Eur J Appl Physiol (2009) 105:715–721 DOI 10.1007/s00421-008-0953-x

O R I G I N A L A R T I CL E

Longitudinal changes in haemoglobin mass and VO2max in adolescents Annette Eastwood · Pitre C. Bourdon · Robert T. Withers · Christopher J. Gore

Accepted: 25 November 2008 / Published online: 16 December 2008 © Springer-Verlag 2008

Abstract This study assessed the relationship between haemoglobin mass (Hbmass) and maximum oxygen consumption (VO2max) in adolescents over 1 year. Twentythree subjects (11–15 years) participated; 12 undertook »12 months of cycle training (cyclists) and 11 were sedentary (controls). Hbmass and VO2max were measured approximately every 3 months. At baseline there was a high correlation (r = 0.82, P < 0.0001) between relative VO2max (ml kg¡1 min¡1) and relative Hbmass (g kg¡1). During 12 months there was a signiWcant increase in relative VO2max of the cyclists but not the controls; however, there was no corresponding increase in relative Hbmass of either group. The correlation between percent changes in relative VO2max and relative Hbmass was not signiWcant for cyclists (r = 0.31, P = 0.33) or controls (r = 0.42, P = 0.19). Training does not increase relative Hbmass in adolescents consistent with a strong hereditary role for Hbmass and VO2max. Hbmass may be used to identify adolescents who have a high VO2max. Keywords Training · Adolescents · Haemoglobin mass · Hereditary · Talent identiWcation

A. Eastwood (&) · P. C. Bourdon Sport Science Unit, South Australian Sports Institute, Brooklyn Park, P.O. Box 219, Adelaide, SA 5032, Australia e-mail: [email protected] A. Eastwood · R. T. Withers · C. J. Gore Exercise Physiology Laboratory, Flinders University, Adelaide, Australia C. J. Gore Department of Physiology, Australian Institute of Sport, Canberra, Australia

Introduction Performance in endurance events is highly correlated with maximum oxygen consumption (VO2max) (Craig et al. 1993; Heinicke et al. 2001). As oxygen supply to the muscle is regarded as a key factor in determining VO2max (Cain 1995; Saltin and Calbet 2006; Wagner 1996, 2006) and oxygen is transported primarily by haemoglobin (Hsia 1998), it follows that the total haemoglobin mass (Hbmass) aVects the oxygen transport capacity of the blood, and therefore VO2max (Gore et al. 1997). In adults, a high correlation between total haemoglobin and VO2max has been reported (Astrand 1952; Gore et al. 1997; Kanstrup and Ekblom 1984), emphasising the importance of the total amount of haemoglobin for obtaining a high VO2max and endurance performance (Kanstrup and Ekblom 1984). A high correlation between Hbmass and VO2max were reported in a group of elite runners and rowers (Gore et al. 1997); however, no changes in Hbmass occurred in the rowers in response to 12 week training despite increases in VO2max. Other studies have also demonstrated increases in VO2max independent of changes in red cell mass with periods of endurance training ranging from 12 days (Green et al. 1991) to 3 months (Glass et al. 1969). It is possible that changes in Hbmass occur over longer periods than that were monitored in these studies. Schmidt and Prommer (2008) showed increases in Hbmass of »6% during 9 months of aerobic training in leisure sportsmen, suggesting the need for more sustained longitudinal monitoring of Hbmass. Currently, very little research exists about the eVects of training on VO2max and Hbmass in adolescents. Hansen and Klausen (2004) investigated longitudinal changes in VO2max, haemoglobin concentration [Hb] and haematocrit (Hct) in 10- to 13-year-old males over a period of 4 years.

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They reported an increase in [Hb] over time, which was highly correlated to the increase in VO2max. However, measures of [Hb] cannot diVerentiate between a change in Hbmass and a change caused by plasma volume Xuctuations (Ashenden et al. 1999; Williams 1995). In order to monitor longitudinal changes in oxygen transport capacity, a reliable method of measuring Hbmass is required. The carbon monoxide (CO) rebreathing procedure of Schmidt and Prommer, (2005) provides an accurate and reliable estimate of Hbmass (Gore et al. 2006), which is safe for repeated measurements. It has been shown that individuals with no history of training may possess a high VO2max due to a naturally occurring high blood volume and Hbmass (Martino et al. 2002). In addition, cross-sectional studies reveal that endurance trained athletes have a larger Hbmass than sedentary subjects or power trained athletes (Brotherhood et al. 1975; Heinicke et al. 2001; Kjellberg et al. 1949). However, it is uncertain whether Hbmass increases as a result of training, whether individuals with a high Hbmass are predisposed to endurance sports, or a combination of both. The purpose of this study, therefore, was to investigate the association between Hbmass and VO2max in adolescents and secondly, to quantify the 12-month longitudinal changes in these variables as a result of growth (control group) compared with growth and training (training group).

Methods Subjects Twelve young cyclists (7 males, 5 females) aged 11– 15 years, and a group of 11 age-matched controls (5 males, 6 females) participated in this study. The cyclists were recruited from the South Australian Sports Institute Talent Search Cycling Program but were not in systematic training at the start of the study. The control subjects were selected randomly from the local community. All experimental procedures were approved by the Australian Institute of Sport Ethics Committee and written parental consent was obtained prior to any testing.

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prising three supervised training sessions per week and one to three unsupervised sessions per week. The control subjects reported that they completed 2.2 § 2.4 h per week of moderate physical activity during this 12-month period. VO2max was determined by an incremental exercise test to exhaustion on a calibrated, air-braked cycle ergometer (Clark et al. 2007). The test started at 100 W and increased by 25 W per minute for males, and 80 W increasing by 20 W per minute for females. The test was terminated when the subject could no longer complete the desired workload despite vigorous verbal encouragement. During the test, expired air was measured using a custom built VO2 system that has been described previously (Clark et al. 2007). The criteria of a plateau in oxygen uptake, a heart rate close to age predicted maximum, and a respiratory exchange ratio (RER) value of >1.1 were used to determine, whether the subjects reached VO2max (Howley et al. 1995). Hbmass was assessed using a modiWed version of the CO rebreathing procedure Wrst described by Schmidt and Prommer (2005). This modiWed version has been described in detail by Prommer and Schmidt (2007) and Eastwood et al. (2008) brieXy, this procedure comprised inhalation of a bolus of 99.5% chemically pure CO (BOC gasses, Sydney, Australia) in doses of 1 ml kg¡1 of body mass for the male cyclists, 0.8 ml kg¡1 for the female cyclists, 0.7 ml kg¡1 for the male controls and 0.6 ml kg¡1 for the female controls. Arterialised capillary blood samples (200
L) were taken from a pre-warmed Wnger tip and analysed in quintuplicate for percent carboxyhemoglobin (%HbCO) using a diode array spectrophotometer (OSM3 Hemoximeter, Radiometer, Denmark) before as well as, 6 and 8 min after commencing the rebreathing (Prommer and Schmidt 2007). Hbmass was calculated according to the method described by Schmidt and Prommer (2005). The duplicate measures of VO2max and Hbmass conducted on all 23 subjects at the commencement of the study were used to calculate the typical error (Hopkins 2000), as the standard deviation of the diVerence scores divided by q2, and the associated 90% conWdence limits (CL). The typical error for relative VO2max (and CL) was 3.6 (2.8–5.7)% and for relative Hbmass was 2.2 (1.6–3.4)%. The typical error for absolute VO2max was 3.0 (2.3–4.7)% and for absolute Hbmass was 2.3 (1.7–3.6)%.

Protocols Statistics VO2max and Hbmass were assessed twice within »2 weeks at the start of the study and once subsequently at each of »3, 6, 9, and 12 months thereafter. Both the cyclists and control subjects, were required to maintain activity log books throughout the study; however, four cyclists and two control subjects did not fully complete their log books. The cyclists reported that they completed 12 months of cycling training for a mean (§SD) »5.9 § 1.4 h per week, com-

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DiVerences between the cyclists and controls at the start of the study were assessed with unpaired, Student’s t tests. Linear regression was used to determine the Pearson product correlation between relative VO2max and relative Hbmass, as well as, between the change in VO2max and the change in Hbmass over 12 months. Linear modelling was used to compare the slope of the regression lines for the cyclists and

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controls at the end of the 12-month period. The changes in VO2max and Hbmass were also assessed using a repeated measures analysis of variance (ANOVA) for group (cyclists vs control) by time (0, 3, 6, 9, 12 months). Tukey post hoc tests were used to evaluate signiWcant diVerences between cell means. Statistica software (version 6.0, StatSoft Inc. Tulsa, OK, USA) was used for all statistical analyses.

Absolute Hbmass increased in both groups as a function of time (Fig. 1e). Relative Hbmass was signiWcantly higher in the cyclists than the controls at the start of the study (P = 0.035); however, in the subsequent 12 months there was not a signiWcant increase in relative Hbmass of the cyclists from 10.6 § 1.1 to 10.6 § 1.2 g kg¡1 (0.3 § 5.4%) or the controls from 9.7 § 1.0 to 9.4 § 1.0 g kg¡1 (¡2.3 § 8.1%) (Fig. 1f). The top six cyclists had a Hbmass of 11.3–12.0 g kg¡1.

Results

Relationship between Hbmass and VO2max

Percent HbCO values

There was a high correlation (r = 0.82) between relative VO2max (ml kg¡1 min¡1) and relative Hbmass (g kg¡1) for all 23 subjects at the start of the study (Fig. 2a). At the end of the 12-month period, there was also a signiWcant correlation between relative VO2max and relative Hbmass in both the cyclists (r = 0.84, P = 0.001) and the controls (r = 0.81, P = 0.003) (Fig. 2b). Comparison of the regression lines for all 23 subjects pre and post training revealed no signiWcant diVerences between the slopes (P = 0.28). The correlation between the percent change in relative VO2max and the percent change in relative Hbmass over 12 months was not signiWcant for either the cyclists (r = 0.31, P = 0.33) or the controls (r = 0.42, P = 0.19).

The raw HbCO values for all 23 subjects at baseline, minute 6 and 8 were 1.64 § 0.39, 6.52 § 0.83, 6.46 § 0.83% (mean § SD), respectively. The corresponding ranges were 0.67–2.60, 4.77–8.57, and 4.60–8.20% for baseline minute 6 and 8. Subject characteristics DiVerences in the subjects’ characteristics at the start of the study are reported in Table 1. Body mass increased signiWcantly (P < 0.0001) in both cyclists (mean § SD, 6.0 § 4.8%) and controls (11.0 § 8.7%) during the study (Fig. 1a), but this increase was not diVerent between groups. There was a signiWcant increase in height in both groups (2.1 § 1.7% cyclists, 3.2 § 1.8% controls), which was also not diVerent between the groups (Fig. 1b). BMI values at the end of the 12 month period were 19.6 § 1.8 and 19.7 § 2.0 kg m¡2 (mean § SD) for the cyclists and controls respectively. There was no signiWcant change in the BMI of all 23 subjects over the 12-month period (P = 0.39). VO2max and Hbmass over 12 months During the 12 months, absolute VO2max increased signiWcantly in the cyclists but not the controls (Fig. 1c). There was a signiWcant increase (11.3 § 8.9%) in relative VO2max of the cyclists from 57.8 § 7.6 to 64.2 § 8.6 ml kg min¡1, but no signiWcant change (¡3.3 § 10.2%) in the controls from 44.4 § 7.6 to 43.3 § 10.7 ml kg min¡1 (Fig. 1d).

Discussion This is the Wrst study, to investigate the relationship between Hbmass and VO2max in adolescents using an agematched control group. Similar to cross-sectional studies on adults (Astrand 1952; Gore et al. 1997; Kanstrup and Ekblom 1984) there was a signiWcant association between relative VO2max and relative Hbmass. This strong association suggests that the CO rebreathing method may be used as a test to identify adolescents who have a high Hbmass (for instance, ¸»11 g kg¡1) and, consequently, have a high VO2max. A key advantage of using the CO rebreathing method as a talent identiWcation test is that it is relatively simple to administer, requiring only 0.6 ml of Wnger tip blood. Furthermore, CO rebreathing is a valid and reliable method of estimating Hbmass (Burge and Skinner 1995; Gore et al. 2006; Schmidt and Prommer 2005) that does not

Table 1 Characteristics of the cyclists and controls at the start of the study Age (years)

Height (cm)

Body mass (kg)

BMI (kg m¡2)

VO2max (ml kg¡1 min¡1)

Hbmass (g kg¡1)

Cyclists (n = 12)

13.8 § 1.0

165.2 § 7.7

52.7 § 8.0

19.2 § 1.8

57.8 § 7.6

10.6 § 1.1

Controls (n = 11)

13.1 § 1.3

158.5 § 5.9

48.2 § 10.2

19.0 § 2.6

44.4 § 7.6

9.6 § 1.0

P value

0.17

0.03

0.25

0.84

0.0004

0.04

Values are mean and SD

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Eur J Appl Physiol (2009) 105:715–721

* * *

50

60

170

* * *

160

* * *

150

2

min -1) -1

VO 2max (L min

3

D

GroupxTime F (4,84) =6.92, p<0.0001 Time F (4,84) =4.73, p=0.002 Group F (1,21) =27.33, p<0.0001

VO 2max (ml kg

* * * *

-1

)

4

VO 2max=7.3xHb mass -23.0 r=0.82, p<0.0001 SEE = 5.85ml kg -1 min -1

A

VO 2max=5.9xHb mass +1.4 r=0.84, p=0.001 SEE=4.94ml kg -1 min -1

B

Controls Cyclists

40

C GroupxTime F (4,84) =6.70, p<0.0001 Time F (4,84) =24.74, p<0.0001 Group F (1,21) =21.24, p<0.0002

80

40 Height (cm)

* * *

B

GroupxTime F (4,84) =2.19, p=0.08 Time F (4,84)=43.19, p<0.0001 Group F(1,21) =4.66, p=0.04

180

60 Body mass (kg)

A

70

VO2max ( m l kg-1 m in-1 )

GroupxTime F (4,84)=1.72, p=0.15 Time F (4,84) =33.43, p<0.0001 Group F (1,21)=1.22, p=0.28

20

0 80

60

*

40

60

20

50

Cyclists Controls

40

VO2max=8.3xHb mass -34.8 r=0.81, p=0.003 SEE=6.65ml kg -1 min -1

0 6

30

8

10

12

14

-1

Hb mass (g kg ) GroupxTime F (4,84) =0.12, p=0.98 Time F (4,84) =7.47, p<0.0001 Group F (1,21) =4.08, p=0.056

E

GroupxTime F (4,84) =0.48, p=0.75 Time F (4,84) =1.67, p=0.16 Group F (1,21) =5.09, p=0.035

12

*

600

500

Hbmass(g kg -1)

700 Hbmass (g)

F

11 10 9

400 8 0

3

6

9

Time (months)

12

0

3

6

9

12

Time (months)

Fig. 1 Changes in body mass a height, b absolute VO2max, c relative VO2max, d absolute Hbmass, e and relative Hbmass, f over the 12-month period in the cyclists and controls. Asterisks denote a statistically signiWcant diVerence within a group relative to their own initial value (P < 0.05)

require the motivation, skill or physical exertion of a laboratory-based VO2max test (Gore et al. 1997) or of a time-trial such as a 1.6 km run (Safrit 1990), which can also be aVected as much by body composition as by aerobic Wtness (Rowland et al. 1999). A limitation of the CO rebreathing test is that it requires use of a hemoximeter with at least one decimal place resolution. Although CO is toxic in suYcient doses (Lilienthal 1950), the symptoms of its toxicity, including headache, drowsiness and lethargy are not appar-

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Fig. 2 a The relationship between relative VO2max (ml kg¡1 min¡1) and relative Hbmass (g kg¡1) for all 23 subjects at the start of the study. b The relationship between relative VO2max (ml kg¡1 min¡1) and Hbmass (g kg¡1) for the cyclists (n = 12) and controls (n = 11) at the end of the 12-month period. The slopes of the regression lines are not diVerent (P = 0.30)

ent until the level of HbCO exceeds 15% (Lilienthal and Pine 1946) or perhaps as high as 25% (Lilienthal 1950). Indeed a level of HbCO of 18% was used during exercise without incident by Gonzalez-Alonso et al. (2001). Smokers have chronic levels of HbCO of »5–6% (Hirsch et al. 1985; Klesges et al. 1992) which indicates that this level of CO, per se, is not detrimental. Moreover, the half-life of CO in the blood stream is »4 h (Roughton and Root 1945), which suggests that a dose of CO which yields a level of »6% as in the present study, is unlikely to be detrimental in adults and adolescents given the rate of clearance after completion of a test. Despite of 11% increase in relative VO2max in the cyclists during a year of training, there was no corresponding change (<1%) in their relative Hbmass during the same time period. Previous studies have reported that acute infusion of red blood cells increases both VO2max and Hbmass (Gledhill et al. 1999). Our results with adolescents contrasting with those of Gledhill et al. (1999) suggest that the cyclists’ increased VO2max was due to factors other than Hbmass, such as improved cardiac output, diVusing capacity of the lungs

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and muscles, oxidative enzyme activity, capillarisation and mitochondrial density (Saltin and Calbet 2006; Wagner 2006). Unfortunately, we conducted none of these more invasive measures on our cyclists so the mechanism(s) of their increase in VO2max is unclear. Nevertheless, our Wndings with adolescents are similar to those of Gore et al. (1997), who reported a 7.8% increase in VO2max in response to 12 weeks of rowing training in elite rowers yet no increase (¡0.2%) in Hbmass over the same time period. The subjects in the study of Gore et al. (1997) were highly trained and may therefore have limited scope for further augmentation of their Hbmass in response to training (Gore et al. 1998). Glass et al. (1969) also reported no change in Hbmass in highly trained cyclists in response to 2 months of intensive training. Despite little evidence of training induced changes in Hbmass in elite athletes, the hypoxia of moderate altitude can provide suYcient erythropoietic stimulus to increase Hbmass even in trained athletes (Heinicke et al. 2001; Wehrlin et al. 2006; Wehrlin and Marti 2006). Whilst the present study showed no increase in relative Hbmass in adolescents following 12 months of aerobic training, an increase in Hbmass of 6.4% was reported in recreational athletes following 9 months of aerobic training (Schmidt and Prommer 2008). This suggests a training dependent erythropoietic stimulation, which may be mediated by anabolic hormones (Hero et al. 2005), and therefore does not occur before puberty. Although absolute Hbmass increased in both cyclists and controls as a result of growth, relative Hbmass did not increase during 1 year in non-systematically trained adolescents who commenced a regular aerobic training program. Similar Wndings for adolescents were reported by von Dobeln and Eriksson (1972) who reported no change in Hbmass and a 17–20% increase in VO2max in a group of 12 untrained boys aged 11–13 years in response to 4 months of aerobic training. They concluded that despite of, the large change in VO2max, absolute Hbmass did not increase more than could be explained by growth. However, because there was no control group in the study of von Dobeln and Eriksson (1972), the eVects of growth were inferred from changes in body size. Collectively, our results and those of von Dobeln and Eriksson (1972) allude to the measurement of Hbmass in adolescents as being a valuable tool for talent identiWcation for endurance sports. It has been shown that adults with no history of training can have a naturally high VO2max, Hbmass and blood volume (Martino et al. 2002), suggesting that these factors are substantially inherited. Indeed, it has been reported that up to »60% of VO2max is inherited (Bouchard et al. 1998) and therefore Hbmass, with a typical error of »2% (Gore et al. 2006), could be a sensitive indicator of a high VO2max regardless of training status. In contrast to the present Wndings, it has been suggested that training increases total red cell mass in previously

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untrained adult subjects (Kjellberg et al. 1949; Remes 1979) and, in his comprehensive review, Sawka et al. (2000) concluded that the magnitude was »8–10% after at least 3 weeks of training. The increase in red cell volume is about a third of the magnitude of increase in VO2max (Green et al. 1991; Parisotto et al. 2001; Remes 1979); and the same relationship holds when Hbmass is artiWcially elevated with recombinant erythropoietin (reanalysis of data from Parisotto et al. 2001). In the current study, the cyclists increased VO2max by »11%, which is well outside of our typical error of »3% for these adolescents. Furthermore, if an increase in VO2max of 11% was associated with a onethird (»4%) increase in Hbmass, the latter should also have been detectable since our typical error for Hbmass was 2.2%. Further studies of the trainability of Hbmass in adolescents appear warranted. Tomkinson and Olds (2007) reported mean relative VO2max values of 43.8 ml kg¡1 min¡1 for 77,366 Australian adolescents aged 11–15 years based on the scores of the 20m shuttle run test, which were used to estimate VO2max using the equation of Leger et al. (1988). The VO2max values of the cyclists in our study (57.8 § 7.6 ml kg¡1 min¡1) correspond to almost the 99th percentile of the Australian population (57.9 ml kg¡1 min¡1) age 11–15 years at the start of the study. Tomkinson and Olds (2007) also reported VO2max values of 218,650 adolescents from 37 diVerent countries worldwide and showed that the scores of Australian adolescents are lower than those internationally. Given the increasing sophistication of international sport, it may be necessary to target individuals with relative VO2max values at or above the 95th percentile internationally for talent identiWcation for endurance sports. Based on the baseline regression equation of the current study, these percentiles would equate to a Hbmass of 11.2 g kg¡1 for males and 10.3 g kg¡1 for females aged 11–15 years. These values could be relevant as targets for young adolescents when measuring Hbmass for talent identiWcation purposes. The mean relative Hbmass values for the subjects at the start of this study (10.7 and 9.5 g kg¡1 for males and females, respectively) were higher than those reported by Astrand (1952) for 12- to 13-year-old males (8.8 g kg¡1), 14- to 15-year-old males (10.0 g kg¡1), 12- to 13-year-old females (7.8 g kg¡1) and 14- to 15-year-old females (7.6 g kg¡1). It has been shown that both absolute and relative values of Hbmass increase with the onset of puberty (Astrand 1952) particularly in males. Hero et al. (2005) reported an increase in Hb concentration and red blood cell volume following testosterone administration in boys with delayed puberty, and suggested that accelerated erythropoiesis was directly due to the eVects of androgens. One of the limitations of the current study is that no indicators of maturation, were obtained therefore the subjects may have been at diVerent maturational stages throughout the study. It is

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possible that the erythropoietic eVect of androgens during puberty may have caused increases in Hbmass in some subjects. Considering the diVerent rates of growth and maturation in adolescents and the resulting changes in body composition, it is also possible that a 12-month period may be insuYcient to detect changes in Hbmass in this population. Changes in body composition were not monitored in the present study and therefore, may confound the results. However, our subjects were not considered overweight their BMI did not change signiWcantly throughout the study. It has been suggested that lean body mass should be used as a reference parameter for Hbmass particularly in obese individuals (Pearson et al. 1995), therefore future studies should use measures of lean body mass when monitoring longitudinal changes in Hbmass. In conclusion, our results of no change of adolescents’ relative Hbmass during 1 year of intensive cycle training are consistent with the strong role of heredity for determining a high VO2max, for which oxygen transport via the total Hbmass is a key component. Therefore, measurement of Hbmass with the optimised CO rebreathing procedure (Prommer and Schmidt 2007; Schmidt and Prommer 2005) may be used as a talent identiWcation screening test to identify individuals who may also possess a high aerobic capacity. Acknowledgments Sincere thanks to Grant Tomkinson for the provision of VO2max percentiles for adolescents. We would also like to thank Julie Hill and Nick Herlihy for their assistance with data collection. This study was jointly funded by the South Australian Sports Institute and the National Elite Sports Council Australia.

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