beginning of the study ([Table 2]). Dietary intake was assessed on 2 weekdays and one weekend day record (one week
before the intervention) by means of a photo album with 134 pictures containing average
raw/cooked food portions. Mean daily food intake was converted to nutrients using
ESHA’s Food Processor Nutrition Analysis software [24]. Dietary intake values prior to the experiment were similar between groups ([Table 2]). Because the groups presented similar nutritional intake before intervention and
firefighters had the same meals during the intervention, they were not tested again
for these variables.
Table 2 Means and standard deviation (±SD) values regarding nutritional intake before supplementation
for both groups.
|
Supplemented (n=12)
|
Placebo (n=12)
|
Paired t-test
|
|
Energy intake (kcal)
|
3050±600
|
2985±580
|
ns
|
|
Carbohydrate (%)
|
51.2±5.8
|
50.8±3.1
|
ns
|
|
Protein (%)
|
18.4±2.5
|
19.2±2.1
|
ns
|
|
Fat (%)
|
30.2±3.3
|
30.8±2.8
|
ns
|
|
Vitamin C (mg)
|
147.2±58.2
|
152.8±47.3
|
ns
|
|
Vitamin E (mg)
|
7.9±2.3
|
8.3±2.8
|
ns
|
|
Beta carotene (μg)
|
165.1±4.3
|
158.3±7.2
|
ns
|
|
Magnesium (mg)
|
431.4±76.8
|
411.9±82.5
|
ns
|
|
Selenium (μg)
|
163.3±22.7
|
161.9±54.2
|
ns
|
|
Zinc (mg)
|
16.9±5.7
|
17.1±4.2
|
ns
|
ns: t-test non-significant (p>0.05);
For serum immunoglobulin concentrations assessments, venous blood samples were drawn
from an antecubital vein in a fasting state always at 8:00 a.m., on the first Monday
after the resting days, at pre- (3 months after the start of training activities)
and post-5 weeks of supplementation. The serum immunoglobulin concentrations were
determined using specific N antiserum to human IgG, IgA, and IgM (code numbers OSAS,
OSAR, and OSAT, respectively). The immune complexes formed were measured in a Behring
nephelometer (Dade Behring Marburg GmbH, Newark, NJ, USA) and the amount of IgG, IgA,
and IgM were calculated by comparison with standards of known concentration. Blood
samples were analysed in the same laboratory following common analytical procedures.
Statistical analysis
A sample size of 24 subjects was deemed adequate (G*Power 3.1.9.2 software; Heinrich
Heine Universität Düsseldorf, Germany), assuming 0.65 of effect size, 85% of statistical
power, and 0.05 α error probability. Data were first tested for distribution normality
and variance homogeneity. The effects of supplementation (experimental vs. placebo), time (pre- vs. post-supplementation), and interaction (supplementation×time) were assessed for each
variable using a mixed ANOVA. A paired measures t-test (post hoc) was used to compare
IgG, IgA, and IgM values, before and after the five-week period, between both supplemented
and placebo groups (between- and within-group). IBM SPSS Statistics for Windows, Version
19.0 (IBM Corp., Armonk, NY, USA) was used with results presented as mean plus standard
deviation (alpha significance level was established at 0.05).
Results
Mean and standard deviation (±SD) values of IgG, IgA, and IgM for supplemented and
placebo groups before and after the training period are displayed at [Table 3]. Interaction (supplementation×time) was not significant for any variable (IgG η2: 0.006, p=0.60, power: 0.08; IgA η2: 0.003; p=0.70; power: 0.06; IgM η2: 0.003; p=0.70; power: 0.06). Supplementation effect was detected for IgG (eta-squared, η2: 0.09; p=0.035; power: 0.56), IgA (η2: 0.24; p=0.001; power: 0.95), and IgM (η2: 0.09; p=0.036; power: 0.56). Time effect was not significant for any immunoglobulin (IgG
η2: 0.003, p=0.73, power: 0.06; IgA η2: 0.003; p=0.71; power: 0.06; IgM η2: 0.015; p=0.42; power: 0.12). Although IgA was different between groups at baseline (mean difference:
42.58; 95%CI: 7.00 to 78.16 mg/dL; p=0.021; d=2.48), within-group analyses (before vs. after five weeks) showed no differences. The IgG and IgM were similar at baseline,
but IgG decreased (mean diff.: 46.4; 95%CI: 6.7 to 86.1 mg/dL; p=0.03; d=0.74) and IgM increased in the control group (mean diff.: −10.7; 95%CI: −15.8 to
−5.5 mg/dL; p=0.001; d=−1.33).
Table 3 IgG, IgA, and IgM for the supplemented (SUP) and placebo (PLA) groups before and
after the training period.
|
Variables
|
Group
|
Before
|
Mean diff (95%CI); Sig.; Cohen’s d
|
After
|
Mean diff (95%CI); Sig.; Cohen’s d
|
|
|
|
Between-Group (Before)
|
|
Within-Group (Before vs. After)
|
|
IgG (mg/dL)
|
SUP
|
909.0±120.7
|
90.50 (−63.9 to 244.9); 0.237; 1.21
|
918.9±171.2
|
−9.9 (−61.8 to 42.0); 0.68; −0.12
|
≈
|
|
PLA
|
818.5±228.0
|
|
772.1±216.1
|
46.4 (6.7 to 86.1); 0.03*; 0.74*
|
↓
|
|
IgA (mg/dL)
|
SUP
|
176.5±42.2
|
42.58 (7.00 to 78.16); 0.021*; 2.48*
|
186.1±48.5
|
−9.6 (−24.5 to 5.4); 0.19; −0.40
|
≈
|
|
PLA
|
133.9±41.8
|
|
133.7±43.3
|
0.2 (−8.8 to 9.3); 0.95; 0.01
|
≈
|
|
IgM (mg/dL)
|
SUP
|
65.9±20.2
|
−16.00 (−41.2 to 9.25); 0.202; 0.01
|
69.7±20.8
|
−3.8 (−7.8 to 0.1); 0.06; −0.61
|
≈
|
|
PLA
|
81.9±37.0
|
|
92.6±40.5
|
−10.7 (−15.8 to −5.5); 0.001*; −1.33*
|
↑
|
*P<0.05, ≈ similar, ↓decreased and ↑increased.
Discussion
The main aim of this study was to verify the influence of multi-micronutrient supplementation
on resting serum immunoglobulins in subjects undergoing an intensive physical training
program. Our results suggest that despite mean values having remained within the reference
values, changes observed for immunoglobulin G and M may reflect some immune protection
for firefighters engaged in recruit training. Different endogenous and exogenous stressors
(e. g. light, temperature, metabolism) constantly challenge human body homeostasis
and induce adaptive immune changes [8]. Immunoglobulins are protective proteins produced by the immune system in response
to foreign substances called antigens. Antigens are viruses and bacteria as well as
deleterious substances produced by the body itself [2]. The reduction of plasma and mucosal levels of immunoglobulins open the window for
the emergence of various opportunistic infections while abnormal increases in immunoglobulins
levels can trigger allergic situations [2]. Physical exercise is one of the stressors which the human body needs to handle.
Thus, people engaged in regular exercise make use of many types of workouts and different
combinations of exercise modes in their training sessions. Each one presents unique
characteristics and challenges regarding its duration, intensity, rest interval, environmental
conditions, and personal abilities [7].
It is described that intensive exercise triggers the release of large amounts of autoantigen
[5], which can promote serum immunoglobulin increase [25] or decrease [26], depending on exercise type. While 2 hours of intensive judo induced an increase
in serum IgG concentration, low- to moderate-intensity exercise did not promote significant
changes in IgG levels [26]. To study the effects of training and acute exercise on immunoglobulin levels, Nieman
et al. [27] matched eleven marathon runners with nine sedentary controls in three moments: baseline,
after a graded maximal treadmill test, and 45 min of recovery. The results showed
that at no point in the study did the groups differ. Conversely, 45–75 km of running
depressed serum immunoglobulin levels for up to two days [8]. It seems that the longer the exercise, the greater the acute decrease in the concentration
of immunoglobulins and the longer the time to return to baseline values.
For standard workouts, 24 h are usually sufficient to recover basal levels of serum
immunoglobulin after a single bout of severe exercise [23]. Acute exercise-induced changes in serum immunoglobulin levels do not appear to
be translated into chronic adaptation [23]. It was observed that a training program that consisted of 3 km jogging, three times
per week for eight weeks did not alter basal IgG, IgA, and IgM serum concentrations
in untrained male subjects, perhaps due to its low intensity. It seems that acute
changes in serum immunoglobulin concentrations are reversible as quickly as exercise
intensity lessens. Although several studies [8]
[27] suggest the reversibility of acute adaptations induced by exercise in serum concentrations
of immunoglobulins, it is debatable whether some chronic adaptations occur.
Baseline values from our study showed differences between groups only for IgA. Nonetheless,
trivial differences observed IgG and IgM must be considered circumstantial because
the values all fall within the normal range. Stability was observed in the supplemented
group for all immunoglobulin values. In the control group, immunoglobulin patterns
were irregular, i. e. IgG decreased and IgM increased whereas IgA remain stable. The
administration of the maltodextrin might have contributed to acute changes during
the training process [7], but our results point to the lack of chronic effects on serum IgA concentrations
because post-values were similar to pre-values.
Although the supplemented group showed stable values for these markers, the subjects’
responses were inconsistent. That said, it is important to highlight that the kinetics
of leukocyte subset changes and their by-products cannot be fully appreciated in studies
where pre-and post-exercise blood sample are compared, i. e. it is not possible to
follow the time-dependent changes. However, as the participants’ inclusion in each
group was randomized, it can be speculated that the observed responses are circumstantial
and define a special immunological trait at the time of the study or possibly the
influence of smoking habits (because two subjects in supplemented group and seven
subjects in placebo group were regular smokers). It seems that smoking reduces secretory
IgA [28]. It was suggested that the raised IgM during the first 12 h after exercise reflects
a secondary antibody response to autoantigens [2]. IgA in serum tends to be stable even after high-intensity training [11], which justifies the stability of our results independent of the difference between
groups. Regardless of the differences observed in the placebo group, we should not
assume that these differences have any physiological significance, because the variations
are within the literature reference values for serum immunoglobulins.
All variable values for both groups were within the normal laboratory range [29]
[30]. They were similar to those found before and after a period of long intensive training
[31] but lower than those observed in orienteers and runners/skiers [32]. Perhaps the severity of firefighter training, by inducing severe physical and psychological
stress, depressed some immunoglobulin serum concentrations, in line with previous
results [33]. However, supplementation seems to have stabilized the immune response to training
in the present study because the placebo group expressed alterations for IgG and IgM.
It seems that training status does not promote significant changes in serum immunoglobulin
concentrations. A comparison of marathoners and sedentary subjects verified that IgG,
IgA, and IgM serum levels did not differ during rest, graded maximal exercise, and
recovery [27]. It was also observed that different exercise types (varying in intensity and duration)
did not alter basal IgG, IgA, and IgM serum concentrations [1], similar to what happens before vs. after a volleyball season [25]. It was previously stated that IgG and IgM are maintained at a higher level in exercised
subjects [9]. However, the early immunoglobulin increase subsequent to the initial exercise loads
is attenuated by hard training continuity [12]. The results from the present study partially conflict with this assumption because
the placebo group suffered a serum IgG reduction at the time of the second evaluation.
Although low- to moderate-intensity training tends to stimulate the production of
IgG [1] and IgA [1]
[12], continued high-intensity exertion promotes a significant decrease of IgG and IgM
without an alteration in IgA serum concentration [11]. Thus, the current results for IgA are supported by other studies. The IgG decrease
in the placebo group is in accordance with Mashiko et al. [11]. However, the IgM increase verified in the placebo group is partially supported
by the literature, which verified that a reduction in IgM serum concentration after
a period of hard training was followed by an increase before competition probably
due to the recovery process in the tapering phase [31]. The IgM increase verified in our placebo group cannot be attributed to tapering
because the training program volume and intensity was maintained without any recovery
phases. It can be speculated that if two days of rest are sufficient to return the
values to their basal expression, changes in the control group and stability in the
supplemented group point to a certain stabilizing effect of the chosen supplementation.
The exercise immune response modulation with nutritional supplementation suggests
some positive effect of micronutrient supplementation in inhibiting eventual exacerbation
of autoimmune inflammatory responses to intensive training continuity [34]. This finding partially corroborates that of Vidal et al. [14], who verified that a milk-based wolfberry formulation increased serum IgG levels
in healthy sedentary elderly. It was also verified that bovine colostrum protein concentrate
prevented a drop in post-exercise serum IgG concentration in highly trained cyclists
[18]. However, bovine colostrum supplementation did not alter serum IgG and salivary
IgA concentrations during periods of strength and speed training [35].
In healthy non-active well-fed subjects, any type of supplementation seems to be ineffective
for improving humoral immune status. For instance, humoral immune response is not
enhanced by vitamin E supplementation in healthy sedentary women [36] and a salmon-rich diet (improving n-3 fatty acid intake) did not change serum IgG,
IgM, and IgA concentrations in healthy subjects [37]. However, conflicting results in relation to the positive effects of vitamin E supplementation
on the immune system have been observed. Even though there is no clear indication
that vitamin E supplementation is necessary to improve the humoral immune functions,
vitamin E supplementation may be beneficial to all adult age groups as a preventive
measure for complications related to oxidative damage [36]
[38].
Moreover, in the absence of nutritional deficiency (even in highly stressed athletes),
nutritional supplementation seems be ineffective. For example, Stroescu et al. [39] observed that basal serum immunoglobulins in elite female gymnasts undergoing strenuous
training did not change after supplementation with SUPRO brand isolated soy protein.
However, supplementation can be effective in situations of plasma micronutrient deficiency.
In asthma patients (characterized by lower levels of selenium, zinc, betacarotene,
and vitamins C and E), a multiple-nutrient supplement recovered normal micronutrient
plasma levels and improved immunoglobulin status [40]. In animal studies, selenium and vitamin E deficiency induced a decrease in IgG
and IgM serum concentrations and IgA remained unchanged [41]. In addition, it was observed that supplementation can improve the immune status
of healthy active subjects undergoing hard physical training when humoral immunity
is depressed. In fact, it was shown that the administration of ectisten-containing
tincture of leuzea and leveton restored the decrease of serum IgG and IgA induced
by intensive running training [42]. Studies analysing the modulation of serum immunoglobulin concentration by multi-micronutrient
supplementation are lacking because single-micronutrient supplementation failed to
improve immune response to exercise in well-trained and well-fed subjects.
Although differences between groups were observed only for IgA, it can be stated that
the stability of the supplemented group clearly contrasts with the changes in the
placebo group. It was observed that selenium supplementation can selectively enhance
human antibody production with increased switching from IgM to IgG production [43]. Therefore, perhaps the absence of supplementation in the placebo group could accentuate
the switch from IgG to IgM production, justifying the current study results. We hypothesise
that supplementation stabilized humoral immunity during hard training and that the
IgM increase in the placebo group was an index of immune fragility. It is known that
natural IgM antibodies induce apoptosis of transformed cells and act to rapidly remove
foreign cells and particles with attenuation of the inflammatory processes [44]. The lack of studies relating to micronutrient supplementation and serum immunoglobulin
status during continued, hard physical exertion does not allow deeper comparisons
with current data.
However, this study points to the association between nutrition and immune changes
induced by heavy physical exertion. This is an open field for scientific research.
Firefighter training is aggressive in many ways, either due to the exercise intensity
or due to the conditions in which it is developed. Thus, the control of immune variables
is important to follow the acute effects and chronic adaptations induced by different
types of exercises. However, it is important to acknowledge several shortcomings and
potential limitations of our study. Although participants reported that they avoided
physical exertion over the weekends during the study period, this was not monitored.
Also, the kinetics of leukocyte subset changes and their by-products cannot be entirely
observed when pre- and post-exercise blood samples are compared.
The results from the present study suggest that in well-fed firefighters, multi-micronutrient
supplementation reduces the variations induced by a five-week strenuous physical training
program in some serum immunoglobulins: IgG and IgM. Although there were significant
differences in some immunoglobulins, which allows us to assume some protective effect
from supplementation (as the values clearly remained within the reference values),
the results obtained may have derived from normal fluctuations and not from the chronic
effect of the chosen supplementation.
