Category: ergogenics


There is widespread anecdotal evidence that growth hormone (GH) is used by athletes for its anabolic and lipolytic properties. Although there is little evidence that GH improves performance in young healthy adults, randomized controlled studies carried out so far are inadequately designed to demonstrate this, not least because GH is often abused in combination with anabolic steroids and insulin. Some of the anabolic actions of GH are mediated through the generation of insulin-like growth factor-I (IGF-I), and it is believed that this is also being abused. Athletes are exposing themselves to potential harm by self-administering large doses of GH, IGF-I and insulin. The effects of excess GH are exemplified by acromegaly. IGF-I may mediate and cause some of these changes, but in addition, IGF-I may lead to profound hypoglycaemia, as indeed can insulin. Although GH is on the World Anti-doping Agency list of banned substances, the detection of abuse with GH is challenging. Two approaches have been developed to detect GH abuse. The first is based on an assessment of the effect of exogenous recombinant human GH on pituitary GH isoforms and the second is based on the measurement of markers of GH action. As a result, GH abuse can be detected with reasonable sensitivity and specificity. Testing for IGF-I and insulin is in its infancy, but the measurement of markers of GH action may also detect IGF-I usage, while urine mass spectroscopy has begun to identify the use of insulin analogues.
Keywords: GH, IGF-I, insulin, sport, abuse
Introduction
It is widely believed that growth hormone (GH) has been used by sportsmen and women since the 1980s to improve their athletic performance (McHugh et al., 2005) despite being banned for many years and appearing on the World Anti-Doping Agency list of banned substances.
The actions of GH that interest athletes are anabolic and lipolytic, leading to an increase in lean body mass and reduction in fat mass. Some of the anabolic GH actions are mediated through the generation of insulin-like growth factor-I (IGF-I), and there is anecdotal evidence that this too is being abused by athletes either alone or in combination with GH. The regulation of protein synthesis involves the synergistic actions of GH and IGF-I stimulating protein synthesis, while insulin simultaneously inhibits protein breakdown (Russell Jones et al., 1993). GH stimulates protein synthesis through a mechanism that is separate and distinct from anabolic steroids and therefore it seems likely that their effects will be additive. This has led many athletes to combine GH with insulin and anabolic steroids (Sönksen, 2001).
The GH doses used by athletes are thought to be up to 10 times higher than those used by endocrinologists, and as such athletes are putting themselves at risk of harmful effects such as hypertension, diabetes and Creutzfeld–Jakob disease.
The detection of GH poses the greatest contemporary challenge to the anti-doping community. The detection of GH abuse has proved difficult for several reasons. Unlike many substances of abuse, such as synthetic anabolic steroids, GH is a naturally occurring substance. The demonstration of exogenous administration must therefore rely on finding concentrations exceeding normal physiological levels while excluding pathological causes such as acromegaly. This is made harder because GH is secreted in a pulsatile manner with exercise and stress being major stimulators of GH secretion (Prinz et al., 1983; Savine and Sönksen, 2000). Consequently, GH concentrations are often at their highest in the immediate post-competition setting when most dope testing occurs. Recombinant human GH is almost identical to pituitary GH, whereas cadaveric GH, which is in plentiful supply on the Internet, is indistinguishable from endogenously produced GH. Blood testing is needed for GH and IGF-I because less than 0.1% is excreted unchanged and even that is erratic, rendering urine testing unfeasible (Moreira-Andres et al., 1993).
The review will explore why athletes abuse GH, IGF-I and insulin and also the methodology that has been developed to catch the cheats.
GH abuse in sport
Growth hormone was first extracted from human pituitary glands in 1945 (Li et al., 1945). It was shown to promote growth in hypopituitary animals and was soon used to restore growth in children with hypopituitarism. How and where GH was first used as a doping agent is unknown, but the earliest publication to draw attention to it was Dan Duchaine’s ‘Underground Steroid handbook’, which emerged from California in 1982 (Duchaine, 1982). Although it contains some fundamental errors, such as the recommendation and advertisement of animal GH for use in humans, the description of the actions of GH in this article was remarkably accurate and pre-dated adult endocrinology experience by about a decade. GH was described as the ‘most expensive, most fashionable and least understood of the new athletic drugs. It has firmly established itself in power-lifting and within a few years will be a commonly used drug in all strength athletics.’
The most famous case of GH abuse in professional athletics came to light in 1988 after Ben Johnson won the 100 m gold medal at the Olympic Games in Seoul. He was subsequently disqualified after stanazolol was detected in his urine, but at a later hearing, both he and his coach Charley Francis admitted under oath that he had taken human GH in addition to anabolic steroids.
It is impossible to determine the precise prevalence of GH abuse among sportsmen and women, as much of our evidence comes from anecdotal reports (McHugh et al., 2005). Although initially advocated for strength disciplines, endurance athletes are also attracted to GH’s lipolytic actions and reduced fat mass; in 1988, a large quantity of GH was found in a team car at the Tour de France.
There is evidence that adolescents are using GH. In a survey of two US high schools, 5% of male students admitted to having taken GH and nearly one-third knew someone who had taken GH (Rickert et al., 1992). Most GH users were unaware of its side effects and reported their first use between 14–15 years of age.
Growth hormone is an expensive drug and this has led some parents to sell GH prescribed to treat their child’s GH deficiency on the black market. Officials have also misappropriated GH for their athletes. At the 1998 World Swimming Championships, Yuan Yuan, a Chinese swimmer, was stopped on entry into Perth with a suitcase full of GH that had been exported to China for therapeutic reasons.
A few athletes have admitted to taking GH. In a death-bed confession, Lyle Alzado, an American football player, admitted that 80% of American footballers have taken GH. In 2000, Australian discus champion Walter Reiterer claimed institutional and supervised usage of GH. With this in mind, it is interesting to note that 6 months before the Sydney Olympic Games, 1575 vials of GH were stolen from an importer’s warehouse in Sydney.
More recently, Victor Conte, the owner of the Bay Area Laboratory Co-Operative, claimed that he had supplied GH to many high-profile American athletes including Tim Montgomery and Marion Jones. This admission came after the raid on the Bay Area Laboratory Co-Operative’s headquarters on 3 September 2003, when evidence of systematic doping was found and many of the top names in athletics, baseball and American football were implicated in the scandal. Although many have denied taking GH, Tim Montgomery allegedly admitted to taking GH before a US Federal grand jury and later faced a 2-year ban for doping offences. Conte was imprisoned for 4 months for his role in the scandal (Fainaru-Wada and Williams, 2006).
The recent conviction of Sylvester Stallone, who was caught with GH in his possession on entering Australia, suggests that GH is readily available in athletic and body-building circles.
IGF-I abuse in sport
The prevalence of IGF-I abuse is probably much lower than for GH because, unlike GH, there is no readily available natural source, and therefore all IGF-I is obtained through recombinant DNA technology. Two companies currently market IGF-I, and these preparations have only recently received approval for use in humans to treat growth failure in children with severe primary IGF-I deficiency or with GH gene deletion who have developed neutralizing GH antibodies. The first product is Increlex or recombinant human IGF-I, manufactured by Tercica, and the second product manufactured by Insmed is Iplex, which differs from Increlex in that the recombinant human IGF-I is supplied bound to its major binding protein, IGFBP-3 (Kemp et al., 2006; Kemp, 2007).
Although in relatively short supply, other companies make IGF-I for cell culture and other uses, and this material could also become available to athletes. The wider availability of IGF-I, together with an appreciation of the efforts to detect GH abuse, is likely to increase its illicit use by athletes, despite being prohibited by the World Anti-Doping Agency.
Abuse of insulin in sport
We have only sketchy details about the use of insulin by professional athletes. It is alleged that short-acting insulin is being used in a haphazard way to increase muscle bulk in body builders, weight lifters and power lifters (Sönksen, 2001). After concerns raised by the Russian medical officer at the Nagano Olympic games, the International Olympic Committee immediately banned its use in those without diabetes. Athletes with insulin-requiring diabetes may use insulin with a medical exemption.
Physiology of the GH–IGF axis
Growth hormone is the most abundant pituitary hormone and is secreted in a pulsatile manner under the control of the hypothalamic hormones, GH-releasing hormone, somatostatin and ghrelin (Figure 1).
Figure 1
Figure 1

The growth hormone (GH)–insulin-like growth factor (IGF)-I axis. GH is secreted from the pituitary gland under the control of the hypothalamic hormones, somatostatin, Ghrelin and GH-releasing hormone (GHRH). GH circulates bound to its binding (more …)
Regulation of GH secretion
Growth hormone-releasing hormone and ghrelin stimulate the synthesis and release of GH, whereas somatostatin is inhibitory in action (Veldhuis, 2003). Although ghrelin is secreted by the hypothalamus, the major source of ghrelin is the stomach and it is thought to be one mechanism that controls the GH response to eating (van der Lely et al., 2004). Circulating IGF-I reduces GH secretion through classical negative endocrine feedback (Carroll et al., 1997).
There are a number of physiological stimuli that increase or inhibit GH release, the most important of which are exercise and sleep (Savine and Sönksen, 2000). The highest GH peaks occur at night within the first hour of sleep during slow-wave sleep (Takahashi et al., 1968). Nutritional status regulates GH secretion both acutely and chronically; hypoglycaemia, reduced circulating free fatty acids (FFA) and higher amino-acid concentrations all increase GH secretion (Ho et al., 1988; Pombo et al., 1999), whereas in the longer term, GH secretion is increased in anorexia nervosa and decreased with obesity (Veldhuis et al., 1991; Argente et al., 1997).
Age and gender are important determinants of GH secretion (Savine and Sönksen, 2000). Secretion is highest during the pubertal growth spurt, and after the mid-20s, GH secretion decreases by 14% every decade (Toogood, 2003), and this decrease may be responsible for some of the age-related body composition changes (Holt et al., 2001). Women have higher baseline GH secretion but the pulses are not as high and are less erratic than in men (van den Berg et al., 1996).
The actions of GH and IGF-I
Growth hormone exerts its multiple metabolic and anabolic actions through binding to specific GH receptors that are found on every cell of the body (Holt, 2004). Following binding to the GH receptor, the tyrosine kinase Janus kinase 2 is activated and multitude of signalling cascades are initiated that result in the wide variety of biological responses, including cellular proliferation, differentiation and migration, prevention of apoptosis, cytoskeletal reorganization and regulation of metabolic pathways (Lanning and Carter-Su, 2006). Although a detailed description of these signalling cascades is beyond the scope of this review, it is worth remarking on the number of signalling proteins and pathways activated by GH, which include JAKs, signal transducers and activators of transcription, the mitogen-activated protein kinase pathway, and the phosphatidylinositol 3′-kinase pathway. Although these pathways are well described, the inter-relationship between the different pathways is not fully understood.
Growth hormone exerts most of its anabolic actions through the generation of circulating IGF-I (the somatomedin hypothesis) (Le Roith et al., 2001), the majority of which is produced in the liver. IGF-I may also act in a paracrine or autocrine fashion in response to GH action at other target tissues. Transgenic animals, in which the IGF-I gene has been selectively deleted in the liver and whose serum IGF-I is marked reduced, have led some to question the somatomedin hypothesis as these animals appear to grow normally (Sjogren et al., 1999; Yakar et al., 1999), even though with the development of insulin resistance (Sjogren et al., 2001; Yakar et al., 2001). It is known, however, that the total amount of circulating IGF-I in an adult is not required for normal growth, as neonates have much lower circulating levels but grow very rapidly. The precise mechanisms regulating IGF-I bioavailability to the tissues are not yet understood but certainly involve IGF-binding proteins (IGFBPs). As IGFBP distribution in liver IGF-I knockout mice, as in newborn humans, is quite different from that in healthy adults, it has been argued that these data do not really challenge the view that endocrine IGF-I has a role in growth regulation.
Effects on whole body physiology
The physiological effects of GH are best examined by considering the condition of adult GH deficiency. Studies of hypopituitary individuals with appropriate replacement of thyroid, steroid and sex steroid replacement have shown that GH plays a pivotal role in body composition, well-being, physical performance and cardiovascular health (Cuneo et al., 1992; Carroll et al., 2000). In the absence of GH, lean tissue is lost and fat accumulates, and correspondingly waist-to-hip ratio increases as visceral fat increases (Table 1).
Table 1
Table 1

Clinical features of GH deficiency and effect of GH replacement
Effects on intermediate metabolism
Protein metabolism

Protein synthesis and degradation are each regulated by multiple hormonal and nutritional factors, and protein turnover in individual tissues and the whole body is in a state of constant flux. Insulin, GH and IGF-I have synergistic anabolic effects on protein metabolism (Figure 2) (Sönksen, 2001).
Figure 2
Figure 2

The synergistic action between insulin, IGF-I and GH in regulating protein synthesis. Without insulin, GH loses much (if not all) of its anabolic action. GH and IGF-I stimulate protein synthesis directly, whereas insulin is anabolic through inhibition (more …)
Growth hormone causes nitrogen retention, as shown by decreased urinary excretion rates of urea, creatinine and ammonium. In healthy humans, acute administration of GH modestly stimulates muscle and whole body protein synthesis (Fryburg et al., 1991). When GH is infused locally into the brachial artery, forearm muscle protein synthesis increases, whereas systemic IGF-I concentrations and whole body protein turnover are unchanged, indicating that GH stimulates protein synthesis directly as well as indirectly through IGF-I (Fryburg et al., 1991).
Insulin-like growth factor-I has an anabolic effect on protein metabolism, inhibiting whole body protein breakdown and stimulating protein synthesis (Fryburg, 1994). This effect is dependent on serum insulin and an adequate supply of amino acids. After IGF-I is administered systemically, serum insulin and amino acids decrease, the former through decreased production and the latter as a result of increased clearance from the blood. This attenuates the stimulation of whole body protein synthesis, but if the amino acids and insulin are replaced during the administration, the effect on protein synthesis by IGF-I is clearly seen (Russell Jones et al., 1994; Jacob et al., 1996).
Protein breakdown is inhibited by physiological concentrations of plasma insulin, the ‘Chalonic’ action of insulin (Umpleby and Sönksen, 1985; Tessari et al., 1986). In contrast, the rates of protein degradation are increased in cardiac and skeletal muscles in situations where insulin concentrations are low such as type 1 diabetes or starvation (Charlton and Nair, 1998). Although the rate of protein synthesis is reduced by 40–50% in young diabetic rats, a physiological anabolic effect of insulin on protein synthesis has not been confirmed in humans (Liu and Barrett, 2002).
The similarities between IGF-I and insulin suggest that these proteins act in a coordinated manner to regulate protein turnover. There are interesting differences, however, in their respective dose–response curves. Low physiological insulin concentrations inhibit protein breakdown and increase glucose disposal into skeletal muscle, whereas higher, non-physiological concentrations are required to stimulate protein synthesis (Louard et al., 1992). In contrast, increases in IGF-I that have no effect on glucose uptake stimulate protein synthesis, and higher concentrations are required to inhibit protein breakdown (Fryburg, 1994). During the last decade, there have been advances in the understanding of the intracellular signalling mechanisms of insulin and IGF-I, many of which are shared, such as insulin receptor substrate 1. The precise mechanism by which these similar but divergent pathways interact is not fully understood, but IGF-I, similar to GH, most likely acts through stimulating amino-acid uptake.
Lipolysis
The administration of GH to humans, either by continuous infusion or by a bolus injection, leads to stimulation of lipolysis and increased fasting FFA concentrations with the peak effect around 2–3 h after the injection (Hansen, 2002). These findings are in keeping with the changes in FFAs following physiological stimuli of GH secretion. In young healthy subjects, the nocturnal or exercise-induced peak of GH precedes the peak of FFAs by 2 h (Moller et al., 1995). Furthermore, during times of fasting or energy restriction, the lipolytic effect of GH is enhanced, although the effect is suppressed by co-administration of food or glucose (Moller et al., 2003). Acromegaly is associated with increased circulating FFAs, increased muscle uptake of FFAs and increased lipid oxidation.
Although GH receptors are abundantly expressed on adipocytes, it has been suggested that GH has a permissive effect on catecholamine-induced lipolysis (Hansen, 2002). In vitro studies have shown that GH has no direct lipolytic effect on human fat cells, but markedly increases the maximal lipolysis induced by catecholamines (Marcus et al., 1994).
Glucose homeostasis
The first observation that GH had an effect on glucose metabolism came in the 1930s when it was observed that hypophysectomy ameliorated the hyperglycaemia of experimental diabetes in dogs (Houssay and Biasotti, 1930). In both healthy subjects and those with type 1 diabetes, GH increases fasting hepatic glucose output, by increasing hepatic gluconeogenesis and glycogenolysis, and decreases peripheral glucose utilization through the inhibition of glycogen synthesis and glucose oxidation (Tamborlane et al., 1979; Bak et al., 1991; Fowelin et al., 1991, 1993, 1995). Patients with acromegaly develop insulin resistance and hyperinsulinaemia (Sönksen et al., 1967), and up to 40% become diabetic (Ezzat et al., 1994; Colao et al., 2000).
Although the effect on glucose homeostasis would appear to be disadvantageous for the athletes, it is worth bearing in mind that long-standing adult GH deficiency is associated with insulin resistance and that any acute effect on glucose homeostasis does not take into account changes in IGF-I, which also affects insulin sensitivity (Salomon et al., 1994).
Intravenous IGF-I causes hypoglycaemia in rats by stimulating peripheral glucose uptake, glycolysis and glycogen synthesis, although having only a minimal effect on hepatic glucose production (Jacob et al., 1989). In dogs, however, IGF-I has been shown to suppress hepatic glucose output, but to a lesser degree than insulin at the doses used, as well as increasing peripheral glucose utilization (Shojaee Moradie et al., 1995). As hepatic expression of the IGF-I receptor is reportedly low (Stefano et al., 2006), it is possible that this effect of IGF-I on hepatic glucose output is by an indirect mechanism. For example, IGF-I may bind the insulin receptor with low affinity (Holt et al., 2003). Alternatively, it may improve whole body insulin sensitivity by inhibiting the secretion of GH.
The effects of an intravenous IGF-I infusion in humans are similar to those described in animals and lead to hypoglycaemia (Zenobi et al., 1992). Insulin sensitivity increases with respect to glucose by IGF-I through increased peripheral glucose uptake and decreased hepatic glucose production (Boulware et al., 1994; Russell Jones et al., 1995). A subcutaneous infusion of IGF-I also causes hypoglycaemia, but the effect is slower in onset than insulin and decreases more slowly after the infusion was stopped, because of the presence of the IGF-binding proteins.
Bone metabolism
Growth hormone has profound effects on bone metabolism. GH deficiency is associated with osteopaenia, which is reversed by GH replacement (Gomez et al., 2000). Male subjects with osteoporosis have reduced GH peaks and low serum IGF-I concentrations, and furthermore, IGF-I concentrations correlate well with estimates of bone mineral density (Patel et al., 2005). In addition to a direct effect on bone stimulating the cycle of bone growth, there is evidence to suggest that GH and IGF-I may modify intestinal calcium absorption and serum 1,25 hydroxy vitamin D concentrations. Patients with acromegaly have increased intestinal calcium absorption (Lund et al., 1981), and GH therapy in pigs increases intestinal calcium absorption, probably through increased production of serum 1,25 hydroxy vitamin D (Chipman et al., 1980). GH replacement therapy in adult GHD leads to a short-term increase in serum 1,25 hydroxy vitamin D concentrations (Burstein et al., 1983). These data suggest that GH increases intestinal calcium absorption by its effects on renal 25 (OH)D 1-α-hydroxylase activity, but other mechanisms may also be involved (Halloran and Spencer, 1988).
Why do athletes abuse GH?
The importance of GH in adult physiology as well as in children was confirmed beyond doubt in 1989 by two independent studies undertaken in the UK and Denmark (Jorgensen et al., 1989; Salomon et al., 1989). Both groups undertook double-blind, placebo-controlled trials in adults with hypopituitarism given appropriate replacement therapy with everything except GH. The studies showed remarkably congruous results after 6 months of GH treatment.
The most impressive finding was a change in and normalization of body composition with an average 6 kg increase in lean body mass, largely accounted by an increase in skeletal muscle, and a concomitant loss of fat. The body composition changes were accompanied by improvements in quality of life, particularly in the area of ‘increased energy’ and performance enhancements (McGauley et al., 1990; Cuneo et al., 1991a, 1991b). Longer studies over the first 3 years of GH replacement showed that exercise performance continued to improve (Jorgensen et al., 1994).
Does GH enhances performance in normal healthy young adults?
There has been considerable debate about the ability of GH to translate these body composition and other metabolic changes into improved performance (Rennie, 2003). Despite the theoretical benefits of GH, sceptics point to the condition of acromegaly and negative clinical studies as evidence of a lack of benefit.
Acromegaly—nature’s experiment of GH excess
It has been argued that acromegaly, where there is over-secretion of GH usually from a pituitary adenoma, provides evidence that excess GH is not performance enhancing, as it is not associated with athletic prowess. Indeed, acromegaly is usually associated with muscle weakness rather than excessive strength (Table 2) (McNab and Khandwala, 2005). It needs to be appreciated, however, that acromegaly frequently remains undiagnosed for many years and the clinical presentation at diagnosis may not reflect earlier stages of the disease. Many patients if questioned carefully will give a history of increased strength in the first few years of their condition. Indeed, we know a rower who competed at an elite level during the early stages of his acromegaly. Not only was he one of the strongest crew, but he could also tolerate harder training sessions than his colleagues and recovered more quickly afterwards (PHS, unpublished data).
Table 2
Table 2

Clinical features of acromegaly
Although only a clinical case report, it illustrates that the timing and degree of GH excess are important for the physiological effect. Prolonged massive GH excess coupled frequently with deficiencies of other pituitary hormones, such as ACTH, may tell us little about the effects of lesser GH excess earlier in the natural history of the illness.
Can clinical trials provide us with the answer?
Although there have been many negative studies in this area, traditional randomized controlled trials that have the power to examine differences of 20–30% are ill suited to detect the much smaller differences that determine whether an individual wins a gold medal or not. Despite these difficulties, one trial showed that, in healthy, elderly men, the combination of GH and testosterone led to a 20% improvement in fitness as measured by maximal oxygen uptake (Giannoulis et al., 2006), which was larger than with either compound alone. Very recently, a well designed and executed study in past abusers of anabolic steroids showed for the first time an ergogenic effect of GH in healthy young athletes (Graham et al., 2008).
A further problem with the designs of our clinical trials is that they are designed to test one or at most two interventions. In reality, for the reasons described above, GH is frequently used by athletes in combination with insulin and anabolic steroids in varying concentrations during differing training and dietary regimens. It is impossible to control for all these variables within a single trial, and therefore it seems likely that the athletes using the ‘n=1′ design are probably best placed to address the question whether GH is performance enhancing. This certainly appeared to be a potent weapon when abused by the former East German coaches (Franke and Berendonk, 1997).
A definitive answer to this question will probably never be found, as it would be difficult to obtain ethics approval for a suitable study. It should be remembered, however, that there were similar arguments about anabolic steroids 20 years ago that have subsequently been shown to have performance benefits.
Pharmacology of GH, IGF-I and insulin administration
The half-life of endogenously secreted GH is around 13 min (Sohmiya and Kato, 1992). It is rapidly cleared from the body by the liver, kidney and peripheral tissues through interaction with GH receptors. If recombinant human GH (rhGH) is administered intravenously, the half-life is similar to endogenously secreted GH (Refetoff and Sönksen, 1970; Haffner et al., 1994). In practice, however, the pharmacokinetics of exogenously administered GH differs, as it is given by intermittent, usually daily, subcutaneous injection. Following injection, GH concentrations increase and reach a maximum concentration after about 2–6 h, depending on the age and gender of the recipient (Kearns et al., 1991; Janssen et al., 1999). The estimated bioavailability of rhGH is 50–70%, because of degradation at the site of injection. This may explain why intramuscular injection results in a higher maximal and area-under-the-curve GH concentration than subcutaneous injection (Keller et al., 2007). Thereafter, rhGH is rapidly cleared and GH is usually undetectable in women 12 hours after injection, even after high doses, while men have only low levels of GH. (Giannoulis et al., 2005). There is quicker clearance of GH in women, probably reflecting their extra body fat that contains a high density of GH receptors (Vahl et al., 1997). Longer-acting GH preparations are currently being developed.
The pharmacokinetics of IGF-I is complicated by the presence of a family of highly specific binding proteins (IGFBPs) that coordinate and regulate the biological functions of IGF-I. Less than 5% of serum IGF-I is free and most is bound in a ternary complex of IGF-I, IGFBP-3 and an acid-labile subunit (Figure 1). The half-life of free IGF-I is only a few minutes, whereas the half-lives of IGF-I bound in a binary and ternary complex are 20–30 min and 12–15 h, respectively (Guler et al., 1989). When subcutaneous IGF-I is administered to healthy volunteers, the maximum concentration is achieved at about 7 h and half life is 20 h (Grahnen et al., 1993). The half-life is prolonged when IGF-I is administered as a complex with IGFBP-3. In patients with severe GH insensitivity syndrome, IGF-I concentrations peaked between 15 and 19 h after the injection of the complex, and a single injection was effective in increasing IGF-I concentrations in these patients for a 24 h period (Camacho-Hubner et al., 2006).
The half-life of intravenous insulin is only 4 min, but apart from the treatment of diabetic emergencies and possibly the replenishment of glycogen using an insulin clamp (Sönksen and Sönksen, 2000), pharmacologically administered insulin is through subcutaneous injection (Matthews et al., 1985). Insulin manufacturers have developed many preparations of insulin including insulin analogues in the attempt to provide insulin replacement to people with diabetes in the most physiological way (Peterson, 2006). Consequently, the shortest-acting insulin analogues appear in the circulation within 5–10 min of injection and cleared within 4–6 h, whereas longer-acting insulins are present for over 24 h.
Potential adverse effects of GH administration
The side effects of GH administration to adults with GH deficiency are well documented, and any athlete receiving GH will potentially be at risk of these side effects (Powrie et al., 1995). It is believed, however, that many athletes are using doses that are up to 10 times higher than those used therapeutically. The effects of chronically administering this dose of GH are unknown, but it would be reasonable to expect that athletes may develop some of the features of acromegaly with prolonged use (Table 2).
Sodium and fluid retention
Growth hormone causes fluid retention through its action on the kidney to promote sodium reabsorption (Powrie et al., 1995; Moller et al., 1999). This may be manifest as ankle swelling, hypertension and headache.
Diabetes
Patients with acromegaly develop insulin resistance and hyperinsulinaemia, and up to 40% become diabetic (Sönksen et al., 1967; Ezzat et al., 1994; Colao et al., 2000).
Cardiomyopathy
Cardiovascular complications are a major cause of morbidity and mortality in patients with acromegaly (Colao et al., 2001). The excess of GH and IGF-I causes a specific derangement of cardiomyocytes, leading to abnormalities in cardiac muscle structure and function, inducing a specific cardiomyopathy. In the early phase, there is a hyperkinetic syndrome, characterized by increased heart rate and systolic output. Two-thirds of patients have concentric cardiac hypertrophy and this is commonly associated with diastolic dysfunction and eventually with impaired systolic function leading to heart failure, if the acromegaly is left untreated. In addition, abnormalities of cardiac rhythm and those of heart valves have also been described. The coexistence of arterial hypertension and diabetes may further aggravate acromegalic cardiomyopathy. It is alleged that the American sprinter, Florence Griffith-Joyner (Flo Jo), purchased GH from fellow sprinter Darrell Robinson. When Flo Jo died at the age of 38 years, her heart was enlarged consistent with cardiomyopathy (Sullivan, 1998).
Cancer
Although controversial, the consensus statement of the Growth Hormone Research Society (2001) was that there is no increased risk of cancer when GH is given at physiological replacement doses (2001). There is evidence to suggest that acromegaly, where GH levels have been much higher than physiological doses for many years, may be associated with increased rates of colorectal, thyroid, breast and prostate cancers (Jenkins et al., 2006).
Creutzfeld–Jakob disease
Initially, the only source of GH came from extracts of human pituitary glands, and tragically, this was discovered to be a source for the prion-induced Creutzfelt–Jacob disease (Brown et al., 1985). As a result, pituitary-derived GH was withdrawal from the market place in 1985 and was replaced with recombinant human GH in 1987. Despite the dangers, supplies of pituitary-derived GH continue to be available on the black market to this day, and athletes continue to use this and thus a case of Creutzfelt–Jacob disease may emerge in an elite athlete at some time in the future.
Potential adverse effects of IGF-I and insulin administration
We have only limited experience with the use of exogenous IGF-I, and so most of the known side effects relate to short-term usage only. It seems reasonable to hypothesize, however, that many of the longer-term effects of GH administration would also occur with IGF-I, as the anabolic effects of GH are closely related to the production of IGF-I in different tissues.
In the clinical trials, the commonest short-term side effects are oedema, headache, arthralgia, jaw pain and hypoglycaemia (Kemp et al., 2006; Kemp, 2007). These appear to be more marked when IGF-I is used alone as the recombinant human IGFBP-3 seems to buffer the acute effects of IGF-I.
The side effects of insulin are well documented from our experience in treating people with diabetes. The most commonly experienced side effect is hypoglycaemia. Weight gain is also a problem in people with diabetes, but this is probably less of an issue for athletes whose diet and training regimens are closely monitored.
Detection of GH abuse
Two complementary approaches have been investigated to detect GH abuse: the first is based on the detection of different pituitary GH isoforms, whereas the second relies on measurement of GH-dependent markers. The very different approaches are viewed as a major strength, as their different properties mean that they are useful in different situations.
The isoform or differential immunoassay method
Growth hormone exists as multiple isoforms; 70% of circulating GH is in the form of a 22-kilo Dalton (kDa) polypeptide, whereas 5–10% occurs as a 20 kDa isoform, from mRNA splicing. Dimers and oligomers of GH exist as do acidic, desaminated, acylated and fragmented forms (Baumann, 1999). The differential immunoassay approach is based on the principle that endogenous GH occurs as a number of isoforms, whereas rhGH contains only the 22 kDa isoform (Figure 3). When rhGH is administered in sufficiently high doses, there is a suppression of endogenous GH secretion through negative feedback to the pituitary, and therefore the ratio between 22 kDa GH and non-22 kDa GH is increased (Bidlingmaier et al., 2003). This change in ratio can be detected with specific immunoassays that distinguish the different isoforms.
Figure 3
Figure 3

Principle of the isoform method. rhGH contains 22 kDa and this is specifically recognized by assay 1. Pituitary GH contains multiple isoforms and these are recognized by assay 2. When rhGH is administered, endogenous production of pituitary GH (more …)
The isoform method was first established by Christian Strasburger and Martin Bidlingmaier in Germany by employing one assay that specifically measured 22 kDa GH and another permissive assay that measured all GH isoforms (Figure 3). A slightly different approach has been adopted by an Australian Japanese Consortium that developed assays that specifically measure either 22 or 20 kDa GH (Momomura et al., 2000).
When the German method is applied to a normal population, the ratio between 22 kDa and total GH is less than 1 with a normal distribution of values, whereas individuals receiving GH have values that are greater than one (Figure 4) (Wu et al., 1999). Age, sex, sporting discipline, ethnicity and pathological states do not seem to affect the relative proportions of GH isoforms (Holt, 2007), but exercise causes a transient relative increase in the 22 kDa isoform, thereby lowering the sensitivity of the test if samples are taken immediately after competition (Wallace et al., 2001a, 2001b).
Figure 4
Figure 4

Ratio between 22 kDa-hGH assay and total hGH assay in serum samples obtained from 125 controls and 30 individuals treated with rhGH. Mean values are 1.43±0.21 for treated individuals and 0.50±0.12 for controls (P<0.0001). (more …)
The short half-life and rapid clearance of rhGH, even when injected subcutaneously, means that the ‘window of opportunity’ for detection of GH doping with this test is less than 36 h (Keller et al., 2007). As GH is usually administered in the evening, GH is frequently undetectable in a blood sample taken the following day (Giannoulis et al., 2005). The 20 kDa GH remains suppressed for 14–30 h in women depending on the dose used, whereas in men, 20 kDa GH remains undetectable for up to 36 h (Keller et al., 2007). Spontaneous GH secretion returns 48 h after the last dose of rhGH treatment (Wu et al., 1990). Consequently, any athlete who stops administering GH several days before the competition will not be detected. Thus the isoform method is unlikely to catch a GH abuser in the classical ‘post competition’ dope testing scenario, and the optimal use of this method must be in unannounced ‘out of competition’ testing. This method does not detect individuals receiving cadaveric GH, IGF-I or GH secretagogues.
This test was introduced at the Athens and Turin Olympic Games and no positives were detected in samples taken ‘post competition’. One of the World Anti-Doping Agency rules for the use of immunoassays is that two antibodies recognizing different epitopes are needed for each analyte. The German group had carefully characterized their assays and antibodies, and this may have been an overriding factor in the decision to implement the test.
The GH-dependent marker method
Growth hormone administration leads to the alteration of the concentrations or ratios of several serum proteins, and this change may be used as a means of detecting exogenous GH. An ideal marker or combination of markers would have well-defined reference ranges, would change in response to GH administration and would remain altered after GH has been discontinued (Holt, 2007). The marker should be largely unaffected by other regulators of GH secretion such as exercise or injury and should be validated across populations.
This approach was pioneered by the large multi-centre GH-2000 project coordinated by Peter Sönksen with funding from the European Union under their BIOMED 2 initiative, with additional funding from the International Olympic Committee and the rhGH manufacturers Novo Nordisk and Pharmacia. The aim was to develop a test in time for the Sydney Olympic Games. It had three main components, the first of which was a cross-sectional study of elite athletes at National or International events to establish a reference range of selected markers of GH action (Healy et al., 2005). Blood samples taken within 2 h of competitions showed that the markers were dependent on age as is the case in the general population, but in contrast, sporting discipline, gender and body shape had little effect. The findings of this study were subsequently confirmed by the Australian Japanese consortium led by Ken Ho. In a study of 1103 elite athletes sampled out of competition, less than 10% of the total variance of the markers was explained by gender, sporting discipline, ethnicity and body mass index, whereas age contributed to between 20 and 35% of the variance (Nelson et al., 2006).
The second component of the GH-2000 project was the ‘wash-out’ study (Wallace et al., 1999, 2000). When the project was conceived, 25 potential markers of GH action were considered. The aim of the ‘wash-out’ study was to narrow this number down to the most suitable markers for a more in-depth analysis. rhGH was administered to recreational male athletes for 1 week, with blood samples collected during and after the GH administration. Subjects also undertook exercise tests to assess the potential effect of ‘competition’ on the markers. Nine markers, either members of the IGF–IGF-binding protein axis or markers of bone and soft tissue turnover, were then selected for analysis in the third component of the GH-2000 project. This was a 28-day GH administration study involving self-administered rhGH at two doses to 102 recreational athletes under double-blind, placebo-controlled conditions to evaluate the potential markers for their ability to discriminate active drug from placebo and to assess the ‘window of opportunity’ when the test remained positive after rhGH was stopped (Dall et al., 2000; Longobardi et al., 2000).
From these studies, the GH-2000 project proposed a test based on IGF-I and type 3 pro-collagen (P-III-P) (Powrie et al., 2007) (Figure 5). These markers were chosen because they provided the best discrimination between individuals receiving GH or placebo during the randomized controlled trial. They exhibit little diurnal or day-to-day variation and are largely unaffected by exercise or gender (McHugh et al., 2005). In the wash-out study, IGF-I and P-III-P increased 20 and 10.2%, respectively, following exercise, but this increase was small in comparison with the larger 300% increase in the markers with GH (Wallace et al., 1999, 2000). Although discrimination was the prime reason for the selection, it is important to note that these proteins are produced by different tissues, thereby reducing the number of pathological conditions that could lead to an elevation in both markers and potential false-positives.
Figure 5
Figure 5

Change in IGF-I (a) and P-III-P (b) following the administration of GH or placebo for 28 days to 50 healthy male volunteers.
It is known that there is sexual dimorphism in the GH–IGF axis. There are small differences in IGF-I and P-III-P concentrations in elite male and female athletes (Healy et al., 2005), although gender explained around 1% of the overall variance in these markers (Nelson et al., 2006). Women are known to be inherently more resistant to the actions of GH and so the increase in markers is less pronounced in women than in men (Dall et al., 2000; Longobardi et al., 2000). This potential disadvantage may be offset because women may need to receive higher doses to obtain a performance-enhancing benefit.
Although a single marker could be used, by combining markers in conjunction with gender-specific equations, ‘discriminant functions’, the sensitivity and specificity of the ability to detect GH abuse can be improved compared with single-marker analysis (Powrie et al., 2007).
The procedure used to generate the discriminant functions involves splitting the available data into two: a ‘training’ set of data is used to calculate the discriminant function and a ‘confirmatory’ set is then used to validate the sensitivity and specificity of the discriminant function. The confirmatory set required to ensure the model is applicable to the general population and not just the ‘training’ set.
The sensitivity of any test is dependent on the specificity. Standard medical practice accepts as ‘normal’ values those being within two standard deviations of the mean, but by definition, 5% of the population lie outside the ‘normal range’. This creates an unacceptably high false-positive rate if applied to athletes. The specificity to be used has not been determined by the anti-doping authorities, but nevertheless the GH-2000 formulae show reasonable sensitivity even up to false-positive rates of 1 in 10 000 and beyond (Figure 6). The formula has been modified more recently to take into account the effect of age to prevent younger athletes from being placed at a disadvantage (Powrie et al., 2007).
Figure 6
Figure 6

Change in GH-2000 score in men (a) and women (b) following 28 days of GH administration. Dotplot of the standardized scores for each visit day of the studies by group and data set. The mean of a normal population is 0 and the standard deviation is 1. (more …)
The results of the GH-2000 project were presented at an International Olympic Committee workshop in Rome in March 1999 to review critically and assure the quality of the results. The conclusion of the workshop was strong support for the methodology, but it was felt that several issues needed to be addressed before the test could be fully implemented at an Olympic games. The biggest issue was related to potential ethnic effects of GH, as the vast majority of volunteers in the GH-2000 study were white Europeans. It was felt that injury could confound the test and further work was needed to develop immunoassays owned by International Olympic Committee and subsequently World Anti-Doping Agency to prevent arbitrary changes being made to commercially owned immunoassays.
Apart from the assay development, these issues have largely been addressed by the GH-2004 study. A further cross-sectional study of elite athletes has shown that although there are small differences in the mean values between ethnic groups—for example, the IGF-I concentrations in Afro-Caribbean men are approximately 8.2% lower than white European men—nearly all the values lie within the 99% prediction intervals for white European athletes, regardless of ethnic background. A further double-blind GH administration study suggests that the response to GH in other ethnic groups is similar to white European amateur athletes. The effect of injury was systemically examined by the GH-2004 team who followed 143 men and 40 women following a sporting injury. There was no change in IGF-I over the 12-week follow-up, but P-III-P increased by approximately 20%, reaching a peak 2–3 weeks after injury. This, however, did not cause any false-positive readings in the proposed test combining IGF-I with P-III-P.
Several other groups have also examined the use of GH-dependent markers, the first of which pre-dated the GH-2000 study. In this first study, the ratio of IGFBP-2 to IGFBP-3 was found to discriminate between those taking GH or placebo (Kicman et al., 1997). These findings were not supported by the GH-2000 study, although several other groups have confirmed the utility of IGF-I and P-III-P. The Institut für Dopinganalytik und Sportbiochemie in Kreischa, Germany, undertook a 14-day GH administration study in amateur male athletes and derived a discriminant function based on IGF-I, P-III-P and IGFBP-3 (Kniess et al., 2003). Most recently, the Australian Japanese Consortium presented the results of an 8-week GH administration study at the American Endocrine Society meeting in Toronto in June 2007. This also confirmed the value of IGF-I and P-III-P, although this suggested that an alternative bone marker (carboxyterminal cross-linked telopeptide of type I collagen) might provide better discrimination during the wash-out phase. This study also examined the effect of co-administration of anabolic steroids in males and showed that there were additive effects on P-III-P.
These confirmatory studies are important because it is unknown how well these GH-2000 formulae will perform in ‘real life’, where the patterns and doses of GH abused by athletes are unclear. When the male GH-2000 formula was applied to an independent data set obtained from the Institut für Dopinganalytik und Sportbiochemie Kreischa, 90% of the individuals who had received GH were correctly identified and there were no false-positives, findings that were identical to those of the GH-2000 data when the formula was used (Erotokritou-Mulligan et al., 2007).
Although this methodology has been rigorously tested, the development of World Anti-Doping Agency-owned immunoassays has lagged behind the science underpinning the method, despite the International Olympic Committee having been made aware of the need for these assays before the worldwide introduction of this test.
Future technologies to detect GH
Surface plasmon resonance
Surface plasmon technology is a non-labelled optical methodology that measures the refractive index of small quantities of a material absorbed onto a metal surface allowing measurement of mass. This technology is being applied for the detection of GH and dependent markers by the Barcelona anti-doping laboratory, but at present does not yield the same sensitivity as conventional immunoassays.
Mass spectrometry
Surface-enhanced laser desorption/ionization–time-of-flight mass spectrometry is a proteomic technique in which proteins are bound to proprietary protein chips with different types of adsorptive surfaces. It can be used to analyse peptide and protein expression patterns in a variety of clinical and biological samples, and biomarker discovery can be achieved by comparing the protein profiles obtained from control and patient groups to elucidate differences in protein expression. This technique has been applied to the detection of GH abuse to find potential new markers of GH abuse, such as the haemoglobin α-chain (Chung et al., 2006), but the sensitivity is insufficient to deal with analyses of IGF-I and P-III-P.
The detection of IGF-I
At present, there are no technologies to detect IGF-I abuse, but it is reasonable to adopt a similar approach as the GH-dependent marker test, and this is currently being evaluated by the GH-2004 team. Similarly, this approach should detect athletes abusing GH secretagogues.
The detection of insulin

The challenges of detecting insulin are in many ways similar to GH in that insulin is a naturally occurring pulsatile peptide hormone. At present, there are no methods to detect endogenous insulin abuse but urinary mass spectroscopy may be useful to detect the presence of analogue insulin. This technique involves concentration of the urine and followed by isolation by immunoaffinity chromatography. The eluate may then be analysed using microbore liquid chromatography/tandem mass spectrometry that produces characteristic product spectra obtained from the analogues that are distinguishable from human insulin (Thevis et al., 2006; Thomas et al., 2007). Some insulin analogues are handled differently from insulin and excreted in much higher quantities, which may facilitate this approach (Tompkins et al., 1981).
Future challenges

A major challenge for the future is the use of gene doping, where DNA is incorporated into target tissues, such as skeletal muscle, with or without the aid of a vector, such as an adenovirus. Expression of the gene may then lead to enhanced local production of an anabolic substance such as IGF-I.
Proof-of-concept experiments have been undertaken in animals in which injection of a recombinant adeno-associated virus genetically manipulated to induce myocyte overexpression of IGF-I in young mice induced a 15% increase in muscle mass and a 14% increase in muscle strength without inducing a systemic increase in IGF-I (Barton-Davis et al., 1998).
It is unclear whether this technology is being used by athletes; certainly, it has not been used in clinical practice, where it is highly needed despite significant investment. Anecdotal evidence, however, suggests that athletes are considering its use and this would provide new challenges to the anti-doping community. Traditional blood and urine testing may be of no benefit if gene doping causes no change in serum concentrations of the relevant proteins. Although the detection of vectors may be possible and changes in blood markers may occur, new technologies will be needed to catch this new form of doping.
Conclusion

Doping with GH and its related protein IGF-I remains a major challenge for those working in anti-doping. Anecdotal evidence suggests that abuse with GH and insulin is common, whereas the abuse of IGF-I is set to increase. There are compelling physiological reasons to explain why GH may have performance benefits. The athletes are risking long-term harm by using these drugs. Over the last decade, there have been major advances in methodologies to detect GH, and this should mean that once World Anti-Doping Agency has established suitable assays for IGF-I and P-III-P in its worldwide network of laboratories, athletes will no longer be able to cheat by taking GH without being caught.
Acknowledgments
The GH-2004 project is funded by the United States Anti-Doping Agency and the World Anti-Doping Agency. Our thanks go to the rest of the GH-2004 team: Eryl Bassett, Ioulietta Erotokritou-Mulligan, David Cowan, Christiaan Bartlett and Cathy McHugh. The GH-2004 study was undertaken in the Wellcome Trust Clinical Research Facility (WT-CRF) at Southampton General Hospital and we acknowledge the support of the WT-CRF nurses and Southampton medical students who have supported the study. We also pay tribute to our scientific collaborators, Astrid Kniess, Ken Ho, Anne Nelson, Christian Strasburger and Martin Bidlingmaier.
Abbreviations
FFA free fatty acid
GH growth hormone
IGF-I insulin-like growth factor-I
IGFBP IGF binding protein
P-III-P type 3 pro-collagen
rhGH recombinant human growth hormone

Ingestion of creatine (Cr) and glycerol (Gly) has been reported to be an effective method in expanding water compartments within the human body, attenuating the rise in heart rate (HR) and core temperature (Tcore) during exercise in the heat. Despite these positive effects, a substantial water retention could potentially impair endurance performance through increasing body mass (BM) and consequently impacting negatively on running economy (RE). The objective of the present study was to investigate the effects of a combined Cr and Gly supplementation on thermoregulatory and cardiovascular responses and RE during running for 30 min at speed corresponding to 60% of maximal oxygen uptake (VO2max) in hot and cool conditions. Methods: Cr * H2O (11.4 g), Gly (1 g * kg-1 BM) and Glucose polymer (75 g) were administered twice daily to 15 male endurance runners during a 7-day period. Exercise trials were conducted pre- and post-supplementation at 10 and 35 degreesC and 70% relative humidity. Results: BM and total body water increased by 0.90 +/- 0.40 kg (P < 0.01; mean +/- SD) and 0.71 +/- 0.42 L (P < 0.01), respectively following supplementation. Despite the significant increase in BM, supplementation had no effect on VO2 and therefore RE. Both HR and Tcore were attenuated significantly after supplementation (P < 0.05, for both). Nevertheless, thermal comfort and rating of perceived exertion was not significantly different between pre- and post-supplementation. Similarly, no significant differences were found in sweat loss, serum osmolality, blood lactate and in plasma volume changes between pre- and post-supplementation. Conclusions: Combining Cr and Gly is effective in reducing thermal and cardiovascular strain during exercise in the heat without negatively impacting on RE.

Low dose exogenous erythropoietin elicits an ergogenic effect in standardbred horses

Source

Equine Science Center, Department of Animal Sciences, Rutgers the State University of New Jersey, 84 Lipman Drive, New Brunswick, New Jersey, 08901-8525, USA.

Abstract

REASONS FOR PERFORMING STUDY:

Recombinant human erythropoietin (rhuEPO) causes an increase in red blood cell production and aerobic capacity in other species; however, data are lacking on effects in the horse.

HYPOTHESIS:

This study tested the hypothesis that rhuEPO administration would alter red cell volume (RCV), aerobic capacity (VO2max) and indices of anaerobic power.

METHODS:

Eight healthy, unfit mares accustomed to the laboratory and experimental protocols were randomly assigned to either a control (CON, n = 4; 3 ml saline 3 times/week for 3 weeks) or EPO group (EPO, n = 4, 50 iu/kg bwt rhuEPO/3 ml saline 3 times/week for 3 weeks). Exercise tests (GXT) were performed on a treadmill (6% incline), 1 week before and 1 week after treatment. The GXT started at 4 m/sec, with a 1 m/sec increase every 60 sec until the horse reached fatigue. Oxygen uptake was measured via an open flow indirect calorimeter. Blood samples were collected before, during (each step) and 2 and 15 min post GXT to measure packed cell volume (PCV), haemoglobin concentration (Hb), blood lactate concentration (LA) and plasma protein concentration (TP). Plasma volume (PV) was measured using Evans Blue dye. Blood volume (BV) and RCV were calculated using PCV from the 8 m/sec step of the GXT.

RESULTS:

There were no alterations (P>0.05) in any parameters in CON horses. By week 3, EPO produced increases (P<0.05) in resting PCV (37 +/- 2 vs. 51 +/- 2) and Hb (37%). RCV (26%) and VO2max (19%) increased, but BV did not change (P>0.05) due to decreased PV (-11%, P<0.05). There was a significant increase in velocity at VO2max and LApeak for horses treated with rhuEPO and substantial decrease (P<0.05) in VO2 recovery time when the pretreatment GXT was compared to the post treatment GXT. No differences (P<0.05) were detected for TP, VLA4, run time or Vmax.

CONCLUSIONS:

Low dose rhuEPO administration increases RCV and aerobic capacity without altering anaerobic power.

POTENTIAL RELEVANCE:

This study demonstrates that rhuEPO enhances aerobic capacity and exercise performance, a question relevant to racing authorities.

PMID:
17402424
[PubMed – indexed for MEDLINE]

Braz J Med Biol Res, December 2011, Volume 44(12) 1194-1201

Abstract

Recent biotechnological advances have permitted the manipulation of genetic sequences to treat several diseases in a process called gene therapy. However, the advance of gene therapy has opened the door to the possibility of using genetic manipulation (GM) to enhance athletic performance. In such ‘gene doping’, exogenous genetic sequences are inserted into a specific tissue, altering cellular gene activity or leading to the expression of a protein product. The exogenous genes most likely to be utilized for gene doping include erythropoietin (EPO), vascular endothelial growth factor (VEGF), insulin-like growth factor
type 1 (IGF-1), myostatin antagonists, and endorphin. However, many other genes could also be used, such as those involved in glucose metabolic pathways. Because gene doping would be very difficult to detect, it is inherently very attractive for those involved in sports who are prepared to cheat. Moreover, the field of gene therapy is constantly and rapidly progressing, and this is likely to generate many new possibilities for gene doping. Thus, as part of the general fight against all forms of doping, it will be necessary to develop and continually improve means of detecting exogenous gene sequences (or their products) in athletes. Nevertheless, some bioethicists have argued for a liberal approach to gene doping.

Key words: Genetic manipulation; Erythropoietin; Vascular endothelial growth factor; Insulin-like growth factor-1; Myostatin; Bioethics


Introduction

In the domain of genetics, scientific knowledge has made many remarkable advances in recent years. In the last decade, the publication of the human genome (1,2), together with much molecular information existing at the time, has permitted genetic manipulation (GM) techniques to be used in the treatment of various diseases. In the past, doping and cheating in sports were enabled by advances in pharmacology and physiology. Now, advances in molecular genetics have given rise to the potential to improve various non-medical human features including sports performance by the use of GM technology (3,4).

Gene expression is regulated primarily by two main mechanisms: a) changes in DNA structure and b) direct control over the processes of transcription and translation. The first includes epigenetic modifications and mutations. Through epigenetics, the degree of gene transcription is altered, without causing changes in DNA sequence (5). Also of importance, mutations promote changes in the nucleotide sequence of the gene and can derail the process of transcription or generate a new product, different from the original (6). The second mechanism consists of repressor and inductor molecules, transcription factors, enhancers and post-transcriptional modifiers (7). Thus, some changes in the regulatory process may result in an increased or decreased concentration of gene product.

The World Anti-Doping Agency (WADA) considers gene doping as the use of pharmacological or biological agents that alter gene expression, or the transfer of cells or genetic elements (DNA and RNA) (8). As explained by Sharp (9), some changes in gene expression are potentially able to increase sports performance in a number of ways (Figure 1). Such expression changes have the potential to up-regulate cellular functions in a wide variety of organs and tissues, leading to potential performance benefits. Examples include increased erythrocyte production and enhanced oxygen transfer via bone marrow targeting (10); liver targeting to increase Cori-cycle lactate-removing kinetics and energetic metabolism (11), erythropoietin (EPO) synthesis via kidney targeting (12), targeting of the myocardium to enhance cardiac output, and skeletal muscle targeting to influence fiber-type quality, percentage, sarcomere structure, mitochondrial number, glycolytic/glycogenesis enzymes, and muscle capillary numbers (13,14). It might even be possible to target neurologic areas in order to increase pain tolerance (15).

The rapid improvement of biotechnology inadvertently supplied fuel to drive the development of doping in all its modern forms. From recombinant protein production in GM microorganisms to gene doping, detection of athletes who have benefitted from such abuse remains a major technological challenge (16). Moreover, the new opportunities generated by recent scientific advances require bioethical analysis. What, if any, limits should be placed on the use of science to promote athletic performance? Should sportspeople be permitted to use gene therapy to treat disorders even if this results in improved performance? Does GM technology threaten to undermine the very spirit of sport? In this review article, we will provide a current overview of gene doping possibilities, including their development and detection techniques.

Candidate genes for use in gene doping

Erythropoietin: increase in energy production by aerobic metabolism

In adults, EPO is produced mostly by interstitial cells within the kidney, and in small quantities by the liver, where fetal synthesis occurs (17). Erythropoiesis is regulated by the concentration of circulating oxygen, and thus differs in conditions of normal oxygen tension (normoxia) and low oxygen tension (hypoxia) (18). The enzymes prolyl hydroxylase and asparaginyl hydroxylase are sensors of the level of intracellular oxygen. In normoxia, the hypoxia-inducible transcription factor 1α (HIF-1α) has its proline residues 402 and 567 hydroxylated at O2-dependent sites of proteosomal degradation. The next step is hydroxylation of asparagine residues in the carbon terminal transactivation domain (C-TAD). These hydroxylations prevent the binding of HIF-1α to the nucleotide sequence (A/G) CGTG, a regulatory region of the EPO gene. The opposite occurs in conditions of hypoxia – the hydroxylations do not occur, resulting in binding of HIF-1α to the regulatory region of the EPO gene. Thus, in hypoxia the up-regulation of gene expression leads to increased levels of intracellular EPO. Other transcription factors are also involved in the regulation of EPO expression, including HIF-1β, HIF-2α/β and HIF-3α/β (19). Following transcription and translation, the resultant EPO polypeptide undergoes glycosylation and other post-translational modifications essential for the establishment of proper in vivo function (20).

The hematopoietic system is regulated by three main groups of hematopoietic growth factors: a) the colony- stimulating factors, b) EPO and thrombopoietin, and c) cytokines, mainly interleukins. The erythroid lineage, which ultimately yields the erythrocytes, is primarily regulated by signaling performed by EPO (21). Erythropoiesis occurs in several steps of cell differentiation: the stem cell produces the pluripotent hematopoietic myeloid progenitor, which differentiates into the colony-forming unit erythroid (CFU-E). EPO stimulates the proliferation and differentiation of this CFU in basophilic, polychromatic and orthochromatic erythroblasts, in that order (22). These last cells differentiate into reticulocytes. Last, but not least, the reticulocytes mature and produce the erythrocytes, cells that contain hemoglobin, the key protein involved in gas exchange during cell respiration (23). Erythrocytes carry oxygen to tissues, where it is used for energy production through oxidative phosphorylation. Thus, an increase in the number of circulating red blood cells leads to an increase in the body’s ability to supply oxygen to tissues and to a concomitantly greater energy production by aerobic mechanisms. This is the basic principle for the use of EPO in gene doping (24).

Bone marrow contains the early stages of the CFU-E progeny. These have a dimerized receptor for EPO, whose intracellular portion has a tyrosine kinase-coupled domain, called Janus kinase 2 (JAK-2). Binding of EPO to the receptor induces interaction of JAK-2 with the SH2 domain of the cytosolic protein STAT 5 (a signal transducer and activator of transcription 5). This interaction promotes phosphorylation of STAT 5, forming a homodimer of phosphorylated STAT 5. This facilitates translocation to the nucleus, whereupon binding to specific nucleotide sequences takes place, resulting in the promotion of transcription of the genes necessary for erythropoiesis differentiation (25).

Recombinant human EPO (rhuEPO) is produced on a large scale by biotechnological processes and has wide application for the treatment of various diseases, such as anemia, chronic renal insufficiency, hematological malignancies, chemotherapy, and premature birth (26,27). Additionally, rhuEPO is used to minimize allogeneic blood transfusions after major surgical procedures. Notoriously, rhuEPO has also been illicitly used for enhancement of sport performance (28).

Fattori et al. (29) analyzed the efficacy of intramuscular injection of the EPO gene with the application of electric pulses to optimize the process of transduction. The gene was electro-injected into mice, rabbits and cynomolgus monkeys to test for protein production and biological effects. The study concluded that the injected EPO gene yields higher levels of circulating EPO and a more pronounced biological effect than the endogenous gene in all the species tested, thus showing great potential in clinically developable gene therapy approaches for EPO delivery.

Vascular endothelial growth factor: increase in oxygen supply

Oxygen is vital for the synthesis of ATP by aerobic respiration (30). Oxygen, as a small molecule, is able to diffuse through the plasma membrane of endothelial cells. Therefore, an increased vascular branching promotes a more rapid and effective diffusion of oxygen to the tissues and a greater availability of it for energy production. Vascular endothelial growth factor (VEGF) promotes the branching of a preexisting vessel, in a process called angiogenesis (31). In gene doping, several copies of the gene coding for VEGF would be inserted into the muscle, probably using viral vectors. Thus, if successful in athletes, the muscular microcirculation would be stimulated and the supply of oxygen to the muscles increased (32).

Insulin-like growth factor type 1: increase in muscle growth and differentiation

Insulin-like growth factor type 1 (IGF-1) is a 70-amino acid polypeptide synthesized primarily in the liver under the control of growth hormone (GH) (33). The hypothalamus produces two peptides, growth hormone-releasing hormone (GHRH) and somatostatin, which control the release of GH by the somatotropes of the anterior pituitary. GHRH, sleep, intense physical exercise, hypoglycemia, and low levels of circulating IGF-1 stimulate the release of GH (34,35). However, this is inhibited by somatostatin, hyperglycemia and increased blood concentrations of IGF-1. GH acts on hepatocytes stimulating production and secretion of IGF-1, a growth factor that stimulates the growth and differentiation of skeletal muscle tissue and the overall growth of bone tissue (36). The availability of IGF-1 is regulated by binding proteins (IGF-BP); IGF-1 is mainly complexed to IGF-BP3. Production of IGF-1 also occurs in skeletal muscle, and acts in autocrine and paracrine pathways (37).

The main route of intracellular signaling of IGF-1 starts with its binding to IGF1R, a receptor formed by dimerization of two glycoproteins with the cytoplasmic domains of tyrosine kinase (36). The interaction of IGF-1 and its receptor induces tyrosine autophosphorylation in the cytoplasmic domains of the receptor, triggering an intracellular signaling cascade that promotes the survival and proliferation of the muscle cell. IGF-1 also has activities beyond muscular effects, including an ability to drive tumor development and progression (36,37).

In gene doping, multiple copies of the gene coding for IGF-1 might be inserted in skeletal muscle, promoting an increase in muscle mass due to hypertrophy of muscle cells. This somatic gene insertion could be accomplished through the use of two alternative vectors: plasmids or viruses. Plasmids represent the least expensive method, but also the least efficient. Viral vectors, widely used in gene therapy, actively insert the exogenous DNA into the genome of the target cell. Viral classes commonly used in relevant gene therapy attempts include: a) the adenoviruses, with double-stranded DNA, and b) the adeno-associated viruses, with single-stranded DNA (9,37).

Myostatin antagonists: increase in muscle hypertrophy and hyperplasia

First described by McPherron et al. (38), myostatin, a member of the superfamily of transforming growth factor (TGF)-β, is a strong negative regulator of skeletal muscle growth and differentiation, where its expression predominates (38,39). The growth of this tissue can be achieved by the inhibition of its negative effects using specific antibodies or drugs that bind to myostatin, a principle that has the potential to be used in gene doping in order to enhance muscle percentage and athletic performance (40). Mosher et al. (41) have shown an association between double-muscle phenotype and a mutation in the myostatin gene in whippet dogs with higher racing performance, thus demonstrating the role of this protein in muscle development.

Several substances block the inhibitory activity of myostatin, promoting muscle growth and differentiation. Important amongst these substances are follistatin and a number of follistatin domain-containing proteins; all have the ability to bind to myostatin, making it unavailable for its natural inhibitory function (42).

Endorphin and enkephalin: increase in pain endurance

Pain during or following physical exertion functions as a sensory mechanism that serves as an alert to the subject that will lead to reduced activity. In evolutionary terms, this mechanism presumably developed as a means to avoid physical or physiological damage. However, the occurrence of pain in an athlete, produced either by injury or metabolic changes (for example lactic acid build-up), reduces the ability to train and perform. Competitors are trained to psychologically handle pain during intense activities, and may consume anti-inflammatory and pain-relieving drugs (43).

The advantage of a higher resistance to pain has brought several candidate genes into focus for potential use in gene therapy (for clinical pain) or gene doping. The genes encoding endorphin and enkephalin are prominent contenders in this context. The expressed molecules are neuropeptides that bind to opioid receptors, promoting analgesic effects and, therefore, pain relief. In order to increase the pain threshold, copies of these genes might be inserted, with expression being targeted to the nervous system (44). This promising possibility has been preliminarily studied using animal models (45). No such data have been reported in humans, suggesting that pain-killing gene therapy will not be available soon for clinical use or for gene doping.

Side effects of gene doping: health risks of performance enhancement

Since 2001, when the improvement of athletes’ abilities using the principles of gene therapy was discussed for the first time, doping has been subject to much discussion, some of it polemic, regarding its prohibition. As gene therapy is a new form of medicine, and until recently was tested only in patients with terminal illnesses, its long-term consequences are unknown. Thus, important questions remain to be answered about the potential use of GM in the context of sport. Perhaps the most fundamental question concerns the theoretical possibility that the transgenes used in gene doping could inadvertently affect the germ cells, producing permanent alterations, which could be transmitted to future generations (46). At present, there are no definitive answers to this question.

Some of the negative effects of gene doping will not be specific to GM, but rather to the nature of the gene product so expressed. In this regard, the risks of gene doping can be analyzed with reference to knowledge (where available) on the conventional use of the product concerned. To take one example: although there are no descriptions of the consequences of gene doping on reproductive parameters in vivo and in vitro, studies have shown that EPO stimulates steroidogenesis in Leydig cells, triggering an increase in testosterone production, leading to a negative feedback on the release of reproductive hormones and, consequently, reducing spermatogenesis and the spermatic potential, which may cause infertility (47).

By contrast, some studies have shown positive side effects of expression products of doping candidate genes, such as the reduction of adipogenesis promoted by myostatin antagonists (48) and neuroprotective properties provided by EPO-based treatments (49,50). Still, it is necessary to learn more about the complex physiology of each transgene product, before it would be safe and acceptable to sanction any such use.

Detection of gene doping: challenges and limitations

The detection of gene doping is likely to be difficult, although it could be accomplished by a number of available approaches, involving either direct or indirect methods (51). Direct methods involve the detection of recombinant proteins or gene insertion vectors (such as viruses or plasmids). Indirect methods depend upon the detection of signature changes associate with gene doping: for example, changes in the immune system following gene transfer, or changes in the transcriptome or proteome of a particular cell type (Table 1).

In gene therapy, the transgene substitutes for a defective gene and thus gene expression will be detected where it was previously missing. However, in gene doping, the expected effect will be an increased concentration of a gene product previously expressed at normal levels, thus making detection more difficult in the latter case (52). Lasne et al. (53) showed some structural differences between endogenous and recombinant EPO, via their different isoelectric behavior. Another possibility for direct detection is based on the use of molecular tests to differentiate the genomic DNA from complementary DNA (cDNA). The sequence of cDNA does not contain introns, so it can be distinguished using techniques such as the polymerase chain reaction (PCR) or Southern blotting (54).

The detection of insertion vectors in blood plasma presents great difficulties, considering the extremely short half-life of circulating plasmids, adenoviruses and adeno-associated viruses. Thus, the only way to detect the insertion vectors in bodily fluids would be through the application of molecular tests with relatively short intervals, with the need to create a regular testing regime (55).

Indirect methods are based on studies of gene doping effects on cells, tissues or the entire organism. This strategy includes examining for potential immune responses to gene insertion vectors or ‘non-self’ peptides encoded by introduced nucleic acid (52). Recently, the use of transcriptomics, which consists of analysis of tissue mRNA transcripts, became a promising potential method to detect gene doping. The quantity and composition of a tissue transcriptome is highly reflective of its metabolic activity. Some tissues can be easily accessed to construct a gene expression pattern. So, it is possible to evaluate some alterations in each expression pattern, supporting the development of doping detection techniques (56).

Bioethics of gene doping

In the ancient Olympic Games, athletes competed for recognition, eternal fame and an olive branch. Today, such motives continue to serve as important reasons for participation in international competitions; however, they have been joined by an additional factor: money. For an athlete, to win a medal is a guarantee of lucrative contracts in the future. Thus, multiple factors conspire to place pressure upon athletes to a hitherto unprecedented degree, generating temptations to resort to extreme measures, including conventional doping and, potentially, gene doping (57).

The main arguments used by the WADA to justify the prohibition of gene doping are 2-fold. Uppermost are concerns over potential health risks from the insertion of genes or the use of substances that interfere in gene expression. Such alterations of gene expression can bring unknown risks to the athlete’s health and, should exogenous sequences reach the germ line cells, some changes might be transmitted to future generations. A second argument of the WADA centers on the issue of fairness. The use of GM to increase sports performance is seen as a violation of the sporting spirit, giving unfair physical advantages to those who have access to the requisite technology (46).

Arguably, there are two ways to analyze the ethical status of gene doping (58). The first holds that sports ethics is subservient to medical ethics. So, if the use of gene therapy for medicine is permitted, any performance increase should be acceptable, essentially as a form of ‘side effect’. In this way, a sports physician could prescribe potentially performance-enhancing substances – or gene therapy – to athletes in order to alleviate a medical condition. However, this matter is not straightforward and it is not an easy task for a physician to answer the question: how best to treat the athlete-patient – more as an athlete or more as a patient? Furthermore, some patients might have more interest in receiving a treatment that makes them well for sports performance, rather than well for life.

The second way to approach sports doping as an ethical issue is to consider sports ethics as representing a separate entity from medical ethics. In other words, sport is seen as a moral practice which, while not requiring a rejection of the concepts of medical ethics, depends more on the sporting context than on the medical context (59,60).

Viewpoints favorable towards liberalizing the use of performance-enhancing agents in sport have been expressed by some ethicists, albeit a minority thereof (see for example, Refs. 58 and 61). The essential argument here is that since athletes legitimately strive to improve their performance (for example, by use of training methods, nutrition, and psychological conditioning), there are no obvious reasons to exclude performance-enhancing agents (e.g., anabolic steroids) from the set of methods that may be used to enhance athletic performance. While it may seem that this would give an unfair advantage to athletes able to access these agents, it can be argued that specialist gym apparatus, advanced nutrition, and psychological coaching are also expensive and thus – like performance-enhancing drugs – not available to all athletes. So, it can be argued that performance-enhancing drugs should not be singled out for prohibition on the basis of a lack of fairness. Similarly, health risks are inherent in many forms of sports training; thus, banning performance-enhancing agents on grounds of safety is arguably inconsistent, as the basis for such prohibition implies a similar ban on forms of training, such as lifting heavy weights, or practicing gymnastic moves, that might conceivably endanger the health of the athlete. Logically, any prohibition should be based on an objective assessment of risk, which would evaluate drugs and training methods on an equal basis, as opposed to prohibiting simply on the basis of categorizations.

Such pro-enhancement arguments, if accepted with respect to conventional performance-enhancing drugs, would logically also apply to gene doping (58). At present, the risks of human gene therapy are inadequately understood in the context of application to healthy individuals, but if a GM method could be shown to carry an acceptably low health risk (including any risk of inter-generational transmission of introduced gene sequences), then there would be no logical grounds for prohibition. Because gene therapy is at the cutting edge of medical science, its use for performance enhancement would be expensive and thus restricted to well-funded athletes, again raising the issue of fairness. However, it is an economic rule that technologies (e.g., computers, medical devices, etc.) drop steeply in price as they come to be used more widely, and this rule is very likely to apply to human GM technology. Thus, it may be that inexpensive commercial gene therapy ‘kits’ will become available in the future. If so, gene doping could become widely accessible to athletes regardless of their financial situation.

Those who reject conventional doping on ethical grounds will also reject gene doping. However, an acceptance of conventional doping, as advocated by a minority of bioethicists, would rationally permit the full use of GM technology in sports. If this were to happen, then there can be little doubt that world sporting records (many of which have reached a plateau) would tumble, and athletic performance would reach hitherto unprecedented levels. As GM technology advances, athletes, regulatory bodies and society in general will need to decide whether to open the door to the ‘brave new world’ of gene doping.

Conclusions

Recent developments in molecular biotechnology have provided new approaches to the treatment of several diseases, but have also generated new opportunities for cheating in sports. Most recently, these discoveries have enabled the potential use of gene doping, a strategy that promises (or threatens) to radically enhance athletic performance using GM approaches that will be hard to detect. Sport-regulatory organizations will need to remain vigilant for signs that gene doping starts to be used by athletes; if this does happen, scientists will need to rise to the challenge of entering an ‘arms race’ to develop effective means to detect such abuse. Meanwhile, bioethicists need to promote an active debate on an important emerging question: should gene doping be banned, controlled or liberalized?



Figure 1. Targeted tissues and organs for gene doping. Main aims of gene doping to enhance sports performance: improvement of pain tolerance (endorphin/enkephalin genes), muscle quality and vascularization (VEGF gene and myostatin antagonists) and erythrocyte number (EPO gene). With more genomic understanding, other organs will be targeted in the future, such as heart and kidneys, to increase cardiac output and EPO production, respectively. VEGF = vascular epithelial growth factor; EPO = erythropoietin.

Chronic oral ingestion of l-carnitine and carbohydrate increases muscle carnitine content and alters muscle fuel metabolism during exercise in humans

Non-technical summary

After 30 years of endeavour, this is the first study to show that muscle carnitine content can be increased in humans by dietary means and, perhaps more importantly, that carnitine plays a dual role in skeletal muscle fuel metabolism that is exercise intensity dependent. Specifically, we have shown that increasing muscle total carnitine content reduces muscle carbohydrate use during low intensity exercise, consistent with an increase in muscle lipid utilisation. However, during high intensity exercise muscle carnitine loading results in a better matching of glycolytic, pyruvate dehydrogenase complex and mitochondrial flux, thereby reducing muscle anaerobic energy generation. Collectively, these metabolic effects resulted in a reduced perception of effort and increased work output during a validated exercise performance test. These findings have significant implications for athletic performance and pathophysiological conditions where fat oxidation is impaired or anaerobic ATP production is increased during exercise.

Abstract

We have previously shown that insulin increases muscle total carnitine (TC) content during acute i.v. l-carnitine infusion. Here we determined the effects of chronic l-carnitine and carbohydrate (CHO; to elevate serum insulin) ingestion on muscle TC content and exercise metabolism and performance in humans. On three visits, each separated by 12 weeks, 14 healthy male volunteers (age 25.9 ± 2.1 years, BMI 23.0 ± 0.8 kg m−2) performed an exercise test comprising 30 min cycling at 50% Graphic, 30 min at 80% Graphic, then a 30 min work output performance trial. Muscle biopsies were obtained at rest and after exercise at 50% and 80% Graphic on each occasion. Following visit one, volunteers ingested either 80 g of CHO (Control) or 2 g of l-carnitine-l-tartrate and 80 g of CHO (Carnitine) twice daily for 24 weeks in a randomised, double blind manner. All significant effects reported occurred after 24 weeks. Muscle TC increased from basal by 21% in Carnitine (P < 0.05), and was unchanged in Control. At 50% Graphic, the Carnitine group utilised 55% less muscle glycogen compared to Control (P < 0.05) and 31% less pyruvate dehydrogenase complex (PDC) activation compared to before supplementation (P < 0.05). Conversely, at 80% Graphic, muscle PDC activation was 38% higher (P < 0.05), acetylcarnitine content showed a trend to be 16% greater (P < 0.10), muscle lactate content was 44% lower (P < 0.05) and the muscle PCr/ATP ratio was better maintained (P < 0.05) in Carnitine compared to Control. The Carnitine group increased work output 11% from baseline in the performance trial, while Control showed no change. This is the first demonstration that human muscle TC can be increased by dietary means and results in muscle glycogen sparing during low intensity exercise (consistent with an increase in lipid utilisation) and a better matching of glycolytic, PDC and mitochondrial flux during high intensity exercise, thereby reducing muscle anaerobic ATP production. Furthermore, these changes were associated with an improvement in exercise performance.

Abbreviations 

CHO
carbohydrate
CoASH
free co-enzyme A
CPT1
carnitine palmitoyl-transferase 1
PDC
pyruvate dehydrogenase complex
TC
total carnitine
Graphic
maximal oxygen uptake
Introduction

More than 95% of the body’s carnitine pool is confined to skeletal muscle, where it fulfils two major metabolic roles. Firstly, in mitochondrial fatty acid translocation carnitine is a substrate for carnitine palmitoyl-transferase 1 (CPT1) (Fritz & McEwen, 1959; Fritz & Yue, 1963). Secondly, during high intensity exercise, the formation of acetylcarnitine is essential for the maintenance of a viable pool of free co-enzyme A (CoASH), thereby enabling PDC and TCA flux to continue (Childress & Sacktor, 1966; Harris et al. 1987; Constantin-Teodosiu et al. 1991a). Not surprisingly therefore, oral carnitine feeding has been advocated as an ergogenic aid, the main premise being that increasing muscle carnitine content will increase muscle fat oxidation and delay muscle glycogen depletion. However, we are aware of no study that has unequivocally shown carnitine feeding can impact on muscle fuel metabolism or exercise performance, which is undoubtedly attributable to carnitine ingestion (or indeed i.v. carnitine infusion) per se failing to increase muscle carnitine content (Barnett et al. 1994; Vukovich et al. 1994; Wächter et al. 2002; Stephens et al. 2006a).

In a series of i.v. infusion studies, we demonstrated that elevating serum insulin concentration in the presence of hypercarnitinaemia (550–600 μmol l−1) acutely increased muscle total carnitine (TC) content by ∼15% in humans (Stephens et al. 2006a,b). Furthermore, this increase in carnitine retention occurred only when serum insulin concentration was elevated above 50 mU l−1, which we confirmed could be achieved by combined carbohydrate (CHO) and l-carnitine feeding (94 g and 3 g, respectively), albeit at a much lower rate of retention (equating to a projected ∼0.1% increase in the muscle TC pool per day; Stephens et al. 2007). Assuming this effect of combined CHO and carnitine feeding on carnitine retention is sustainable and cumulative, it was calculated that about 24 weeks of feeding would be needed to increase skeletal muscle TC content to the same extent as acute i.v. carnitine and insulin infusion.

During low intensity exercise, when PDC activation (PDCa) and flux are relatively low, the principal role of carnitine will most likely be mitochondrial fatty acid translocation. Although it has been suggested that free carnitine only limits fat oxidation at exercise intensities >70% of maximal oxygen consumption (Graphic; van Loon et al. 2001), we demonstrated that an acute 15% increase in muscle carnitine content reduced insulin-mediated muscle glycolytic flux and PDCa compared to control. Furthermore, this was accompanied by a subsequent increase in muscle glycogen and long-chain acyl-co-enzyme A accumulation (Stephens et al. 2006b), pointing to a carnitine-mediated increase in muscle fatty acid oxidation and CHO storage. Free carnitine availability may therefore be limiting to mitochondrial fatty acid translocation even at rest and during low intensity exercise, and an increase in skeletal muscle TC content would be expected to augment fatty acid oxidation and decrease PDCa and glycogen use during low intensity exercise.

During high intensity exercise, the primary functional role of carnitine shifts towards acetyl group buffering (i.e. forming acetylcarnitine) and maintaining a pool of free CoASH which is essential for mitochondrial flux to continue (including the PDC reaction). However, during exercise of this nature there is still an increase in the acetyl-co-enzyme A (acetyl-CoA)/CoASH ratio, possibly due to the significant depletion (to <6 mmol (kg dry muscle)−1) of the free carnitine pool caused by acetylcarnitine formation (Harris et al. 1987; van Loon et al. 2001). Therefore, it is plausible that an increase in skeletal muscle TC content would provide more effective buffering of acetyl-CoA production during high intensity exercise, offsetting the increase in the acetyl-CoA/CoASH ratio, and thereby increasing PDC flux and mitochondrial ATP production. This in turn would reduce the contribution from glycolysis and PCr hydrolysis to ATP production, particularly during the rest to exercise transition period when inertia in mitochondrial ATP production is known to reside at the level of PDC activation and flux (Timmons et al. 1997, 1998; Howlett et al. 1999; Roberts et al. 2002, 2005). In addition, increasing PDC flux during high intensity exercise would be expected to decrease muscle lactic acid production which could translate to a positive effect on exercise performance by reducing muscle acidosis (Sahlin, 1992). In support of this stance, previous work from our laboratory has shown that pharmacological activation of PDC at rest using the pyruvate dehydrogenase kinase inhibitor, dichloroacetate, markedly reduced muscle lactate production and PCr hydrolysis during a subsequent 20 min period of intense muscle contraction in isolated and perfused canine skeletal muscle, resulting in a substantial improvement in tension development (Timmons et al. 1997).

Based upon the above information, it is logical to conclude that increasing skeletal muscle TC content could alter muscle fuel metabolism in at least two different ways during exercise, with the dominating role being dictated by the exercise intensity employed. With this in mind, we first aimed to determine whether chronic l-carnitine and CHO feeding to healthy male volunteers could increase skeletal muscle TC content in a manner similar to that which we have observed acutely under i.v. l-carnitine infusion and hyperinsulinaemic clamp conditions. Secondly, we hypothesised that any increase in muscle TC content would result in a blunting of PDCa and flux during low intensity exercise, causing a corresponding decrease in muscle glycogen utilisation. Thirdly, during high intensity exercise, when the primary functional role of carnitine switches to acetyl group buffering, we proposed that increasing muscle carnitine content would increase muscle PDC flux (and mitochondrial ATP delivery), thereby reducing anaerobic ATP production and muscle lactic acidosis during exercise. Finally, we hypothesised that these positive metabolic effects of muscle carnitine loading would improve high intensity exercise performance.

Methods

Human volunteers

Fourteen healthy, non-smoking, non-vegetarian recreational athletes (Graphic 51.6 ± 2.5 ml (kg body mass)−1, training 3–5 times per week in triathlon, cycling, running or swimming), aged 25.9 ± 2.1 years and with a body mass index (BMI) of 23.0 ± 0.8 kg m–2 participated in this study. Moderately trained recreational athletes were recruited as they were accustomed to ingesting CHO supplements. The study was approved by the University of Nottingham Medical School Ethics Committee in accordance with the Declaration of Helsinki. Prior to the study, each subject completed a routine medical screening and a general health questionnaire to ensure their suitability to take part. All gave their informed written consent to participate in the study and were aware that they were free to withdraw from the experiment at any point.

Pre-testing

Fourteen days before the trial, each subject’s Graphic was measured using an electronically braked cycle ergometer (Lode NV Instrumenten, Groningen, the Netherlands) and a continuous and incremental, exhaustive exercise protocol. Oxygen consumption was measured using an online gas analyser (Vmax; SensorMedics, Anaheim, CA, USA) and Graphic was confirmed during a repeat test 3 days later. Graphic was accepted when a plateau in oxygen consumption was achieved despite a further increase in workload. Once Graphic had been obtained, workloads to be used in subsequent experimental visits were calculated that would elicit 50% and 80% of Graphic. Subjects were familiarised with the experimental exercise protocol (which also allowed confirmation that the workloads were at the appropriate intensity to elicit 50% and 80% Graphic) at least 1 week prior to the beginning of subsequent experimental visits. A repeat Graphic test was also performed at least 1 week after the completion of the study to confirm no significant change in aerobic capacity had occurred over the course of the study.

Experimental protocol

Volunteers reported to the laboratory at 08.30 h on three occasions over a 24 week period, each visit being separated by 12 weeks. Subjects arrived after an overnight fast having abstained from strenuous exercise and alcohol consumption for at least 48 h, and caffeine for at least 24 h. On each visit, subjects performed the following experimental protocol. On arrival at the laboratory, subjects were weighed and then rested in a semi-supine position whilst a resting blood sample was collected from an antecubital vein (venipuncture) for blood glucose, serum insulin and plasma TC concentration measurements. Volunteers then exercised for 30 min on the cycle ergometer at a workload corresponding to 50% Graphic followed immediately by 30 min of exercise at a predetermined workload of 80% Graphic. During both bouts of exercise, a rating of perceived exertion (Borg Scale) was obtained every 10 min. Finally, immediately following the completion of exercise at 80% Graphic, subjects performed a 30 min work output performance test. This ‘all-out’ performance test involved using the ergometer hyperbolic mode function, where work output is dependent upon volitional cycling cadence. This performance test has been shown to be a more reliable measurement of endurance exercise performance than cycling at a fixed exercise workload to volitional exhaustion (Jeukendrup et al. 1996), and has been used previously in our laboratory to measure performance (Stephens et al. 2008).

Supplementation protocol

After the first experimental visit, subjects were allocated in a randomised, double blind manner to two experimental treatment groups. One group (n = 7) was instructed to consume 700 ml of a solution containing 80 g of orange-flavoured CHO polymer (Vitargo; Swecarb AB, Stockholm, Sweden) on two occasions each day for 168 days (Control), whilst the remaining group consumed 80 g of orange-flavoured CHO polymer containing 2.0 g of l-carnitine tartrate (1.36 g of l-carnitine; Carnipure™, Lonza Group Ltd, Basel, Switzerland) in the same volume of solution and at the same frequency (Carnitine). Volunteers were instructed to ingest the first supplement at breakfast time and the second 4 h later. This feeding protocol was based upon a regimen we have previously shown to increase whole-body carnitine retention over a 14 day period (Stephens et al. 2007). Volunteers were informed of the caloric content of the drinks (∼600 calories per day) and advised to replace their customary CHO supplement with the prescribed supplement and/or amend their diet accordingly to try and avoid weight gain. Volunteers were requested to record any side effects associated with supplementation over the 24 week protocol. None were reported by either group.

Sample collection and analysis

On each experimental study day, venous blood samples were collected whilst subjects rested in a semi-supine position. Following collection, blood glucose concentrations were determined immediately using an autoanalyser (YSI 2300 STATplus, Yellow Springs Instruments, Yellow Springs, OH, USA). Two millilitres of each basal resting blood sample was collected into lithium heparin containers, and following centrifugation (22,000 RCF at +4°C for 2 min) the plasma was snap frozen in liquid nitrogen and stored at −80°C until used to determine plasma TC concentration using a radioenzymatic assay (Cederblad et al. 1982). Finally, a further 2 ml of each basal resting blood sample was allowed to clot and following centrifugation (1,400 RCF at +4°C for 10 min) the serum was stored frozen at −80°C until used to determine insulin concentration using a commercially available radioimmunoassay kit (Coat-a-Count Insulin, Diagnostics Products Corporation, Los Angeles, CA, USA).

On each experimental visit, muscle biopsy samples were obtained from the vastus lateralis muscle at rest and within 5 s of the end of exercise at 50% and 80% Graphic (whilst subjects were seated on the cycle ergometer) using the percutaneous needle biopsy technique (Bergström, 1975). Muscle samples were immediately snap frozen in liquid nitrogen after removal from the limb. One portion of each biopsy sample was freeze dried and stored at −80°C, whilst the remainder was stored ‘wet’ in liquid nitrogen. Freeze-dried muscle was dissected free of visible blood and connective tissue, powdered and used for the determination of muscle free carnitine, acetylcarnitine and long-chain acylcarnitine using the radioenzymatic method described previously by Cederblad (Cederblad et al. 1990). Muscle ATP, phosphocreatine (PCr), free creatine, lactate and glycogen were also determined on freeze-dried muscle using the spectrophotometric method of Harris (Harris et al. 1974). Muscle TC was calculated as the sum of the three carnitine moieties, and was normalised for the highest total creatine content from each individual’s three biopsies of that visit, a procedure routinely carried out to minimise variability from non-muscle constituents (Stephens et al. 2006a,b). Muscle total creatine content was calculated as the sum of free creatine and PCr. Approximately 10 mg of the ‘wet’ muscle was used to determine PDCa, expressed as the rate of acetyl-CoA formation (mmol min−1 (kg wet muscle)−1 at 37°C) using methodology described previously by Constantin-Teodosiu et al. (1991b). In addition, maximal citrate synthase activity was determined spectrophotometrically on whole muscle homogenates based on the methods of Opie & Newsholme (1967); Zammit & Newsholme (1976) and expressed as mmol min−1 (kg wet muscle)−1.

Statistical analysis

A two-way ANOVA for repeated measures (time and treatment effects) was performed to detect differences within and between treatment groups separately for the three conditions (rest, 50% Graphic and 80% Graphic). When a significant time or treatment effect was observed a one-way ANOVA or t test was performed, respectively, to locate individual differences. Statistical significance was declared at P < 0.05. All the values presented in text, tables and figures represent mean ± the standard error of the mean (s.e.m.).

Results

Subject characteristics

Subject characteristics are displayed in Table 1. Body mass was not different between groups before supplementation. However, there was a 2.4 kg increase in body mass from basal in the Control group after 12 weeks of supplementation (P < 0.01), which remained elevated after 24 weeks (P < 0.05). The Carnitine group showed no change in body mass over the course of the study. Fasting venous blood glucose and serum insulin concentrations at baseline were not different between groups and did not change throughout the study (Table 1). Fasting plasma TC concentration was also not different between groups before supplementation. However, plasma TC concentration in the Carnitine group was greater after 12 and 24 weeks of supplementation when compared to the Control group (P < 0.05; Table 1). Perceived exertion during exercise at 50% Graphic did not differ between groups on any visit. The same was also true during exercise at 80% Graphic before and after 12 weeks of supplementation. However, after 24 weeks of supplementation perceived exertion was lower in the Carnitine group when compared to baseline (14.0 vs. 15.0, respectively; P < 0.05) and Control at 24 weeks (14.0 vs. 16.2, respectively; P < 0.05).

View this table:

Table 1. Subject characteristics before (0) and 12 and 24 weeks after twice daily oral ingestion of either 80 g of carbohydrate (Control; n = 7) or 80 g of carbohydrate containing 2 g l-carnitine tartrate (Carnitine; n = 7)

Skeletal muscle total carnitine content

Resting muscle TC content over the course of the study is shown in Fig. 1. There was no difference between or within groups before or after 12 weeks of supplementation. However, after 24 weeks muscle TC content was 30% greater in the Carnitine group compared to Control (P < 0.05), which represented a 21% increase from baseline in the Carnitine group (P < 0.05).

Figure 1  Total skeletal muscle carnitine content (calculated as the mean of 3 biopsies taken from each individual during a given visit) before (0) and 12 and 24 weeks after twice daily oral ingestion of either 80 g of carbohydrate (Control; n = 7) or 80 g of carbohydrate containing 2 g l-carnitine tartrate (Carnitine; n = 7). All values are means ± s.e.m.). Significantly different from Control: *P < 0.05. Significantly different from before supplementation (0): †P < 0.05.

Skeletal muscle metabolites

Absolute muscle metabolite values at rest and during exercise are presented in Table 2. From similar resting values, muscle PCr, glycogen, lactate, acetylcarnitine and free carnitine content changed by a similar magnitude during exercise at 50 and 80% Graphic before and after 12 weeks of supplementation in Control and Carnitine groups. However, following 24 weeks of supplementation there was a trend (P = 0.09) for resting free carnitine content to be 30% greater in the Carnitine group compared to Control, and there was a significant difference between groups in the metabolic response to both low and high intensity exercise. Following exercise at 50% Graphic, muscle glycogen content was 35% greater in the Carnitine group compared to Control (P < 0.05), which equated to 55% less glycogen being utilised during exercise (P < 0.05; Fig. 2A), and free carnitine was 78% greater in the Carnitine group when compared to Control (P < 0.01). Following exercise at 80% Graphic, muscle glycogen content was 71% greater in the Carnitine group compared to Control; however, this was attributable to the reduction in glycogen utilisation during the preceding exercise at 50% Graphic (see above), and accordingly there was no difference between groups in glycogen utilisation during exercise at 80% Graphic (Fig. 2A). However, muscle lactate content was 44% lower in the Carnitine group compared to Control (P < 0.05) following exercise at 80% Graphic, which translated into a marked reduction in muscle lactate accumulation during exercise (P < 0.05, Fig. 2B) and was accompanied by a trend (P < 0.10) for muscle acetylcarnitine and free carnitine content to be greater in the Carnitine group when compared to Control (16% and 63%, respectively). In addition, after 24 weeks of supplementation, the muscle PCr/ATP ratio in the Carnitine group was significantly greater than Control (P < 0.05) and baseline (P < 0.05) following exercise at 80% Graphic.

Figure 2  Skeletal muscle glycogen utilisation (A), lactate accumulation (B) and pyruvate dehydrogenase complex activation status (C) during 30 min of exercise at 50 and 80% Graphic before (0) and 12 and 24 weeks after twice daily oral ingestion of either 80 g of carbohydrate (Control; n = 7) or 80 g of carbohydrate containing 2 g l-carnitine tartrate (Carnitine; n = 7). All values are means ± s.e.m. Significantly different from corresponding Control: *P < 0.05. Significantly different from before supplementation (0): †P < 0.05.

View this table:

Table 2. Skeletal muscle metabolites at rest and following 30 min of exercise at 50 and 80%Graphicbefore (0) and 12 and 24 weeks after twice daily oral ingestion of either 80 g of carbohydrate (Control; n = 7) or 80 g of carbohydrate containing 2 g l-carnitine tartrate (Carnitine; n = 7)

Muscle PDCa

Muscle pyruvate dehydrogenase activation status (PDCa) following exercise is shown in Fig. 2C. Resting muscle PDCa was not different between treatment groups at any time-point, being maintained at ∼0.4 mmol acetyl-CoA min−1 (kg wet muscle)−1. Similarly, muscle PDCa following exercise at 50% Graphic was not different between treatment groups at baseline and 12 weeks; however, at 24 weeks PDCa was 31% lower than baseline in the Carnitine group (P < 0.05). PDCa following exercise at 80% Graphic was not different between treatment groups at baseline or after 12 weeks, but was 38% greater in the Carnitine group at 24 weeks when compared with Control (P < 0.05).

Muscle citrate synthase activity

Muscle citrate synthase activity was not different between Control or Carnitine groups at baseline (5.6 ± 0.9 and 5.0 ± 0.4 mmol min−1 (kg wet muscle)−1, respectively) or after 24 weeks of supplementation (5.4 ± 0.1 and 5.9 ± 0.7 mmol min−1 (kg wet muscle)−1, respectively), and there were also no differences over time.

Exercise performance

Work output (kJ) achieved in the exercise performance test is presented in Fig. 3. Performance was not different between groups before or after 12 weeks of supplementation. However, after 24 weeks work output was 35% greater in the Carnitine group compared to Control (P < 0.05), which represented an 11% increase from baseline (P < 0.05).

Figure 3  Work output generated during a 30 min ‘all-out’ exercise performance test performed immediately following 30 min of exercise at 50 and 80% Graphic before (0) and 12 and 24 weeks after twice daily oral ingestion of either 80 g of carbohydrate (Control; n = 7) or 80 g of carbohydrate containing 2 g l-carnitine tartrate (Carnitine; n = 7). All values are means ± s.e.m. Significantly different from Control: *P < 0.05. Significantly different from before supplementation (0): †P < 0.05.

Discussion

Despite over 30 years of research demonstrating the fundamental role of carnitine in regulating muscle fuel use, attempts to increase skeletal muscle TC content in humans via l-carnitine feeding have been unsuccessful (Barnett et al. 1994; Vukovich et al. 1994; Wächter et al. 2002). The present study is the first to demonstrate that muscle TC content can be increased by 21% in healthy human volunteers when l-carnitine is ingested for 24 weeks in combination with a CHO solution. Moreover, this increase in TC content had a profound effect on muscle fuel utilisation during exercise which was exercise intensity dependent and consistent with the reported dual role of carnitine in muscle fuel metabolism. Namely, during low intensity exercise muscle glycogen utilisation was halved (consistent with an increase in muscle lipid utilisation), whereas during high intensity exercise muscle lactate accumulation was substantially reduced and the muscle PCr/ATP ratio was better maintained, which probably resulted from the carnitine-mediated increase in PDC activation and flux observed at this workload. Finally, increasing skeletal muscle TC content was associated with a 35% improvement in work output over Control, which we propose resulted directly from the observed changes in muscle fuel metabolism.

A major role of carnitine in skeletal muscle is as a substrate for the CPT1-mediated translocation of fatty acids into mitochondria for subsequent β-oxidation (Fritz & McEwen, 1959; Fritz & Yue, 1963). We have previously shown that a 15% increase in muscle TC content resulted in the attenuation of insulin-induced increases in glycolytic flux and PDCa in healthy, resting volunteers, as well as a subsequent overnight increase in muscle glycogen storage. This effect was attributed to a carnitine-mediated increase in acetyl-CoA delivery from fat oxidation, which inhibited PDCa and diverted glucose uptake from oxidation towards storage, and therefore suggests that carnitine availability is limiting to the CPT1 reaction under insulin-stimulated conditions, even at rest (Stephens et al. 2006b). We therefore hypothesised that during low intensity exercise in the present study, when glycolytic and PDC flux are well matched, an increase in muscle free carnitine availability would have a similar effect, i.e. it would augment muscle lipid oxidation thereby blunting PDCa and glycolytic flux. Consistent with this hypothesis, we observed that the increase in muscle TC content after 24 weeks of supplementation was linked to a 55% reduction in muscle glycogen utilisation during exercise at 50% Graphic compared to Control. Furthermore, this was accompanied by muscle free carnitine content being ∼80% greater and PDCa being 31% lower during exercise compared to before supplementation, suggesting that a carnitine-mediated increase in lipid-derived acetyl-CoA inhibited PDCa (Pettit et al. 1975) and thereby reduced muscle CHO flux, which is consistent with The Randle Cycle (Randle et al. 1963). Free carnitine availability has been suggested to limit muscle fat oxidation in vivo in humans during intense exercise when its concentration declines below 6 mmol (kg dry muscle)−1 (∼2 mmol (l intracellular water)−1) (van Loon et al. 2001). However, during exercise at 50% Graphic in the present study muscle free carnitine concentration was 11 mmol (kg dry muscle)−1 (∼3.5 mmol (l intracellular water)−1), which is above the value reported by van Loon et al. (2001) and well above the reported Km of CPT1 for free carnitine (0.5 mmol l−1) (McGarry et al. 1983). Therefore, either the reported Km of carnitine for CPT1, generated via in vitro experiments (McGarry et al. 1983) is not transferable to the in vivo situation or, alternatively, although the cellular carnitine pool is thought to be predominantly (90%) cytosolic (Zammit, 1999), the availability of free carnitine to CPT1 is markedly lower than suggested from determination of free carnitine in whole muscle homogenates. An explanation for this may be that the known catalytic site of CPT1 is located within the contact sites of the outer mitochondrial membrane and therefore not entirely available to the cytosolic carnitine pool. An increase in mitochondrial content over the duration of the study could also explain the apparent increase in fat oxidation and glycogen sparing observed in the Carnitine group during exercise at 50% Graphic after 24 weeks. However, if this was the case it would be expected that citrate synthase activity and/or Graphic would have also increased in this group, which was not observed. These observations, together with the finding that there was no evidence of glycogen sparing during exercise at 80% Graphic in the Carnitine group, makes it highly unlikely that an increase in mitochondrial content occurred over the 24 weeks of supplementation.

Another widely documented function of carnitine is as an acetyl group buffer during conditions of high glycolytic and PDC flux. During high intensity exercise, when acetyl group production by the PDC reaction is in excess of its utilisation by the TCA cycle, free carnitine buffers against acetyl-CoA accumulation by forming acetylcarnitine in a reaction catalysed by carnitine acetyl transferase (CAT), thereby ensuring a viable supply of free CoASH to sustain TCA cycle flux (Childress & Sacktor, 1966; Harris et al. 1987; Constantin-Teodosiu et al. 1991a). In the context of this, another major finding of the present study was the marked reduction in muscle lactate accumulation during exercise at 80% Graphic in the carnitine-loaded state after 24 weeks of supplementation when compared to Control, an effect probably mediated by the greater PDCa (38%) and flux (as evidenced by the 16% greater acetylcarnitine content) observed compared to Control. While it is clear that after 24 weeks muscle lactate did not accumulate in the Carnitine group to any lesser of an extent than seen at baseline, it is important to note that the absence of a change occurred in the face of increased glycogenolysis in both groups, which resulted in an increased lactate accumulation in the Control group only, and is explained by the carnitine-mediated increase in PDCa and flux in the Carnitine group. Furthermore, the magnitude of cellular energy disturbance (as indicated by PCr/ATP ratio) was significantly reduced during exercise at 80% Graphic at 24 weeks in the Carnitine group when compared to baseline and Control at 24 weeks. In keeping with this observation, we, and others, have previously reported that inertia in mitochondrial ATP production during the rest to exercise transition is at least partly limited by PDC activation and flux, resulting in increased anaerobic ATP generation (Timmons et al. 1997, 1998; Howlett et al. 1999; Roberts et al. 2002, 2005).

Pyruvate dehydrogenase activation status is principally regulated by a covalent mechanism of competing pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatases (PDP), which inactivate and activate the PDC, respectively. Pyruvate dehydrogenase kinase and PDP are themselves subject to metabolic regulation, and Ca2+ has been suggested as the principal metabolic activator of PDC during exercise by stimulating PDP (Constantin-Teodosiu et al. 2004). Considering that subjects in the present study exercised at the same work intensity (80% Graphic) during each visit, it can be assumed that the increase in cellular Ca2+ concentration was equal between visits and accordingly a similar stimulatory effect on the PDC was exerted regardless of skeletal muscle TC content. However, PDC activation is also regulated by end-product inhibition, primarily by an increase in the acetyl-CoA/CoASH ratio which stimulates PDK (Cooper et al. 1975; Pettit et al. 1975). Thus, as well as blunting acetyl-CoA accumulation during intense exercise and augmenting PDC flux, increased acetyl group buffering in the Carnitine group may also have modulated a reduction in the acetyl-CoA/CoASH ratio, thereby explaining the greater PDCa in the carnitine-supplemented group at 80% Graphic following 24 weeks of carnitine supplementation compared with Control.

Given that the performance test used in this study is reproducible (Jeukendrup et al. 1996; Stephens et al. 2008), and all the volunteers were recreational athletes familiar with intense exercise, a major finding of the present study has to be that the increase in muscle TC content after 24 weeks of supplementation resulted in a 35% increase in work output compared to Control (and an 11% increase from baseline). We, and others, have previously demonstrated that complete activation of PDC in the resting state, by pharmacological inhibition of PDK, markedly reduced anaerobic energy production in canine and human skeletal muscle during subsequent intense contraction (Timmons et al. 1997, 1998; Howlett et al. 1999; Roberts et al. 2002, 2005), and resulted in a substantial improvement in muscle contractile function (Timmons et al. 1997). It would appear therefore that the 38% greater PDCa and associated flux during exercise at 80% Graphic in the carnitine-loaded state in the present study, coupled with the reduction in muscle lactate accumulation and lower perceived exertion during exercise compared with Control, positively impacted upon work output during the subsequent performance trial. Indeed, it is likely that these metabolic effects observed at 80% Graphic in the carnitine-loaded state continued on into the performance trial given that subjects were attempting to perform as much work as possible in the 30 min of exercise. In keeping with our observations, Brass and colleagues have shown that carnitine loading of rodent soleus muscle reduced fatigue by 25% during electrically evoked contraction (Brass et al. 1993). Furthermore, Coyle (1995) concluded that the ability to maintain a high steady-state Graphic with low muscle lactate content is a prerequisite for enhanced endurance exercise performance in elite athletes. Indeed, muscle lactic acidosis has been suggested as a primary cause of fatigue during high-intensity exercise (Sahlin, 1992), hence the efforts to increase muscle buffering capacity by β-alanine feeding (Hill et al. 2007) or establish pre-exercise metabolic alkalosis by sodium bicarbonate ingestion (Wilkes et al. 1983; McKenzie et al. 1986; Bird et al. 1995) to improve high intensity exercise performance in humans. Finally, given that muscle glycogen content was 147 mmol (kg dry muscle) −1 following exercise at 80% Graphic in the Control group, it is possible that glycogen availability may have limited performance in this group during the 30 min work output trial, which would not have been the case in the Carnitine group where glycogen was 250 mmol (kg dry muscle)−1. It cannot be ruled out therefore that at least some of the positive effect of muscle carnitine loading on exercise performance was attributable to the glycogen sparing that occurred during exercise at 50% Graphic. Whether the beneficial effects of muscle carnitine loading on high intensity exercise and exercise performance led to the better maintenance of body mass in the face of 24 weeks of additional daily caloric intake when compared to control (i.e. via a regular increased energy expenditure during exercise training) is an interesting notion, but cannot be determined from the present study and remains to be explored.

In summary, this is the first study to demonstrate that muscle carnitine content can be increased in humans by dietary means and, perhaps more importantly, that carnitine plays a dual role in skeletal muscle fuel metabolism that is exercise intensity dependent. Specifically, we have shown that increasing muscle TC content spares muscle glycogen during low intensity exercise (consistent with an increase in muscle lipid utilisation), but during high intensity exercise results in a better matching of glycolytic, PDC and mitochondrial flux, thereby reducing muscle anaerobic ATP production. Furthermore, these metabolic changes resulted in positive effects on perception of effort and work output using a validated exercise performance test. Collectively these findings have significant implications for athletic performance and pathophysiological conditions where fat oxidation is impaired or anaerobic ATP production is accelerated during exercise (Noland et al. 2009).

Acute dietary nitrate supplementation improves cycling time trial performance

Source

Sport and Health Sciences, College of Life and Environmental Sciences, St Luke’s Campus, University of Exeter, Exeter, United Kingdom.

Abstract

PURPOSE:

Dietary nitrate supplementation has been shown to reduce the O2 cost of submaximal exercise and to improve high-intensity exercise tolerance. However, it is presently unknown whether it may enhance performance during simulated competition. The present study investigated the effects of acute dietary nitrate supplementation on power output (PO), VO2, and performance during 4- and 16.1-km cycling time trials (TT).

METHODS:

After familiarization, nine club-level competitive male cyclists were assigned in a randomized, crossover design to consume 0.5 L of beetroot juice (BR; containing ∼ 6.2 mmol of nitrate) or 0.5 L of nitrate-depleted BR (placebo, PL; containing ∼ 0.0047 mmol of nitrate), ∼ 2.5 h before the completion of a 4- and a 16.1-km TT.

RESULTS:

BR supplementation elevated plasma [nitrite] (PL = 241 ± 125 vs BR = 575 ± 199 nM, P < 0.05). The VO2 values during the TT were not significantly different between the BR and PL conditions at any elapsed distance (P > 0.05), but BR significantly increased mean PO during the 4-km (PL = 279 ± 51 vs BR = 292 ± 44 W, P < 0.05) and 16.1-km TT (PL = 233 ± 43 vs BR = 247 ± 44 W, P < 0.01). Consequently, BR improved 4-km performance by 2.8% (PL = 6.45 ± 0.42 vs BR = 6.27 ± 0.35 min, P < 0.05) and 16.1-km performance by 2.7% (PL = 27.7 ± 2.1 vs BR = 26.9 ± 1.8 min, P < 0.01).

CONCLUSIONS:

These results suggest that acute dietary nitrate supplementation with 0.5 L of BR improves cycling economy, as demonstrated by a higher PO for the same VO2 and enhances both 4- and 16.1-km cycling TT performance.

Abstract

Exhaustive exercise induces disturbances in metabolic homeostasis which can result in amino acid catabolism and limited L-arginine availability. Oral L-citrulline supplementation raises plasma L-arginine concentration and augments NO-dependent signalling. Our aim was to evaluate the effects of diet supplementation with L-citrulline-malate prior to intense exercise on the metabolic handle of plasma amino acids and on the products of metabolism of arginine as creatinine, urea and nitrite and the possible effects on the hormonal levels. Seventeen voluntary male pre-professional cyclists were randomly assigned to one of two groups: control or supplemented (6 g L-citrulline-malate 2 h prior exercise) and participated in a 137-km cycling stage. Blood samples were taken in basal conditions, 15 min after the race and 3 h post race (recovery). Most essential amino acids significantly decreased their plasma concentration as a result of exercise; however, most non-essential amino acids tended to significantly increase their concentration. Citrulline-malate ingestion significantly increased the plasma concentration of citrulline, arginine, ornithine, urea, creatinine and nitrite (p < 0.05) and significantly decreased the isoleucine concentration from basal measures to after exercise (p < 0.05). Insulin levels significantly increased after exercise in both groups (p < 0.05) returning to basal values at recovery. Growth hormone increased after exercise in both groups, although the increase was higher in the citrulline-malate supplemented group (p < 0.05). L-citrulline-malate supplementation can enhance the use of amino acids, especially the branched chain amino acids during exercise and also enhance the production of arginine-derived metabolites such as nitrite, creatinine, ornithine and urea.

Myo-Inositol trispyrophosphate: a novel allosteric effector of hemoglobin with high permeation selectivity across the red blood cell plasma membrane.

Source

Institut de Science et d’Ingénierie Supramoléculaires, Université de Strasbourg, 8 allée Gaspard Monge, 67000 Strasbourg, France.

Abstract

myo-Inositol trispyrophosphate (ITPP), a novel membrane-permeant allosteric effector of hemoglobin (Hb), enhances the regulated oxygen release capacity of red blood cells, thus counteracting the effects of hypoxia in diseases such as cancer and cardiovascular ailments. ITPP-induced shifting of the oxygen-hemoglobin equilibrium curve in red blood cells (RBCs) was inhibited by DIDS and NAP-taurine, indicating that band 3 protein, an anion transporter mainly localized on the RBC membrane, allows ITPP entry into RBCs. The maximum intracellular concentration of ITPP, determined by ion chromatography, was 5.5×10(-3) M, whereas a drop in concentration to the limit of detection was observed in NAP-taurine-treated RBCs. The dissociation constant of ITPP binding to RBC ghosts was found to be 1.72×10(-5) M. All data obtained indicate that ITPP uptake is mediated by band 3 protein and is thus highly tissue-selective towards RBCs, a feature of major importance for its potential therapeutic use.

BACKGROUND:

beta-Alanine (betaA) has been shown to improve performance during cycling. This study was the first to examine the effects of betaA supplementation on the onset of blood lactate accumulation (OBLA) during incremental treadmill running.

METHODS:

Seventeen recreationally-active men (mean +/- SE 24.9 +/- 4.7 yrs, 180.6 +/- 8.9 cm, 79.25 +/- 9.0 kg) participated in this randomized, double-blind, placebo-controlled pre/post test 2-treatment experimental design. Subjects participated in two incremental treadmill tests before and after 28 days of supplementation with either betaA (6.0 g.d-1)(betaA, n = 8) or an equivalent dose of Maltodextrin as the Placebo (PL, n = 9). Heart rate, percent heart rate maximum (%HRmax), %VO2max@OBLA (4.0 mmol.L-1 blood lactate concentration) and VO2max (L.min-1) were determined for each treadmill test. Friedman test was used to determine within group differences; and Mann-Whitney was used to determine between group differences for pre and post values (p < 0.05).

RESULTS:

The betaA group experienced a significant rightward shift in HR@OBLA beats.min-1 (p < 0.01) pre/post (161.6 +/- 19.2 to 173.6 +/- 9.9) but remained unchanged in the PL group (166.8 +/- 15.8 to 169.6 +/- 16.1). The %HRmax@OBLA increased (p < 0.05) pre/post in the betaA group (83.0% +/- 9.7 to 88.6% +/- 3.7) versus no change in the PL group (86.3 +/- % 4.8 to 87.9% +/- 7.2). The %VO2max@OBLA increased (p < 0.05) in the betaA group pre/post (69.1 +/- 11.0 to 75.6 +/- 10.7) but remained unchanged in the PL group (73.3 +/- 7.3 to 74.3 +/- 7.3). VO2max (L.min-1) decreased (p < 0.01) in the betaA group pre/post (4.57 +/- 0.8 to 4.31 +/- 0.8) versus no change in the PL group (4.04 +/- 0.7 to 4.18 +/- 0.8). Body mass kg increased (p < 0.05) in the betaA group pre/post (77.9 +/- 9.0 to 78.3 +/- 9.3) while the PL group was unchanged (80.6 +/- 9.1 to 80.4 +/- 9.0).

CONCLUSIONS:

betaA supplementation for 28 days enhanced sub-maximal endurance performance by delaying OBLA. However, betaA supplemented individuals had a reduced aerobic capacity as evidenced by the decrease in VO2max values post supplementation.

FULL article:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2887393/?tool=pubmed

 

The effect of acute pre-exercise dark chocolate consumption on plasma antioxidant status, oxidative stress and immunoendocrine responses to prolonged exercise.

Source

Department of Sport and Exercise Science, Aberystwyth University, Ceredigion, Aberystwyth, SY23 3FD, UK, gdd@aber.ac.uk.

Abstract

PURPOSE:

Acute antioxidant supplementation may modulate oxidative stress and some immune perturbations that typically occur following prolonged exercise. The aims of the present study were to examine the effects of acutely consuming dark chocolate (high polyphenol content) on plasma antioxidant capacity, markers of oxidative stress and immunoendocrine responses to prolonged exercise.

METHODS:

Fourteen healthy men cycled for 2.5 h at ~60% maximal oxygen uptake 2 h after consuming 100 g dark chocolate (DC), an isomacronutrient control bar (CC) or neither (BL) in a randomised-counterbalanced design.

RESULTS:

DC enhanced pre-exercise antioxidant status (P = 0.003) and reduced by trend (P = 0.088) 1 h post-exercise plasma free [F(2)-isoprostane] compared with CC (also, [F(2)-isoprostane] increased post-exercise in CC and BL but not DC trials). Plasma insulin concentration was significantly higher pre-exercise (P = 0.012) and 1 h post-exercise (P = 0.026) in the DC compared with the CC trial. There was a better maintenance of plasma glucose concentration on the DC trial (2-way ANOVA trial × time interaction P = 0.001), which decreased post-exercise in all trials but was significantly higher 1 h post-exercise (P = 0.039) in the DC trial. There were no between trial differences in the temporal responses (trial × time interactions all P > 0.05) of hypothalamic-pituitary-adrenal axis stress hormones, plasma interleukin-6, the magnitude of leukocytosis and neutrophilia and changes in neutrophil function.

CONCLUSION:

Acute DC consumption may affect insulin, glucose, antioxidant status and oxidative stress responses, but has minimal effects on immunoendocrine responses, to prolonged exercise.