1. The study examined the relationship between total haemoglobin mass, performance-related genotypes, and VO2max in 4 untrained subjects. 2. Results showed no clear relationship between total haemoglobin mass and VO2max. The subject with the highest endurance genotype score had the lowest VO2max. 3. The subject with the highest sprint genotype score had the lowest endurance genotype score, despite having the highest VO2max, suggesting genotypes may predict sport-specific performance.

1. 1
Determining and predicting athletic performance – the
importance of total haemoglobin mass and genotype
variation.
Iain Christie
1107897
26/03/12

2. 2
Abstract
There is a trend in the world of endurance running where East Africa constantly produces
medal winning athletes. A range of factors to explain this have been investigated, with total
haemoglobinmassandparticular genotypes from candidate genes being important areas of study.
Purpose:To examine the validityandsignificance of these two components, and evaluate whether
either could be used to determine whether or not an athlete could be successful in a particular
sportingactivityatan elite level. Methods:Four healthy but untrained particpants were used, with
age ranging from 22 to 27 (mean 24). Three experimental processes were carried out. Total
haemoglobin mass was calculated via an optimised carbon monoxide rebreathing. Genes were
evaluatedusingabuccal swabtechnique,allowingparticularcandidate genesforperformance to be
analysed. A VO2max test was carried out for each subject also. Results: The subject with the highest
genotype score forsprintinghadthe lowestvalue for endurance, despite having the highest VO2max
of the participants.Resultswere inconclusive astowhethertotal haemoglobin mass was associated
withhigh VO2max or witha particulargenotype combination. Conclusion: Itcannot be said that there
existsaparticulargenotype whichwilldetermine the effectiveness of an athlete’s performance in a
particular sporting discipline. Further research with larger sample size is required to accurately
evaluate whether total haemoglobin mass is a predictor for endurance capacity.
Introduction
In the currentathleticclimate,the traditional trendisforEastAfrica to consistently produce
a multitude of successfulmiddle-andlong- distance runners (Billat et al. 2002). Extensive study has
been carried out as a means to investigate what it is that makes the endurance performance of
these athletessosuperiortotheircounterpartsfromelsewhereinthe world.Itbegs the question, if
a definite mechanism responsible for this was found; could this success be replicated in athletes
from Britain, or mainland Europe? Or in fact does a pre-existing genetic trait give East African
athletesanoverall advantage forendurance capacity?Itismore likelythata myriad of physiological
components combine to create such effective endurance performance. However, two elements
whichare receiving increasing levels of attention could be largely responsible, and these are total
haemoglobin mass and gene variation.
Eastwood et al. (2011) highlight the significance of haemoglobin mass (Hb mass) for
endurance performance becauseof it being an important determinant of oxygen transport – which
isa crucial componentof effective endurance performance. This study also showed how values for
Hb mass vary significantly between genders. It was found that Hb mass of males was 33% higher
than for females, and this was after differences in body mass were accounted for. Body mass is
considered to be strongly associated with Hb mass, and with this value taken into account gender
differencescouldperhapsbe explained by the effects of testosterone in males. Another important
findingthatcame out of thisstudywas the effectsof reducing training levels (ie reducing intensity,
duration or frequency). Total Hb mass is significantly reduced as training loads are decreased.
Notall literature isin agreementoverthe predictive capacityof total Hbmass for endurance
performance. In a study focusing on the elite Kenyan runners, Prommer et al. (2010) state that
althoughtrainedendurance athletesdohave significantlyhighervaluesfor total Hb mass, this along
with blood volume is not responsible for the superior endurance performance of the Kenyan
runners.Aswell assuggestingthisconclusion,the authorsalso offer a separate hypothesis in terms

3. 3
of the effectsof altitude onHbmass; an area that has receivedmuchattentioninrecentyears.They
suggestedthatrunnerslivingandtrainingatgreaterthan2000m have greateroxygentransportthan
those at lower altitude or sea level. Schmidt et al. (2002) conducted a study with a similar
hypothesis. The purpose of this study was to examine if improvements in total Hb mass due to
altitude exposure caused improved endurance performance. The relevance of such a study comes
fromthe authors’suggestionthatthere are minimal structural and functional differences between
athleteswholive ataltitude tothose whodonot. The null hypothesisof thisexperimental procedure
was rejected,anditwasfoundthat altitude exposure does have a positive effect on total Hb mass,
and thisimprovedvalue isdirectlyresponsibleforhighperformance fromthose wholive at altitude.
As well asaltitude andgender,the type of discipline can also affect total Hb mass. Athletes
who compete in anaerobic activities display low values for total Hb mass, similar to those of
untrainedathletes,whereasall activitiesof anaerobicnature require equallyhighlevels(Heinicke et
al.2001). Aerobicactivitiesrely instead on muscle based traits. As a rationale for the high levels of
Hb mass for endurance trained athletes, the authors suggest possible mechanisms. The first being
adaptationsinplasmaand red cell volumes as caused by endurance training, and the second being
that there couldbe genetic predispositions in certain individuals. The latter of these has provoked
much discussion.
With specific reference to the success of East African endurance runners, it has been
suggested that there exists specific genetic determinants of performance, and the notion of
“choosing ones parents well” perhaps carries particular importance. An example of this includes
mitochondrial DNA polymorphisms,whichare saidto play a role in physical performance (Scott and
Pitsiladis, 2006). However in terms of significantly influencing athletic performance, it is
polymorphismsinthe angiotensin-convertingenzyme (ACE) gene that have received more focused
investigation. This enzyme is a component of the renin-angiotensin systems and is an influential
componentinregulatingbloodpressure,sodiumand water homeostasis and tissue growth (Collins
et al. (2004). ACE I/D polymorphism has been associated with cardiovascular disorders, and
conclusive findingsexistlinkingitwithdiabeticnephropathyandAlzheimer’sdisease,howeverthere
is conflicting opinions in regards to its significance for determining physical performance (Sayed-
Tabatabaei et al. 2006). In general terms, it has been hypothesised that the I allele of the ACE
genotype is associated with high endurance performance, and the D allele with greater strength
gains from training (Puthucheary et al. 2011). Gayagay et al. (1998) back suggestions that specific
genotypescandetermineperformance capability,concludingthatthere wasa significantcorrelation
between measurable genetic polymorphism and elite athletic performance, with the ACE I allele
providing competitive advantage for cardiovascular performance. A similar stance was taken by
Collins et al. (2004), in which a study looking performance during the South African Ironman
Triathlons it was found that the I allele was associated with the endurance performance of the
fastest 100 Caucasian male South African-born finishers. However it is important to note that the
same findingswere notreplicatedwiththe foreignbornathletes.Thispointismirroredin a study by
Ash et al. (2011) where it was concluded that ACE genotypes do not determine whether runners
from Ethiopia can perform at an elite level, prompting further study focusing on different
populations.Incurrentstudies,itislimitations like this, as well as such factors as small sample size
and a lack of clearly defined physiological phenotypes (Roth et al. 2012) that cause inconclusive
findings to be produced.

4. 4
The ACE gene isnot the onlygene thatis associatedwithsporting performance however. In
fact there is a whole range that has been examined, each with varying levels of significance for
overall performance. A particular gene type combination could perhaps be the key to elite
performance. Anexampleof othergeneswhichhave receivedattentionin current literature can be
foundina studyby Eynonet al.(2009), wholookedatthe distribution of PPARGC1A and PPARα G/C
in athletic and non-athletic Israeli populations, and found both to be associated with top-level
endurance performance. Gomez-Gallego et al. (2009) suggest the importance of ACTN3, the gene
encoding for the synthesis of α-actinin-3 in skeletal muscle fibres. This study continues to suggest
the relationship between genotype and sprint/power capacity in athletic performance.
Afterclose referenceandexaminationof the literature inthese twopossible components of
endurance performance,anexperimental studywasdesigned.The purpose of whichwastoexamine
how accurately measurements for total haemoglobin mass and genetic variants can be applied to
predictathletic performance. The maximum oxygen uptake (VO2max) was also measured, and since
thisisa keyelementof effective endurance performance,itcouldbe usedtoverifywhetherthe data
obtained from the experiment was a reliable predictor. The null hypothesis was that neither total
haemoglobin mass nor genetic variations provided a way of determining endurance capacity.
Experimental Method
Four untrained,healthysubjectswere usedinthe study(3males,and1 female).All subjects
were briefedonthe experimental protocol before beginning the study, and were made aware that
participationwas voluntary. Body composition data for each subject was recorded (Table 1). There
were three separate experimental procedures in which each other the subjects participated in.
These includedanoptimisedcarbonmonoxide rebreathing (where carbon monoxide was used as a
marker to label haemoglobin), a buccal swab extraction, and a treadmill based VO2max test.
The methodfor the carbon monoxide rebreathing was as outlined by Durussel et al. (2012).
Calculationsforobtainingavalue fortotal Hb masswere alsoinaccordance withthisstudy.Itshould
be noted that for subject 2 capillary blood samples were taken via a finger prick, whereas
intravenous cannulation was used on the remaining three participants.
DNA was extracted from each subject using buccal swabs. A standardized method of DNA
analysis was followed step-by-step.
For the VO2max tests,eachsubjectwasgiventime toadjustto runningonthe treadmill, and a
warm upperiodwasincorporatedintothe test.Due to safetyconcerns,subject3was testedwith an
increasing treadmill gradient, whereas the other three subjects were tested with a gradually
increasing speed.
Once the procedures were completed, results were tabulated for comparison.

5. 5
Results
Table 1 – Subject Data
SubjectID 1 2 3 4 Mean
Age(yr) 27 24 22 23 24
Height(cm) 168.5 180 178 165 170.4
Weight(kg) 68 73 63.4 63 66.9
BMI(kg/m2) 23.9 22.5 20.1 22.6 22.3
Table 2 - VO2max Data
Subject ID 1 2 3 4 Mean
VO2max
(mL/kg/min)
45.63 48.65 53.68 46.38 48.6
Max Heart
Rate (bpm)
192 N/A* 200 201 197.7
*Due to experimental error this value could not be measured properly
Table 3 – Haematological data
Subject ID 1 2 3 4
ctHb 14.7 13.6 14.5 14.5
tHb (g) 783.4 799.5 728.9 688.0
tHb (g/kg) 11.5 11.0 11.6 10.9
Figure 1 – Genotype scores for performance associated genes

6. 6
Table 4 – Performance genotype scores (sprinting)
Subject ID 1 2 3 4
ACE (rs4341) ID (1) ID (1) DD (2) ID (1)
ACTN3
(rs1815739)
RX (1) XX (0) RR (2) RR (2)
Il15RA
(rs2296135)
CC (0) AC (1) AC (1) AC (1)
Total Score (%) 33 33 83 67
Table 5 – Performance genotype scores (endurance)
Subject ID 1 2 3 4
ACE (rs4341) ID (1) ID (1) DD (0) ID (1)
ACTN3
(rs1815739)
RX (1) XX (2) RR (0) RR (0)
BDKRB2
(rs1799722)
CC (0) CT (1) CC (0) CT (1)
PPARD
(rs2267668)
AA (2) AG (1) AA (2) AA (2)
PPARGC1A
(rs8192678)
TT (0) CT (1) CT (1) CC (2)
ADRB2
(rs1042713)
AG (1) AG (1) AD (1) AG (1)
Total Score (%) 42 58 33 58
The VO2max data displayedintable 2is assumedtobe accurate. Thisisbecause at the highest
running speeds (or highest gradient for subject 4) the subjects’ were performing with heart rates
representative of the criteriaformaximal oxygenconsumption. Table 3 displays the measurements
obtained from the carbon monoxide rebreathing. In order to draw fair conclusions via comparison
betweensubjects,the overall valuefortotal haemoglobinmasshadtobe normalisedforbody mass.
As hasalreadybeenestablished,total Hbmassisgenerallylowerforfemales,sothishadto be taken
into account.
With reference to tables 2 and 3, it can be established as to whether or not a relationship
exists between VO2max and total haemoglobin mass. It is unclear if this relationship occurs, as
althoughsubject3 hasa significantlygreater VO2max thansubject1,both have similarvalues for total
Hb mass (once bodymasshas beenaccountedfor),andinfact there is very little variation between
all subjects for this value.
Table 4 shows the performance genotype scores for sprinting for each subject. Subject 3 is
shownto have 83% of the necessary genotype scores for sprint performance, with subjects 1 and 2
havingthe lowestvalues.Asthe previouslymentionedliteraturesuggests,aD allele of the ACE gene
isassociatedwithgreaterstrengthandpowerperformance,and so a DD genotype (as possessed by
subject 2) should be a key component of sprint performance. Endurance performance genotype
scores can be seen in table 5. Subjects 2 and 4 have the highest total score for this genotype, with
subject 3 having the lowest. This contradicts with data obtained from the VO2max tests, where the

7. 7
highest value was recorded for subject 3. Graphical data for these scores can be seen in figure 1,
where a clearer outlook of performance capability is given. Subjects 1 and 4 are shown to be
reasonablybalanced,withsimilarscoresbeingshownforsprintandendurance genotypes. However
significantdifferences are apparent in subjects 2 and 3, who carry more appropriate genotypes for
endurance and for power respectively.
Discussion
Due to the small number of participants involved in the study, there is a low statistical
power.Howeverconclusions can be drawn on an individual basis. VO2max testing was done in order
to compare to data obtained for total haemoglobin mass, as well as to validate the significance of
any findingsof the Hbmassand gene experiments.SchmidtandPrommer (2010) foundthat total Hb
mass isa determinantof VO2max,because of twoparticularmechanisms:increasedtotal Hbmass and
plasma volume causes an increase in cardiac output; and increased total Hb mass alongside
unchanged plasma volume increases haemoglobin concentration thus increasing arteriovenous
oxygen difference. Tables 2 and 3 show that the same trend did not occur in this experiment. A
numberof factors couldbe responsibleforthis.Again,the low samplesize makesit difficult to draw
accurate conclusionsontrendsandgrouppatterns.Howeverfromexaminingthe literature it can be
seenthatthere isverylittle evidence currentlywhichsupportsthe findingsof Schmidtand Prommer
(2010). It wouldmake sense tohypothesize that improved haemoglobin mass would be associated
withhigh VO2max howeveruntil furtherresearch is carried out in this area such a statement can only
be an assumption.One areathat hasreceivedattentionisthatof responsesto altitude training, and
elementsof thesestudies could possibly be applied to evaluate the relationship between total Hb
mass andVO2max. Wehrlin et al (2006) investigated the effects of the “Live High-Train Low” method
of altitude exposure. Looking at orienteering athletes, after 24 days of LHTL there was a significant
increase intotal haemoglobinmass,and this was associated with improved VO2max. However again,
there is conflicting opinions on this matter, and in fact VO2max may well increase due to other
performance components, and not due to a relationship with total Hb mass. This is suggested by
Saundersetal.(2004) who notednochange in total Hb mass aftera period of “Live High-Train low”,
concludingthatitwas improvedrunningeconomywhichmayhave caused improvements in VO2max.
There isalso debate overthe relationshipbetween VO2max and specific genotypes. A review
article by Puthucheary et al. (2011) highlights how opinions are mixed in this area, and any studies
that have beencarriedoutremain relatively inconclusive. Although there are a number of studies,
particularlyfocusingonthe ACE genotype, offering opinions both for and against this relationship,
Puthucheary and colleagues state that “any association between ACE genotype and peak VO2max
remain unproven”. Due to the complex and varied nature of performance gene combinations, a
clearerideaof the value of genotypes to determine athletic performance can perhaps be gained at
lookingata range of performance genesandtheirimplications.Suchinformation can be taken from
tables4 and 5. For sprintperformance genes,both subjects 3 and 4 display the RR allele for ACTN3,
whichisassociatedwithfastcontractile abilityof muscles,which suggests these subjects are suited
for powerbasedactivities.Subject3’sDD allele of the ACEgene strengthensthissuggestion. To give
these findings a context in the field of sporting activity, MacArthur and North (2005) state that the
ACE D and ACTN3 R allele favourperformance insprint or power events. They go on to suggest that
such genetic factors in fact do not predict whether a young athlete could reach elite level, but
instead offer evidence for which event or activity the most success could occur in. Therefore the

8. 8
results from our study are closer to being a guide, rather than hard facts regarding the subjects’
sporting capability. As previously discussed, Eynon et al. (2009) state that PPARGC1A is associated
with effective endurance performance. Subject 4 possessed the CC allele of this gene, and so by
Eynon and colleagues’ findings this would make this subject more likely to have the highest
endurance capacity. Howeverthe VO2max scoresdonotclearlyrepresentthis. All participants except
from subject 2 displayed the AA allele of PPARD, which is associated with metabolic rate and
endurance performance. Subject 3 only displayed the ideal polymorphism for one of the
performance candidate genesforendurance,and had the lowest overall genotype score. This does
not correlate with VO2max, scores, where subject 3 had the highest value.
Lookingat the geneticevidence presented in these results, no accurate conclusions can be
drawnwithout examining in detail the mechanisms of the genotypes and any adaptations that are
suggestedto be associated with them. However the lack of evidence to support any theories from
this study, and others like it may indicate that in fact specific genotypes in candidate genes play a
less important role in athletic success than socio-economic factors (Williams and Folland (2008).
Conclusion
Experimental results for both measured components of athletic components remained
largely inconclusive, thus making it difficult to reject the null hypothesis. From analysis of the
literature itcanbe saidthat the possibility of either total haemoglobin mass or genetic variation to
determine athleticperformance mayexist, however with current levels of evidence on the subject
there are no proven hypotheses. The importance of total Hb mass on endurance performance can
perhapsbe examinedbyapproachingthe subjectfromadifferentangle,inregards tothe risingissue
of blood doping. A rising number of cases are being examined regarding the blood manipulation,
highlightingthe importance of total Hbmassto overall endurance performance, and as Prommer et
al. (2008) suggest, this component is the “main performance limiting factor in elite endurance
athletes”. However the capacity for specific genotypes as a predictor for athletic performance is
evenmore difficulttoascertain. At this current moment in time, due to the subject still being in its
earlystages,evidence islackingtosupportanyhypothesis that there is a particular genotype in any
candidate gene which determines success in the athletic disciplines (Scott and Pitsiladis, 2007). At
thisstage all that can be said with any certainty is that the making of an athlete is complicated and
multi-facetted,andisaffectedbyanumberof environmentalandbehavioural factors(Gayagayetal.
1998). Evidence gathered from these experiments is rather representative of the topic in general,
where no valid practical conclusions can be drawn due to scarce data. Therefore for future
directions,it would be useful to examine genetic variations over different populations, and larger
sample size should be used when looking at total haemoglobin mass.

9. 9
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