Determining and predicting athletic performance – the importance of total haemoglobin mass and genotype

variation.

Iain Christie

1107897

26/03/12

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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 haemoglobin mass and particular genotypes from candidate genes being important areas of study. Purpose: To examine the validity and significance of these two components, and evaluate whether either could be used to determine whether or not an athlete could be successful in a particular sporting activity at an 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 evaluated using a buccal swab technique, allowing particular candidate genes for performance to be analysed. A VO2max test was carried out for each subject also. Results: The subject with the highest genotype score for sprinting had the lowest value for endurance, despite having the highest VO 2max of the participants. Results were inconclusive as to whether total haemoglobin mass was associated with high VO2max or with a particular genotype combination. Conclusion: It cannot be said that there exists a particular genotype which will determine 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 current athletic climate, the traditional trend is for East Africa to consistently produce a multitude of successful middle- and long- 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 athletes so superior to their counterparts from elsewhere in the world. It begs 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 athletes an overall advantage for endurance capacity? It is more likely that a myriad of physiological components combine to create such effective endurance performance. However, two elements which are 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 because of it being an important determinant of oxygen transport – which is a crucial component of 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 differences could perhaps be explained by the effects of testosterone in males. Another important finding that came out of this study was the effects of reducing training levels (ie reducing intensity, duration or frequency). Total Hb mass is significantly reduced as training loads are decreased.

Not all literature is in agreement over the predictive capacity of total Hb mass for endurance performance. In a study focusing on the elite Kenyan runners, Prommer et al. (2010) state that although trained endurance athletes do have significantly higher values for total Hb mass, this along with blood volume is not responsible for the superior endurance performance of the Kenyan runners. As well as suggesting this conclusion, the authors also offer a separate hypothesis in terms

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of the effects of altitude on Hb mass; an area that has received much attention in recent years. They suggested that runners living and training at greater than 2000m have greater oxygen transport than 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 from the authors’ suggestion that there are minimal structural and functional differences between athletes who live at altitude to those who do not. The null hypothesis of this experimental procedure was rejected, and it was found that altitude exposure does have a positive effect on total Hb mass, and this improved value is directly responsible for high performance from those who live at altitude.

As well as altitude and gender, 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 untrained athletes, whereas all activities of an aerobic nature require equally high levels (Heinicke et al. 2001). Aerobic activities rely 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 adaptations in plasma and red cell volumes as caused by endurance training, and the second being that there could be 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, which are said to play a role in physical performance (Scott and Pitsiladis, 2006). However in terms of significantly influencing athletic performance, it is polymorphisms in the angiotensin-converting enzyme (ACE) gene that have received more focused investigation. This enzyme is a component of the renin-angiotensin systems and is an influential component in regulating blood pressure, sodium and water homeostasis and tissue growth (Collins et al. (2004). ACE I/D polymorphism has been associated with cardiovascular disorders, and conclusive findings exist linking it with diabetic nephropathy and Alzheimer’s disease, however there 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 genotypes can determine performance capability, concluding that there was a significant correlation 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 findings were not replicated with the foreign born athletes. This point is mirrored in 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. In current studies, it is limitations 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.

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The ACE gene is not the only gene that is associated with sporting 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. An example of other genes which have received attention in current literature can be found in a study by Eynon et al. (2009), who looked at the distribution of PPARGC1A and PPAR α 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.

After close reference and examination of the literature in these two possible components of endurance performance, an experimental study was designed. The purpose of which was to examine how accurately measurements for total haemoglobin mass and genetic variants can be applied to predict athletic performance. The maximum oxygen uptake (VO2max) was also measured, and since this is a key element of effective endurance performance, it could be used to verify whether the 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, healthy subjects were used in the study (3 males, and 1 female). All subjects were briefed on the experimental protocol before beginning the study, and were made aware that participation was 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 included an optimised carbon monoxide rebreathing (where carbon monoxide was used as a marker to label haemoglobin), a buccal swab extraction, and a treadmill based VO2max test.

The method for the carbon monoxide rebreathing was as outlined by Durussel et al. (2012). Calculations for obtaining a value for total Hb mass were also in accordance with this study. It should 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, each subject was given time to adjust to running on the treadmill, and a warm up period was incorporated into the test. Due to safety concerns, subject 3 was tested with 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.

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Results

Table 1 – Subject Data

Subject ID 1 2 3 4 MeanAge (yr) 27 24 22 23 24Height (cm) 168.5 180 178 165 170.4Weight (kg) 68 73 63.4 63 66.9BMI (kg/m2) 23.9 22.5 20.1 22.6 22.3

Table 2 - VO2max Data

Subject ID 1 2 3 4 MeanVO2max

(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 4ctHb 14.7 13.6 14.5 14.5tHb (g) 783.4 799.5 728.9 688.0tHb (g/kg) 11.5 11.0 11.6 10.9

Figure 1 – Genotype scores for performance associated genes

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Table 4 – Performance genotype scores (sprinting)

Subject ID 1 2 3 4ACE (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 4ACE (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 displayed in table 2 is assumed to be accurate. This is because at the highest running speeds (or highest gradient for subject 4) the subjects’ were performing with heart rates representative of the criteria for maximal oxygen consumption. Table 3 displays the measurements obtained from the carbon monoxide rebreathing. In order to draw fair conclusions via comparison between subjects, the overall value for total haemoglobin mass had to be normalised for body mass. As has already been established, total Hb mass is generally lower for females, so this had to 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 although subject 3 has a significantly greater VO2max than subject 1, both have similar values for total Hb mass (once body mass has been accounted for), and in fact 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 shown to have 83% of the necessary genotype scores for sprint performance, with subjects 1 and 2 having the lowest values. As the previously mentioned literature suggests, a D allele of the ACE gene is associated with greater strength and power performance, 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

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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 reasonably balanced, with similar scores being shown for sprint and endurance genotypes. However significant differences 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. However conclusions 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 findings of the Hb mass and gene experiments. Schmidt and Prommer (2010) found that total Hb mass is a determinant of VO2max, because of two particular mechanisms: increased total Hb mass 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 number of factors could be responsible for this. Again, the low sample size makes it difficult to draw accurate conclusions on trends and group patterns. However from examining the literature it can be seen that there is very little evidence currently which supports the findings of Schmidt and Prommer (2010). It would make sense to hypothesize that improved haemoglobin mass would be associated with high VO2max however until further research is carried out in this area such a statement can only be an assumption. One area that has received attention is that of responses to altitude training, and elements of these studies could possibly be applied to evaluate the relationship between total Hb mass and VO2max. 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 in total haemoglobin mass, 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 Saunders et al. (2004) who noted no change in total Hb mass after a period of “Live High-Train low”, concluding that it was improved running economy which may have caused improvements in VO2max.

There is also debate over the relationship between 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 been carried out remain relatively inconclusive. Although there are a number of studies, particularly focusing on the 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 clearer idea of the value of genotypes to determine athletic performance can perhaps be gained at looking at a range of performance genes and their implications. Such information can be taken from tables 4 and 5. For sprint performance genes, both subjects 3 and 4 display the RR allele for ACTN3, which is associated with fast contractile ability of muscles, which suggests these subjects are suited for power based activities. Subject 3’s DD allele of the ACE gene strengthens this suggestion. 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 favour performance in sprint 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

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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. However the VO2max scores do not clearly represent this. 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 genes for endurance, and had the lowest overall genotype score. This does not correlate with VO2max, scores, where subject 3 had the highest value.

Looking at the genetic evidence presented in these results, no accurate conclusions can be drawn without examining in detail the mechanisms of the genotypes and any adaptations that are suggested to 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 it can be said that the possibility of either total haemoglobin mass or genetic variation to determine athletic performance may exist, however with current levels of evidence on the subject there are no proven hypotheses. The importance of total Hb mass on endurance performance can perhaps be examined by approaching the subject from a different angle, in regards to the rising issue of blood doping. A rising number of cases are being examined regarding the blood manipulation, highlighting the importance of total Hb mass to 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 even more difficult to ascertain. At this current moment in time, due to the subject still being in its early stages, evidence is lacking to support any hypothesis that there is a particular genotype in any candidate gene which determines success in the athletic disciplines (Scott and Pitsiladis, 2007). At this stage all that can be said with any certainty is that the making of an athlete is complicated and multi-facetted, and is affected by a number of environmental and behavioural factors (Gayagay et al. 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.

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