exertional rhabdomyolysis ( er ) affects 1 in 10,000 people per year   and is a significant threat for military personnel and civilian fire and medical service responders during training , particularly when the training is under warm - to - hot environmental conditions [ 24 ] .
importantly , the incidence of er in the military population has more than tripled between 2006 and 2011 ; thus , it is a concern for military readiness .
further , exertion under various environmental and physical conditions induces multiple key proteins as either indicators of stress , signals for metabolic pathways , or protective mechanisms .
proteins known to be increased by exertional stress include creatine kinase , heat shock protein 70 ( hsp70 ) , and interleukin 6 ( il-6 ) [ 510 ] .
high plasma creatine kinase ( ck ) activity levels are considered a clinical marker of exercise - induced muscle damage   with some individuals demonstrating extreme elevations following strenuous exercise [ 5 , 12 ] .
likewise , baseline ck levels may have significant variability ranging from 50 to 1,200  u / l .
heat shock protein 70 ( hsp ) , a highly conserved family of stress - responsive proteins , is induced by both exercise and heat exposure [ 8 , 10 , 14 , 15 ] .
interleukin 6 ( il-6 ) , a cytokine and stress - responsive myokine , is released from contracting skeletal muscles , perhaps acting as an energy sensor , to exert both local and endocrine metabolic effects .
both baseline and exercise - induced levels of these proteins can be influenced by multiple factors including diet , exercise fitness , overall health status , sex , medications , ethnicity , and genetics .
importantly , baseline protein concentrations may be the key to understanding how such proteins change in response to environmental exposures and extreme physical exercise .
studies have shown that baseline ck levels differ by sex and ethnicity   and that both baseline   and exercise - induced ck [ 5 , 12 ] have genetic determinants .
variations in constitutive and inducible hsp70 levels have been demonstrated among individuals of different athletic abilities [ 8 , 10 ] and appear to be upregulated in response to heat acclimation .
likewise , many factors affect baseline and stress - induced levels of il-6 , including hypothalamic pituitary adrenal axis function , immune status , and possibly diet [ 9 , 20 ] .
thus , identifying confounding factors that affect baseline levels of key stress proteins is important for understanding subsequent responses to single and multiple stressors .
the genes that code for these three important stress - associated proteins have well - described polymorphisms that have been examined with regard to various phenotypes . the muscle - specific creatine kinase gene ( ckmm )
has 3 a to g single nucleotide polymorphism ( snp ) , which has been studied with regard to performance and muscle damage [ 21 , 22 ] .
this 3 sequence has been associated with mrna localization of the ck protein , and differential localization of ck could potentially alter metabolic balance .
one hsp70 gene , hspa1b , has a synonymous variant , + a1538 g ( or + a1267 g ) , which has been studied with regard to heat tolerance , high - altitude pulmonary edema , and clinical outcomes after severe injury [ 2426 ] .
the gene that codes for il-6 , il6 , has a well - described snp located within its promoter , g-174c , which may determine baseline and/or il-6 levels in response to stress [ 2730 ] . in an effort to identify biomarkers for er , base line values in a healthy athletic population
the objective of this study was to determine the contribution of genetic variation to baseline plasma concentrations of ck , hsp70 , and il-6 .
we also examined differences in protein levels as a function of sex , ethnicity , oral contraceptives , exercise regimen , supplements , and genetics in a group of marine corps officer candidates .
participants were apparently healthy , physically active students in marine corps officer candidate school , aged 1833 years ( n = 567 ) .
the study was approved by the institutional review boards of the national naval medical center and uniformed services university of the health sciences ( usuhs ) , bethesda , md , usa . participants filled out one - page questionnaire covering demographics ( age , sex , ethnicity , height , and weight ) , along with relevant questions about current health status and health behaviors .
these questions included items about sleep , recent illnesses , medication ( including use of oral contraception ) , exercise history , exercise regimen , exercise related injuries ( including ehi and rhabdomyolysis ) , supplementation ( caffeine or nutritional supplements ) , menstrual status / cycle questions ( women ) , and sct .
blood was drawn in the morning on the first day of medical in - processing and before any physical training for the candidates .
it was collected in edta - containing tubes and stored on ice for transport ( ~1 hour ) . upon arrival
plasma was aliquoted for protein analysis and stored at 80c until being ready for use .
buffy coats were transferred to 1.5  ml centrifuge tubes and stored ( < 48 hours ) at 4c until further processing .
dna was extracted from the buffy coat using the qiaamp dna mini kit 250 as per package protocol ( catalog # 51106 qiagen , valencia , ca , usa ) .
dna concentrations were measured via spectrophotometry at 260  nm ( nanodrop nd-1000 , thermofisher scientific , wilmington , de ) .
dna purity was assessed by 260/280  nm ; ratios within 1.80 to 1.85 were considered acceptable .
portions of dna stock solutions were subsequently diluted to 25  ng/l to create standardized working stock solutions .
analysis of single nucleotide polymorphisms ( snp ) for ckmm nco1 a to g ( rs1803285 )   n = 453 , hspa1b + a1538 g ( mp1 assay )   ( rs1061581 ) n = 455 , and il6 g-174c ( rs1800795 )   n = 454 was accomplished as previously described [ 5 , 30 , 31 ] .
amplicons could not be generated from all samples presumably because polymorphisms existed within the primer locations or dna was no longer available .
plasma hsp70 concentrations were assayed by using the hsp70 high sensitivity eia kit ( catalog # eks-715 , enzo life science , ann arbor , mi , usa ) as per package protocol .
detection range of the hsp70 assay was 0.2012.50  ng / ml ; the intra- and interassay coefficients of variation were < 10 and < 6% , respectively .
plasma il-6 protein ( il-6 ) concentrations were assayed using the quantikine hs human il-6 kit ( catalog # ss600b , r&d systems , minneapolis , mn , usa ) as per package protocol .
detection range of the il-6 assay was 0.156 to 10  pg / ml ; the intra- and interassay coefficients of variation were < 10 and < 7% , respectively .
any sample above the detection limit for the hsp70 or il-6 assay was rediluted to be within the detection limit and reassayed as per assay instructions .
all hsp70 and il-6 assays had the same duplicate control sample to validate interassay results .
fisher 's exact test and  were used to compare the allele distribution with other published studies .
the categorical influence on protein concentrations was performed by anova and/or independent samples t - test when applicable .
protein values generally exhibit a nonnormal distribution and thus all data were log - transformed for analysis ; however data were reported as mean  standard error ( se ) .
samples defined as outliers ( values  3 standard deviations ) were removed from analyses ; this included 6 ck , 8 hsp70 , and 4 il-6 with cutoff values of 520  u / l , 48  ng / ml , and 7.40  pg / ml , respectively .
body mass index ( bmi ) was calculated as weight in kg/(height in m ) .
all statistical analyses were verified by usuhs biostatics consulting center ; statistical power was 80% or greater for all analyses .
the sample population ( n = 567 ) was predominately male ( 78.5% ) relative to females ( 10.1% ) ; 65 participants did not report their sex .
overall , 76% were caucasian ( ca ) ( n = 433 ) , 8% hispanics ( hi ) ( n = 44 ) , 6% asians ( as ) ( n = 32 ) , 4% african americans ( aa ) ( n = 20 ) , 3% mixed race / other ( n = 18 ) , 2.8% nonreporting ( n = 16 ) , and < 1% native americans ( n = 4 ) . ethnic samples identified as native american , mixed race , other , and those nonreporting were excluded from ethnic analyses because of the small sample size .
bmi was significantly different by sex ( f(1,482 ) = 20.6 , p < 0.00 ) and revealed a trend by ethnicity ( f(3,462 ) = 2.3 , p = 0.07 ) with values for aa , as , ca , and hi being 25.4 , 23.6 , 24.4 , and 25.0  kg / m , respectively .
bmi also demonstrated a significant difference ( p < 0.05 ) by il6 genotype cc : 24.2 , cg : 24.0 , and gg : 25.0 .
as expected , ck was significantly higher in aa ( f(3,484 ) = 8.79 , p < 0.00 ) .
plasma il-6 was also significantly different with regard to ethnicity ( f(3,236 ) = 4.39 , p < 0.00 ) with ca having the lowest and aa having the highest concentrations .
in contrast , hsp70 ( p > 0.05 ) did not differ significantly by ethnicity .
however , hsp70 values for aa were approximately 33% lower relative to other ethnic groups .
the influence of bmi on ck , il-6 , and hsp70 concentration values was not significant ( p > 0.05 ) .
mean ck concentrations were significantly higher ( f(1,463 ) = 9.1 , p < 0.00 ) for males
( 144  4  u / l ) than for females ( 106  10  u / l ) . in women ,
the use of oral contraceptives was associated with a significant increase ( 39% ) in ck ( 125  17 versus 90  13 
u / l ) ( f(1,49 ) = 4.5 , p < 0.05 ) as compared to those that reportedly did not use them .
there was no significant difference in ck concentrations between females that used oral contraceptives and males ( t(434 ) = 0.689 , p > 0.05 ) .
mean il-6 concentrations were significantly lower ( f(1,237 ) = 11.8 , p < 0.01 ) in males ( 1.92  0.08  pg / ml ) as compared to females ( 2.69  0.23  pg / ml ) .
mean hsp70 did not differ significantly ( p > 0.05 ) by sex ( male 6.6  0.5 versus female 6.0  0.5  ng / ml ) .
distributions of the genotypes for the ckmm , hspa1b , and il6 polymorphism were all in hardy - weinberg equilibrium ( hwe ) conditions , with  values of 0.28 , p = 0.59 ; 0.003 , p = 0.96 ; and 1.79 , p = 0.18 , respectively .
all ethnic specific genotype frequencies were also in hwe ( p > 0.05 ) .  
the genotypic distributions for the hspa1b and il6 genes differed such that the percentages of gg genotypes for both snps were significantly higher in aa than as , ca , and hi ( hspa1b : 56% versus 14% , 17% , and 27% ; il6 : 80% versus 62% , 32% , and 46% , resp . ) .  
no significant differences in protein levels were noted for ckmm or hspa1b p > 0.05 by genotype .
however , il6 displayed a trend by genotype ( f(2,249 ) = 3.027 , p = 0.05 ) , and mean plasma il-6 levels were significantly lower for c+ genotypes ( 1.87  0.09  pg / ml ) than the gg genotype ( 2.26    0.14  pg / ml ) ( t(250 ) = 2.5 , p < 0.05 ) .
those participants that answered yes ( n = 49 ) to the question  do you drink more than three caffeinated beverages per day ?  revealed a trend for less ck ( 119  12 versus 140  4  u / l ) as compared to those that reported no ( t(505 ) = 1.9 , p = 0.06 ) .
they also had significantly reduced hsp70 ( 4.5  0.7 versus 6.6  0.6  ng / ml ) ( t(186 ) = 2.1 , p < 0.05 ) .
those that reported protein supplementation ( n = 18 ) for this population had significantly higher hsp70 ( 14.6  8.4 versus 6.3  0.4  ng / ml ) ( t(189 ) = 2.0 , p < 0.05 ) than others . no significant difference was found for il-6 and ck . those that reported the use of vitamins (
n = 28 ) or creatine supplementation ( n = 10 ) also did not have significantly different ck , hsp70 , or il-6 levels than the others .
no significant differences of ck , hsp , and il-6 protein levels were found for those who reported performing aerobic exercise ( 95% ) only or strength training ( 92% ) only or both aerobic exercise and strength training together as compared to those nonreporting ( p > 0.05 ) .
no protein differences ( p > 0.05 ) were found for those ( > 30% ) that reported  exercising strenuously with in [ sic ] the past 2 - 3 days  .
we initially sought to determine whether baseline concentrations of three proteins found in plasma with important functions relative to exertional stress differed by sex , ethnicity , and well - characterized snps in a population of healthy , young adults .
we further explored the influence of exercise history , oral contraceptives , and dietary supplementation for this cohort on these proteins .
as expected , plasma ck concentration differed significantly by sex and ethnicity , but not by the ckmm genotype .
females represent 11% of this study 's population and their mean ck levels were significantly lower than the male population ( ~20% ) , which is in agreement with other studies [ 32 , 33 ] . however , if we added the influence of oral contraceptives , females who used oral contraceptives had similar ck values to males . while these values are consistent with the literature [ 34 , 35 ] , this is an area which requires more investigation .
likewise , our ethnic differences are similar to those reported by others [ 18 , 3638 ] , with aas having the highest levels followed by as , ca , and hi . our mean concentrations are higher than others have reported , but our population was both young and beginning military training , as compared to older populations in other studies [ 18 , 33 ] .
u / l ) than another published study , but our study 's average age for hi was ~22 years as compared to ~47 years in their study .
further mean differences between studies may reflect the physical activity   of this population , as > 92% of our participants reportedly exercise at least twice per week .
we are currently exploring the use of caffeine with exercise and heat in a larger sample size to dissect the physiological effects .
no significant differences for hsp70 concentrations were noted across sex , ethnicity , or genotype with our mean hsp70 protein levels in agreement with previous studies . to our knowledge , variations in hsp70 by ethnicity
further , the genetic distribution for hspa1b is in agreement with previous work , but aa had a significantly greater percentage who were homozygous for the minor allele .
again we are currently exploring this phenomenon for a more complete picture of caffeine 's physiological effects .
we also present a new finding that protein supplementation was associated with a significant increase in hsp70 and no other covariable was associated or could explain this phenomenon .
increases in hsp70 are associated with variable health outcomes . on the one hand , elevated hsp70 concentrations have been associated with increased longevity , positive postsurgical outcomes , and cardiac and brain protection from ischemia . on the other ,
increases have been associated with inflammation , preeclampsia , and enhanced cancer progression [ 4345 ] . again
plasma il-6 levels differed by ethnicity , sex , and genotype , with aa and those with the gg genotype having the highest values relative to other ethnicities and the c+ genotypes , respectively .
our results were not altered as expected after adjusting for bmi ( p > 0.05 ) .
our mean il-6 concentrations overall and by sex are consistent with other control populations [ 29 , 46 ] as were values for ca and aa [ 29 , 47 ] . further ,
our mean il-6 concentrations for as are higher than previously reported by ognjanovic et al .   and higher for all ethnicities when compared to those reported by wong et al .
, but these differences may reflect participant differences in age , physical fitness , and other characteristics .
importantly , persons homozygous for the g allele had significantly higher il-6 levels than those with the c allele .
this finding is consistent with two other studies [ 28 , 50 ] and somewhat consistent with one other report that found that persons with the cc genotype had significantly lower il-6 levels than those with the gg or cg genotypes .
however , it is in contradiction to the finding of ognjanovic et al .   who found no significant differences .
clearly multiple factors affect baseline values : inflammation , menstrual status , obesity , physical activity , and age [ 46 , 48 , 51 ] .
it is important to note that participants in this study were young , normal - weight , healthy , and physically fit , so the baseline proteins may not be comparable to other studies examining older more sedentary populations . further research will be required to address this disparity .
it is well established that exercise alters ck , hsp70 , and il-6 protein levels [ 5 , 8 , 10 , 12 , 14 , 16 ] .
however , no protein differences were found in this population in response to reported baseline exercise regimens or recent strenuous exercise .
this may be in part because those that completed this portion of the questionnaire ( 97.5% ) reported they exercised regularly , while the remainder ( 2.5% ) left this section blank .
further , approximately 93% of the cohort reported performing aerobic exercise in combination with strength training regularly ; thus this population is fairly homogenous with regard to exercise regimen so it is not surprising that the protein levels reflect this .
moreover , sample size was not sufficient to detect differences in protein levels for those that reportedly performed aerobic exercise only ( 7% ) or strength training only ( 0.5% ) . limitations to the study include that we did not control for age , mental status , diet , and menstrual status .
we looked exclusively at genotype , sex , ethnicity , oral contraceptive use , dietary supplementation ( caffeine , protein , and creatine ) , and plasma protein concentrations .
in addition , while the population had yet to begin the rigors of officer candidate basic training , we were unable to control activity levels in the previous days prior to medical in - processing .
concentrations of plasma proteins often associated with er are affected by a variety of factors , including sex , ethnicity , genetic variants , diet , exercise , and medications .
this study documents the baseline protein concentrations of ck , hsp70 , and il-6 and the effect of sex , genotype , and ethnicity as well as exercise , oral contraceptives , and dietary supplements on these concentrations from a young , healthy , physically fit , and diverse adult population . in a cohort of young male and female marine corps officer candidates , the major allele frequencies of polymorphisms for ckmm , hspa1b , and
il6 were 70%  a , 57%  a , and 60%  g , respectively .
african americans and asians had 57% and 30% higher ck levels , respectively , than the study mean .
females that used oral contraceptives have ck values similar to their male counterparts in this population .
regular caffeine consumption coincided with reduced plasma ck and hsp70 values , while reported dietary protein supplementation increased hsp70 levels in the blood .
plasma il-6 was 30% higher in females and almost 40% higher among african americans as compared to the study mean .
this report establishes normal protein concentration ranges which can be used as reference guidelines for future er studies on the effect of heat , exercise , nutraceuticals , and other physiological modulators .
 variations of baseline plasma concentrations of creatine kinase ( ck ) , heat shock protein 70 ( hsp70 ) , and interleukin 6 ( il-6 ) have been reported . 
 we report categorical associations which may influence these protein levels . 
 methods . 
 blood was harvested for dna and plasma protein analysis from 567 adults . 
 mean protein levels of ck , hsp70 , and il-6 were compared by sex , ethnicity , genetic variants  
 ckmm nco1 ( rs1803285 ) , hspa1b + a1538 g ( rs1061581 ) , and il6 g-174c ( rs1800795)self - reported history of exercise , oral contraceptive use , and dietary supplement use . 
 results . 
 snp major allele frequencies for ckmm , hspa1b , and il6 were 70% a , 57% a , and 60% . 
 mean ck statistically differed by sex , ethnicity , oral contraceptives , and caffeine . 
 plasma hsp70 differed by caffeine and protein . 
 mean il-6 concentration differed by sex , ethnicity , and genotype . 
 plasma il-6 was significantly lower ( 29% ) in males ( 1.92  0.08  pg / ml ) and higher ( 29% ) among african americans ( 2.85  0.50  pg / ml ) relative to the others . 
 il6 g-174c gg genotype ( 2.23  0.14  pg / ml ) was 19% greater than cg or cc genotypes . 
 conclusion . 
 differences in baseline ck and il-6 plasma protein concentrations are associated with genetics , sex , ethnicity , and the use of oral contraceptives , caffeine , and protein supplements in this young and athletic population . 
1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusion