WO2006079829A1 - Phosphoglycerides for use in improving heart rate recovery and increasing exercise capacity - Google Patents

Abstract

There is described the use of phosphoglycerides, such as phosphatidyl serine, optionally in combination with docosahexaenoic acid, for improving heart recovery and for increasing exercise capacity in a subject.

Description

PHOSPHOGLYCERIDES FOR USE IN IMPROVING HEART RATE RECOVERY AND INCREASING EXERCISE CAPACITY

The present invention relates to the use of certain phosphoglycerides in improving heart rate recovery and in increasing exercise capacity.

The present specification makes reference to various publications. These are indicated by a number in square brackets. The publications are listed at the end of the description and are incorporated by reference into this specification in their entirety.

It is well known that during physical exercise or under conditions of stress a subject's heart rate increases. It is desirable for the heart rate to return to normal as quickly as possible. This is especially true for subjects who are suffering from, or are at risk of suffering from, heart disease or angina or those taking part in sports requiring a succession of sprints or other bouts of intense exercise. It is also well known that during exercise a subject fatigues. It is desirable to decrease the rate of fatigue as much as possible or to delay the onset of fatigue as long as possible, thereby increasing exercise capacity.

It has been reported [1] that, in humans, when 50 or 75 mg of phosphatidylserine was administered intravenously, the ACTH and Cortisol responses to the physical stress associated with riding a bicycle ergometer were reduced. It has also been reported [2] that 800 mg/day of phosphatidylserine administered intravenously for ten days also reduced the ACTH and Cortisol responses. However, neither report mentions any effect on heart rate, or on the ability to increase exercise capacity. (We have also investigated the Cortisol response after riding a bicycle but we were unable to replicate the previous findings.)

It is an aim of the present invention to provide a means for improving heart rate recovery. It is a further aim of the present invention to provide a means for improving exercise capacity.

In the present description and claims, the term "a phosphoglyceride" has the meaning "phosphatidyl serine, phosphatidyl choline, phosphatidyl inositol, phosphatidyl ethanolamine, or any precursor thereof which is directly metabolized thereto in vivo, either alone or in any combination with one another". The present invention provides a phosphoglyceride or a pharmaceutical composition containing a phosphoglyceride for use in improving heart rate recovery.

The present invention also provides a phosphoglyceride or a pharmaceutical composition containing a phosphoglyceride for use in improving exercise capacity. The present invention further provides a method for improving heart rate recovery comprising treating a subject with a phosphoglyceride.

The present invention further provides a method for improving exercise capacity comprising treating a subject with a phosphoglyceride. The present invention also provides the use of a phosphoglyceride in ergogenesis.

The phosphoglyceride may be administered to any mammal and is preferably administered to a human subject.

The preferred phosphoglyceride is phosphatidyl serine.

The phosphoglyceride may be used in combination with docosahexaenoic acid (DHA).

The phosphoglyceride may be provided as a pharmaceutical composition in combination with a pharmaceutically acceptable carrier. The pharmaceutical composition may be a liquid, tablet, capsule or caplet, among others. Suitable pharmaceutical compositions are disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Company, New Jersey, USA, 1991.

A preferred pharmaceutical composition comprises a capsule containing a carrier and the phosphoglyceride. A preferred carrier is lecithin, which is predominantly phosphatidyl choline but which may also contain phosphatidyl inositol and phosphatidyl ethanolamine. Preferably, the pharmaceutical composition also contains DHA.

The phosphoglyceride may be administered before, during or after a subject carries out physical exercise or is subject to stress. If the phosphoglyceride is being administered to increase exercise capacity, the phosphoglyceride is preferably administered prior to exercise. In such a case, the phosphoglyceride may be administered up to one or more days prior to exercise (i.e. administration of the phosphoglyceride may cease 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days etc. prior to exercise beginning, but not more than 100, preferably not more than 75, preferably not more than 50, preferably not more than 40, preferably not more than 30, preferably not more than 20 days prior to exercise beginning).

If DHA is also administered, it may be administered before, at the same time as, or after the administration of the phosphoglyceride.

The phosphoglyceride and, if also used, the DHA may be administered in a single dose or in a number of doses at regular intervals. The amount of phosphoglyceride and, if present, DHA in each dose will vary depending on whether single or multiple dose administration will be used. The dose will also depend on the age, weight, physical condition and previous medical history of the subject and the physical exercise or stress which the subject will experience. Other factors, as are well known to those skilled in the art, may also need to be taken into account.

The dose may be a single dose of from 1.5 to 12 mg/kg/day, preferably 2 to 10 mg/kg/day, most preferably about 4 mg/kg/day for the phosphoglyceride. Preferably, the phosphoglyceride is administered as a number of doses throughout the day in amounts to give the same total daily dose as set out above. If desired, a larger dose may be given initially, followed by smaller maintenance doses thereafter. A larger dose of phosphoglyceride may be administered, if required, to increase exercise capacity. For instance, a total of 500-1000 mg/day, preferably 600-900 mg/day, preferably 700-800 mg/day may be administered. Preferably 750 mg/day is administered to increase exercise capacity. The phosophoglyceride may be administered for one or more days (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 etc. days). Preferably the phosphoglyceride is administered for 10 days.

For DHA, a single dose will be of 5 to 25 mg/kg/day.

It has surprisingly been found that administration of a phosphoglyceride to a subject leads to an increase in the recovery of heart rate. This will reduce the demand on the heart induced by physical exercise or stress. This will be of particular use for subjects suffering from, or at risk of suffering from, a heart condition or angina, or for subjects taking part in activities, for example soccer or tennis, which involve successive bouts of explosive exercise interspersed with periods of recovery, as it will reduce the demand on the heart. It will also be of use for athletes or non-athletes who "work out", as it will allow them to reduce their recovery times.

It has also been found that administration of a phosphoglyceride makes the subject more relaxed.

It has also been found that administration of phosphoglyceride increases a subject's exercise capacity. By "increase in exercise capacity" we mean that following administration of phosphoglyceride, a subject has demonstrated the ability to exercise for longer and/or at a higher intensity compared to their ability prior to administration. In other words, the rate of fatigue is decreased as much as possible or the onset of fatigue is delayed as long as possible For instance, this may be measured by assaying the exercise time to exhaustion at 85% VO_{2 max}.

The beneficial effects of the present invention are shown, by way of example only, by the experiments reported below. The description below refers to: Figure 1, which shows the heart rate while exercising for 10 and 20 minutes;

Figure 2, which shows the recovery of heart rate after exercise;

Figure 3, which shows the first two minutes of heart rate recovery;

Figure 4, which shows Cortisol levels during exercise and recovery;

Figure 5, which shows a schematic diagram of the exercise capacity experimental design;

Figure 6, which shows exercise times to exhaustion at 85% VO_{2 max} completed at the end of each main exercise trial. Individual data are presented as open shapes and mean data are presented as filled shapes. PS: phosphatidylserine group; P: placebo group (supplementation group x trial interaction, P=O.007); Figure 7, which shows (a) Energy expenditure; (b) Carbohydrate oxidation rates; and (c) Fat oxidation rates during each main exercise trial. Values represent mean ±

SEM (N=7). PS: phosphatidylserine group; P: placebo group. Trial 1: pre- supplementation; Trial 2: post-supplementation;

Figure 8, which shows (a) On-kinetic mean response times; and (b) Off-kinetic mean response times during moderate and very heavy exercise stages. Values represent mean ± SEM (N=7). PS: phosphatidylserine group; P: placebo group.

Trial 1 : pre-supplementation; Trial 2: post-supplementation; and

Figure 9, which shows serum Cortisol concentrations throughout each exercise trial.

Values represent mean ± SEM (N=7). PS: phosphatidylserine group; P: placebo group. Trial 1: pre-supplementation; Trial 2: post-supplementation.

Example 1 : Improvements in heart rate recovery

One definition of stress is that it is a bodily response that occurs when faced with intense demands. In this context, the release of the adrenal hormones adrenaline and Cortisol may be viewed as evidence that the body has been subject to stress. Similarly indices of autonomic nervous system reactivity, for example heart rate or blood pressure, can be used as evidence of a stress-related response. The results set forth below show the impact of ingestion of the phosphoglyceride phosphatidylserine (PS) on mood, Cortisol release and the increase in heart rate when faced with a stressful situation.

Twenty male undergraduates, average age 20.6 years, were recruited when they responded to a poster. Of these, three dropped out for reasons unrelated to the study. The subjects were non-smokers, not currently taking medication and did not have a history of chronic physical or psychiatric illness. They gave written informed consent. The procedure was approved by the local ethics committee.

The 20 subjects were randomly, and under a double-blind procedure, allocated to one of two treatment groups that daily took either 300 mg PS or a placebo. Initial heart rate and blood pressure were measured and subjects reported their mood over the previous month. After thirty days, the subjects returned to the laboratory.

In the laboratory: baseline blood pressure, pulse rate and mood were monitored - a cannula was implanted to allow a blood sample to be taken to determine baseline Cortisol values; subjects were fitted with a heart-rate monitor and rode an ergonomic bicycle for ten minutes, after which mood was monitored and a second blood sample was taken; after exercise for a further ten minutes, mood was monitored for a third time, blood was sampled and blood pressure and heart rate recorded; and subjects rested for a total of forty minutes and after twenty and forty minutes, mood was assessed, further blood samples were taken and blood pressure and heart rate were recorded.

The pharmaceutical compositions were manufactured by Lucas Meyer, Hamburg, Germany. Each PS-containing capsule contained 500 mg of Lecithin-PS that provided 100 mg PS. Three capsules a day were consumed, providing a total of300 mg PS per day. The placebo capsules contained 500 mg of hard fat. The capsules were made from gelatin and were coloured red with iron oxide. When the double-blind was broken, six were found to have taken PS and eleven the placebo.

Subjects rated their mood using 100 mm visual analogue scales. The baseline rating and the ratings prior to mental arithmetic reflected how they felt "over the previous month". The other mood ratings reflected how they felt at that moment. Six of the scores of mood reflected the basic dimensions of the Profile of Mood Scale [3]: Composed -

Anxious; Hostile - Agreeable; Elated - Depressed; Unsure - Confident; Energetic - Tired; Confused - Clear-headed. A final mood scale, Relaxed - Stressed, was used as it reflected the major interest of the study.

Blood pressure was measured using a digital blood pressure monitor (Omron,

Tokyo, Japan). Heart rate was measured using a Polar Accurex Plus Heart Rate Monitor (Polar,

Kemple, Finland). Heart rate was recorded for twenty minutes while exercising and for forty minutes after exercising.

Subjects worked at 2 watts per kilogram body weight on a bicycle ergometer

(Monark-Crescent, Varberg, Sweden) for the first ten minutes and then at 2.5 watts per kilogram body weight for a second ten minute period. A sub-maximal estimate of VO_{2 max} was calculated based on the average pulse rate at the fifth and sixth minutes of the first exercise session [4].

Two millilitres of blood were collected in "Hemogard" Vacutainers (Becton

Dickinson, UK) from a cannula implanted in the antecubital vein. Samples were taken before exercise, after ten and twenty minutes of exercise and after twenty and forty minutes of the recovery period. Blood was allowed to clot for sixty minutes at room temperature and then centrifuged at 1200 g for twelve minutes. 0.3-0.5 ml aliquots of serum were stored at -20⁰C. Cortisol was measured using a commercially available ¹²⁵I radioimmunoassay (CORTCTK-125, DiaSorin Ltd). Levels were determined from a standard curve prepared by the use of standards containing 0 and 800 ng of authentic cortisol/100 microlitres. The sensitivity of the assay was less than 5 ng/ml serum.

Statistical Package for the Social Sciences (SPSS) was used to calculate appropriate analysis of variance designs. As uneven sample sizes prevented the calculation of simple main effects, t-tests were used to explore significant interactions. When blood pressure was compared before and after ingesting the capsules but before exercise, it was found that systolic blood pressure (F(1, 15) = 0.12, n.s.) was not influenced by PS . Diastolic blood pressure increased with time (F(1, 15,) = 6.65, p<0.02), possibly as a response to the impending cannulation and exercise. The interaction with ingestion of PS was, however, not significant (F(1, 15) = 1.65, n.s.) Similarly there was a trend for heart rate to be higher just before exercise, when compared with the baseline

(F(1, 15) = 3.20, p<0.09), although the interaction with ingestion of PS was not significant

(F(l,15) = 0.07, n.s.). During exercise there was no evidence that either diastolic (F(1, 15) = 0.33, n.s.) or systolic blood pressure (F(1, 15) = 1.75, n.s.) differed, although with heart rate the interaction Type of supplement x Test session was significant (F(3,42) = 2.93, p<0.04). Table 1 shows that the heart rate of those taking PS declined more quickly after exercise Examination of the individual sessions found that twenty minutes after exercise the difference in heart rate of the two groups approached statistical significance (p<0.07). The VO_{2 max} of those receiving PS did not differ significantly (F(1, 15) = 0.33, n.s.; Placebo 2.0 litres/min +/-0.7; PS 3.4 litres/min +/- 0.4). The measures of VO_{2 max} indicated that the subjects were of slightly below average fitness for their age. Ingesting PS did not influence the heart rate during the first ten minutes of exercise

(Figure 1; F(1, 15) = 1.73, n.s.). In the second ten minutes of exercise, the interaction Supplement x Minute reached statistical significance (F(9,135) = 4.87, p<0.001). Figure 1 illustrates that this reflected a greater heart rate recovery, while a blood sample was taken after ten minutes, in those who had taken PS. When PS has been consumed, heart rate was significantly lower during the first (p<0.01) minutes of the second half of the exercise session and approached significance during the second (p<0.09).

TABLE 1 BLOOD PRESSURE BEFORE AND AFTER PS INGESTION The data are means +/-S.D. Units for blood pressure (B.P.) are millmetres of mercury and for heart rate are beats per minute.

Diastolic Systolic Heart B.P. B.P. Rate

Placebo PS Placebo PS Placebo PS

Before 72 78 122 125 69 81 supplementation +/-7 +/-10 +/-10 +/-11 +/-12 +-26

Before exercise 75 86 122 128 78 88 +/-8 +/-14 +/-10 +/-19 +/-10 +/-19

After exercise 74 85 125 134 120 114 +/-14 +/-18 +/-11 +/-26 +/-18 +/-13

After 20 minutes 71 79 113 109 91 80 rest +/-11 +/-12 +/-17 +/-13 +/-10 +/-9

After 40 minutes 71 82 115 122 78 74 rest +/-8 +/-11 +/-12 +/-20 +/-14 . +/-8 The analysis of the first twenty minutes of recovery following the exercise found that the main effect of ingesting PS just missed statistical significance (F(1, 15) = 3.53, p<0.08), that is the heart rate of those taking PS tended to recover more rapidly (Figure 2). The interaction Supplement x Time was non-significant (F(19,285) = 0.85, n.s.). There was no suggestion that ingestion of PS influenced heart rate during the following twenty minutes (F(1, 15) = 0.60, n.s.). When a more detailed analysis of heart rate in the first two minutes of recovery was made there was an interaction between ingestion of PS and time (F(1, 105) = 2.24, p<0.04). Figure 3 illustrates that the heart rate of those taking PS fell more rapidly than those taking the placebo. For comparison the last two minutes of the exercise period is shown when ingestion of PS did not influence heart rate (F(I₅15) = 0.05, n.s.).

When the seven mood dimensions were examined, in no instance was there a significant difference at baseline between those who eventually consumed the PS and those who consumed the placebo. Similarly when mood at baseline and immediately before exercising was compared, in no instance was there a significantly Supplement x Time interaction.

The baseline measures of the various measures of mood on arrival in the laboratory were used as a co-variate and changes in mood during exercise and recovery were considered using analysis of variance. Neither "agreeable" (F(1, 13) = 2.36, n.s.) "clearheaded" (F(1, 13) = 1.02, n.s.), "elated" (Fl,13) = 0.00,N.S.), "energetic" (F(1, 13) = 0.84, n.s.) nor "relaxed" (F(1, 13) = 1.62, n.s.) were influenced by the ingestion of PS. Similarly the Supplement x Time interactions failed to reach statistical significance. However, with "confident" (F(1, 13) = 5.68, ρ<0.03), the main effect of ingestion of PS reached statistical significance. Those who had taken PS felt more confident throughout, particularly immediately after exercise. With "composed", the interaction Supplement x Time reached statistical significance (F(2,28) = 3.82), p<0.03). Those taking PS felt more composed, particularly immediately after exercise. These results are shown in Table 2.

When the Cortisol values were considered, they changed over time (F(4,56) = 3.37 p<0.015). As can be seen from Figure 4, they increased during exercise and declined in the recovery period. However, neither the Supplement main effect (F(1, 14) = 0.68, n.s.) nor the interaction Supplement x Time (F(4,56) = 0.88, n.s.) reached statistical significance. The finding that mood was improved following PS ingestion (Table 2) supports previous reports. In the elderly, the taking of PS has been found to associated with better mood [5], [6] and [7]. In the first study of the present series the taking of 300mg PS each day for a month was associated with reports of feeling less stressed and having a better mood, especially in those with a more neurotic personality.

The previous reports that PS ingestion blunted the Cortisol response to the physical stress associated with cycling [1] and [2] were not confirmed (Figure 4). It is unclear to what extent differences in methodology proved to be critical. In the two previous studies of this topic, PS in doses of either 50 or 75 mg were administered intravenously [1] or alternatively 800 mg/day PS was taken orally for ten days [2]. Although the present study used a different protocol, the taking of 300 mg/day for 30 days, the positive findings using different parameters (Figures 1-3, Tables 1-2) suggests that the procedure was appropriate.

TABLE 2 THE INFLUENCE OF PHOSPHATID YLSERINE ON MOOD WHILE EXERCISING

The data are means (S.D.) where a higher value reflected a more positive mood

Agreeable Clearheaded Composed Confident Elated Energetic Relaxed

Plac PS Plac PS Plac PS Plac PS Plac PS Plac PS Plac PS

Before 68 70 65 72 78 66 59 74 55 59 56 65 52 65

Supple (24) (28) (16) (21) (15) (21) (15) (24) (16) (18) (29) (24) (29) (26) mentation

Before 77 92 57 82 56 61 58 82 55 54 43 60 56 70 exercise (19) (10) (21) (14) (25) (36) (23) (6) (15) (17) (17) (33) (21) (22)

After 67 86 51 69 62 85 62 84 54 62 25 38 53 71 exercise (16) (9) (21) (12) (16) (12) (19) (11) (11) (15) (22) (25) (21) (17)

After 20 80 87 66 84 79 86 72 86 60 50 52 57 75 83

Min rest (12) (9) (17) (10) (9) (8) (9) (8) (11) (3) (15) (17) (16) (18)

After 40 82 88 75 85 82 89 77 84 68 69 64 69 82 88

Min rest (10) (11) (16) (10) (11) (10) (13) (7) (16) (25) (14) (12) (13) (13) Although previously the lipid composition of the diet has been reported to change the phospholipid profile of the heart [8], the finding that PS supplementation increased the rate of heart-rate recovery after exercise is a novel and unexpected finding. Phospholipids are a rich store of fatty acids in muscles [9] and [10]. However, as the total phospholipid fraction in skeletal muscles remains stable, even after prolonged starvation or exercise, it appears that phospholipids do not donate their fatty acids for energy purposes. Exercise training has been found to increase the phospholipid content of human and rat muscles [11], [12], [13] and [14].

Tibbits et al [15] examined the lipid composition of purified plasma membranes isolated from rat heart. Compared to sedentary controls, the total phospholipid increased by 23%. The mechanisms responsible for the changes in cardiac PS are uncertain. Tibbits et al. [15] speculated that because PS is a major binding moiety of sarcolemmal Ca^+"1" [16], PS influences the sarcolemmal control of excitation-contraction coupling.

As PS comprises 10-20% of total phospholipids in the cell membrance bilayer, they have a range of functions. Membrane phospholipids play an important role in cell to cell communication and the transfer of biochemical messages into the cell. Although without appropriate physiological studies it is only possible to speculate as to the mechanism by which PS acted in the present study, various possibilities exist. Amongst several membrane proteins known to require PS are those associated with NADPH-cytochrome reductase [17] and calcium and magnesium ATPase [18].

The present finding, that PS ingestion increased the rate of heart rate recovery following exercise, is of considerable interest. On the basis of this study, it is reasonable to predict that phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine have similar actions. A mixture of various phospholipids would also be advantageous. The impact of PS, or other phosphoglyceride, ingestion on athletic performance and in those with heart disease will be of clinical use.

Example 2: Improvements in exercise capacity Subjects Fourteen healthy male subjects (Table 3) volunteered to participate in this study and completed all the study requirements. All subjects were informed about the potential risks of the study and gave written informed consent for their participation in the study, which was approved by a university ethics committee. In addition, all subjects were informed that they might be asked to consume a supplement rich in PS; however, there are currently no reported, or inferred, side effects associated with PS supplementation [19, 20]. The experimental procedures were in accordance with the policy statement of the American College of Sports Medicine. No subject had prior history of cardiovascular or respiratory disease and all subjects were non-smokers. Potential subjects attended an interview prior to undertaking the study and were subsequently excluded if they had taken nutritional supplements in the last 8 weeks.

Table 3: Subject characteristics for phosphatidylserine supplementation group (PS) and Placebo group (P).

Characteristic PS (N=7) P (N=7) P-value

Age (dee , years) 23,4 ± 1.9 22,2 ± 1,1 0.638

Mass (kg) 84,9 ± 3, 8 87.7 ± 3.2 0,630 H ei ght (m) 1.79 ± 0,01 1,81 ± 0,02 0.528

Maximal oxygen uptake (ml- kg^-1-min^-1) 43, 8±2.0 42.4 ± 1,7 0.645

Values are mean SEM, P-value calculated usingindependent sample 1-test

Experimental design

Prior to the main exercise trials, all subjects visited the laboratory on two occasions in order to complete an incremental exercise test and a familiarisation trial. Subjects then performed two main exercise trials, which consisted of staged intermittent cycling, separated by 16.0 ± 1.3 d. Approximately five days after completion of the first main exercise trial the subjects were assigned, in a randomised double-blind fashion, to either a PS group or a placebo (P) group and instructed to take supplements for the 10 days prior to trial two. The experimental design is illustrated in Figure 5.

The PS group ingested 750 mg/d PS and the P group ingested a weight matched glucose polymer. The PS supplements were manufactured using the method of specific transesterification of soybean lecithin and then blended with additional soybean lecithin to provide a concentration of 20% PS (Lucas Meyer; Hamburg, Germany). Both supplements were administered in capsules and placed in generic packaging. Subjects were instructed to maintain their normal diet and activity patterns throughout the study. Food was weighed and recorded by the subjects for two days prior to each exercise trial and for the day afterwards. Analysis using commercial software (CompEat v5.8.0; Nutrition Systems, UK) revealed that there were no differences in energy intake or dietary composition between supplementation groups or trials. The daily diet comprised of 11.2 ± 0.6 MJ/d , of which 52 ± 3%, 30 ± 1%, 16 ± 1% and 2 ± 1% of energy intake was obtained from carbohydrates, fat, protein and alcohol, respectively. In addition, the subjects were instructed to abstain from strenuous exercise for three days prior to and two days following each trial. At the completion of the study all subjects gave their verbal assurance that they had complied with all instructions.

Procedures

During their initial visit to the laboratory the subjects completed an incremental exercise test on an electromagnetically braked cycle ergometer (Lode Excalibur Sport; Lode, Holland). The work rate began at 60 W and thereafter increased in 30 W increments every 3 min until volitional exhaustion. Subjects were instructed to pedal at a constant cadence between 75 and 85 rpm. Heart rate (Polar S810; Polar Electro Oy, Finland) and breath-by- breath respiratory parameters (Oxycon Pro; Jaeger, Germany) were simultaneously recorded. The test was used to identify maximal oxygen uptake (VO_{2 max}) and also to calculate, using linear regression, the work rates required for the staged intermittent exercise protocol.

The subjects then completed a staged intermittent exercise protocol on 3 separate occasions. The intermittent protocol required subjects to complete three 10-min stages of cycling at work rates calculated to elicit 45, 55 and 65% VO_{2 max} followed by a final exercise bout at 85% VO_{2 max} that was continued until exhaustion (an inability to maintain a cadence of 60 rev/min despite verbal encouragement). All exercise stages were interspaced with 5-min passive rest periods. The first exercise trial served to familiarise the subjects with the intermittent exercise protocol and the subjects remained blind to their exercise times to exhaustion throughout the study in order to prevent an anticipated trial- order effect. The subsequent main exercise trials (Tl and T2) were completed 14-21 days apart. Breath-by-breath respiratory data (Oxycon Pro; Jaeger, Germany) and heart rate (Polar S810; Polar Electro Oy, Finland) were monitored throughout Tl and T2. Exercise- induced feeling inventory (EFI: [21]) was completed at Pre-exercise and following each exercise stage. Blood samples were taken by venipuncture (Vacutainer system; Becton- Dickinson Ltd, UK) from an antecubital vein at Pre-exercise, on completion of the 55% VO₂ max (Post-55%) and 65% VO_{2 max} (Post-65%) exercise stages, 20 min after the completion of the trial (Post-exercise) and 24 hours after the trial (Post-24 hr). An additional capillary blood sample was taken immediately after exhaustion (Post-85%) for the analysis of blood lactate and glucose concentrations.

Analysis of oxygen uptake and heart rate Breath-by-breath oxygen uptake, carbon dioxide production and 5 s heart rate data were averaged for the last minute of exercise for all exercise intensities. Subsequently nonprotein respiratory exchange ratios were used to calculate the rates of carbohydrate and fat oxidation for Pre-exercise and during each exercise stage up to and include 65% VO_{2 max} as previously described [22], using equations derived by Peronnet and Massicotte [38]. Linear regression was applied to the final 3 min of oxygen uptake data for each of these stages and the 95% confidence interval for the slope was inspected; steady state was assumed if the 95% confidence interval included zero.

In addition, breath-by-breath oxygen uptake data were initially edited to remove occasional errant breaths (from coughs, sighs or swallows) when values were greater than four standard deviations from the local mean [23]. The data for all exercise stages and rest periods were interpolated, using a cubic spline, in 1-s intervals. The data for moderate intensity exercise (45-65% VO_{2 max}) and subsequent rest periods during each trial were normalised (normalised to the respective last minute Vθ₂_values), time-aligned and averaged. The data for the final bout (85% VO_{2 max}) were modelled separately as the oxygen uptake response to different intensity domains (below and above lactate threshold) have been suggested to differ [24]. The traditionally defined phase II oxygen uptake on- kinetic response [25] was determined for 45-65% VO₂ max and 85% VO₂ m_ax- The end of phase I was set to 20 s after the onset of constant-load exercise and a mono-exponential function (Eq. 1) was fitted between the end of phase I and 3 min of exercise [26] using iterative non-linear regression techniques (SPSS version 12.0; SPSS Inc., IL). Similarly, the off-kinetic response to each exercise stage was determined using a mono-exponential function (Eq. 2) after omission of the first 20 s of post-exercise data

VO₂(t) = VO₂(B) + G (l-e-^(t-^TD)/τ) _ (1) VO₂Ct) = VO₂(EE) + G (_e-^(t-^TC')/τ') _ (2) where VO₂(t) is oxygen uptake at time t, VO₂ (B) is baseline oxygen uptake (average VO₂_during the minute prior to the onset of exercise), VO₂(EE) is end of exercise oxygen uptake (average VO₂_during the last minute of exercise), G is the primary gain (i.e. the calculated change in oxygen uptake), TD is the on-kinetic time delay and τ is the time constant (prime mark designates off-kinetics parameters). Mean response times for the on- kinetic responses (MRτon) and off-kinetic responses (MRroff) were calculated as TD + τ and TD' + τ', respectively. Blood sampling and analysis

Venous blood was collected in a 5 mL container (Becton-Dickinson Ltd, UK) containing the anticoagulant ethylenediaminetetra-acid (EDTA). Several small aliquots were removed for the triplicate determination of blood lactate and glucose concentration (YSI 2300 Stat, Yellow Springs Instruments, USA), blood hemoglobin concentration (Hemocue Ltd, UK), hematocrit (Micro hematocrit MK IV, Hawksley, England) and changes in plasma volume as previously described [27]. The remaining blood was centrifuged at 3000 g for 15 min to obtain plasma, which was subsequently dispensed and frozen at -70 ⁰C. Two additional 7 mL blood sample were collected in serum separation tubes (Becton-Dickinson Ltd, UK), left to stand for 15 min then centrifuged at 3000 g for 15 min to obtain serum. The serum was transferred to appropriate containers and subsequently frozen at -70 ⁰C. Serum Cortisol concentrations were determined using an automated time-resolved fluoroimmunoassay (AutoDELFIA™ Cortisol kit, Perkin Elmer, Life Sciences, UK).

Perceived feeling states

Subjects were instructed to respond to the EFI as described in detail by Gauvin and Rejeski [21]. Briefly, the subjects rated their feelings using the 12-item adjective scale on an analogue scale from 0 (do not feel) to 5 (feel very strongly). The appropriate adjectives were averaged to obtain four perceived feeling states (Positive Engagement, Revitalisation,

Tranquillity and Physical Exhaustion) at each time point as previously described [21]. The

EFI was specifically developed to assess distinct feeling states that occur during stages of exercise and psychometric studies have indicated concurrent and discriminant validity

[21]. Moreover, the EFI has been demonstrated to be sensitive to interventions involving recreational active individuals [28].

Statistical analysis

Statistical analysis was carried out using SPSS software (version 12.0; SPSS Inc., IL). Group data were expressed as mean + SEM and statistical significance was set at the P <

0.05 level. Subject characteristics were compared under supplementation groups using independent samples t-tests (Table 3). The exercise times to exhaustion during familiarisation, Tl and T2 were assessed using mixed-model repeated measures ANOVA (within subject factors: trials; between subject factor: supplementation groups) followed by simple main effect analysis. The remaining data, which contained multiple time points during each trial, were analysed using mixed-model repeated measures ANOVA (within subject factors: trial x time of sample; between subject factor: supplementation groups). Mauchly's test was consulted and Greenhouse-Geisser correction was applied if the assumption of sphericity was violated. If a significant P value was identified for the 3-way interaction effect (supplementation group x trial x time of sample), supplementation was deemed to have a significant effect. If a significant P value was identified for the main effect of time (time of sample), multiple pairwise comparisons were made using Bonferonni confidence interval adjustment, with statistical significance set at P < 0.01.

RESULTS

Exercise times to exhaustion at 85% VO_{2 max} were similar between groups during familiarisation and Tl (PS, P: 8:02 ± 1:37, 7:46 ± 0:41; 7:51 ± 1:36, 8:09 ± 0:54 mins). A significant interaction effect (supplementation group x trial, P=0.007) indicated that supplementation had a significant effect on time to exhaustion; post hoc analysis revealed no differences between trials for P (P=0.670) and significant differences between trials for

PS (P=0.001). The magnitude of change in exercise times to exhaustion (individual T2 values minus Tl values) in PS were 2:00 ± 0:28 min:s while P remained similar (0:07 ±

0:13 mins) (Figure 6).

Supplementation did not significantly effect last minute heart rates during the protocol (supplementation group x trial x time of sample, P=O.058). Mean last minute heart rates increased throughout the stages of the protocol (Table 2), being 119 ± 2, 137 ± 3, 156 ± 3 and 183 ± 2 beats/min, respectively at 45, 55, 65 and 85% VO_{2 max}.

Last minute oxygen uptake data are presented in table 2. These data increased progressively with exercise intensity (47 ± 1, 58 ± 1, 70 ± 1 and 97 ± 2%VO_{2 max}; time of sample effect, PO.001) and the supplement x trial x time of sample interaction was not significant (P=O.160). Steady state was confirmed in all stages at exercise intensities up to and including 65% VO_{2 max}. Mean carbohydrate oxidation, fat oxidation and total energy expenditure were: 1.40 ± 0.06, 1.95 ± 0.11, and 2.49 ± 0.14 g/min; 0.28 ± 0.02, 0.27 ± 0.02, and 0.27 ± 0.02 g/min; 34.0 ± 0.8, 42.3 ± 1.0, and 51.1 ± 1.1 kJ/min, respectively at each exercise stage (Figure 3). Supplementation had no effect on carbohydrate oxidation (supplementation group x trial x time of sample, P=O.596), fat oxidation (supplementation group x trial x time of sample, P=O.187) and calculated energy expenditure (supplementation group x trial x time of sample, P=0.595). The mean response times for the on-kinetic response (MRT_0n) were significantly higher at 85% VO_{2 max} than at 45-65% VO_{2 max} (time of sample effect, P=0.019); however, the 3-way interaction was not significant (P=0.069) (Figure 8). Supplementation had no effect on MRτoff (supplementation group x trial x time of sample, P=0.449) and exercise intensity had no significant effect (time of sample effect, P=O.055) (Figure 8). Estimated plasma volume fell by 7-11% during the first two exercise stages and remained below Pre-exercise values throughout exercise (Table 5); supplementation had no effect (supplementation group x trial x time of sample, P=0.392) and no statistical differences were identified between Tl and T2 (trial effect: P=0.491).

Blood lactate concentrations were significantly elevated from Pre-exercise values from Post-65% (Table 6) and peaked at Post-85% (mean value for all trials was 7.70 ± 0.33 mmol/L). Blood glucose concentrations did not differ significantly from Pre-exercise values at any time point (Table 6) and no differences were identified between groups or as a result of supplementation.

Table 4: Last minute heart rate and oxygen uptake during both exercise trials for phosphatidylserine supplementation group and placebo group.

Table 5: Estimated percentage changes in plasma volume throughout both trials for phosphatidylserine supplementation group and placebo group.

Table 6: Blood lactate and glucose concentrations during both exercise trials for phosphatidylserine supplementation group and placebo group.

Table 7: Exercise-induced feeling inventory (EFI) subscale scores throughout both trials for phosphatidylserine supplementation group and placebo group.

Serum Cortisol concentrations were significantly elevated by exercise (time of sample effect, P<0.001) from Pre-exercise values of 378 ± 21 nmol-L -i to Post-exercise values of 554 ± 32 nmol/L (Figure 9). Serum Cortisol concentrations were not significantly affected by supplementation (supplementation group x trial x time of sample, P=O.118). Significant temporal changes were observed in all of the EFI subscales; subjects reported decreases in revitalisation, positive engagement and tranquillity and increases in physical exhaustion through exercise (Table 7). The 3 -way interactions were not significant in any of the subscales (Table 7).

DISCUSSION The primary finding of this investigation was that oral supplementation with 750 mg/day PS for 10 days significantly affected exercise capacity in a group of recreationally active subjects during a staged intermittent cycling protocol. Furthermore, the enhancements in exercise capacity in PS ranged from 0:15 to 3:47 mins, while the exercise times to exhaustion remained unchanged in P. It was originally hypothesised that PS would influence the primary oxygen uptake kinetic response and thereby increase exercise capacity. However, supplementation did not significantly affect MRT_0n- Furthermore, no differences were observed between trial or supplementation group in MRT_Off; therefore, the current data present insufficient evidence to support any change in the primary oxygen uptake kinetic response following supplementation.

The causes of fatigue during cycling at 85% VO_{2 max} (within an intensity domain that has been previously classified as very heavy exercise [24]) have not been fully elucidated and may include central and peripheral components. Exercise during the final bout was associated with near maximal oxygen uptakes and heart rates in addition to relatively high blood lactate concentrations; therefore, it can be assumed that heavy demands were placed on both oxidative and non-oxidative phosphorylation. Metabolic acidosis has been implicated as a mechanism of peripheral fatigue either through direct effects on the contractile proteins or through inhibition of key regulatory enzymes such as phosphofructokinase [29]; however, it may be more likely that other ionic imbalances contribute to fatigue in this exercise model [30]. In-vitro studies have demonstrated that low concentrations of PS are effective in activating (Na⁺-K⁺)-dependent ATPase in mammalian kidney [31] and brain [32] preparations. Similarly, Ca²⁺ -ATPase, an enzyme primarily responsible for Ca²⁺ re-uptake from the muscle cyctosol into the sarcoplasmic reticulum, is known to require PS [18, 33]. Therefore, it is plausible that exogenous PS delayed the onset of fatigue by maintaining ionic homeostasis for longer during exercise.

In addition, it has been reported [15] that extended exercise training increased the levels of phospholipids, especially PS content, in rat cardiac sarcolemma. This adaptation to training might suggest that additional PS within the heart muscle has functional benefits during exercise. An increase in membrane bound PS may have the potential to enhance myocardial excitation-contraction coupling, potentially through the activation of different protein kinase C isoforms [34] and/or enhanced calcium uptake [35]. Thus, it is plausible that these mechanisms may have also contributed to delaying fatigue in the present study.

However, without corroborating data from further studies that investigate the in-vivo pharmacological actions of PS the proposed mechanisms remain speculative.

The significant rise in serum Cortisol concentration that followed the final bout of exercise suggested that the protocol activated the HPA axis [2]. However, supplementation with PS did not significantly influence serum Cortisol concentrations (Figure 5). This finding does not concur with other results [36] which found that PS, using a similar supplementation regime, attenuated serum Cortisol concentrations following resistance training. Furthermore, it has been reported [1] that 800 mg/day BC-PS resulted in significant reductions in plasma Cortisol and adrenocorticotrophic hormone (ACTH) concentrations during submaximal cycle exercise in untrained subjects.

The elevation in blood Cortisol is a generic response to stress from both psychological and physical origin; consequently, there is considerable inter-individual variability in response to exercise. Although the choice of experimental design in the current study investigated individual changes in response (pre- to post-supplementation) and, therefore, reduced the possible effect of subject selection related bias, the possibility exists that the current dose may have been insufficient to attenuate the Cortisol response in these active individuals. Alternatively, the current exercise protocol required that all participants continued the final exercise bout until exhaustion in both trials; therefore, it remains plausible that any effect of PS supplementation on Cortisol concentrations were masked as the PS group completed significantly more work in T2 when compared with Tl .

Blood glucose concentrations remained unchanged throughout all trials. This finding was in agreement with previous studies using similar exercise protocols [1, 2]. The concomitant effects of reduced insulin and elevations in ACTH, Cortisol and epinephrine are responsible for controlling blood glucose during exercise [4]. Therefore, any effects that PS supplementation may have had on blood ACTH and Cortisol did not appear to have over challenged blood glucose homeostasis during exercise. Furthermore, the calculated rates of carbohydrate, fat and combined fuel oxidation during the steady state stages of exercise were similar in all trials, suggesting that any change in the HPA axis induced by PS supplementation did not affect substrate oxidation during moderate exercise stages.

AU subscales of the EFI were sensitive to change during the exercise. However, the 3-way interaction did not reach significance in any subscale; therefore, there was no evidence to suggest that feeling states differed following supplementation in either supplementation group. The participants in the current study provided baseline responses that were similar to those of other recreationally active populations prior to exercise training [28], indicating that the testing procedures did not induce large changes in feeling states prior to exercise in these subjects. It has been reported [37] that improvements in mood occur after mental stress within a sub-group of young healthy adults following chronic PS supplementation. Nevertheless, these improvements were only identifiable in a sub-group of subjects who scored higher than the median for neuroticism; people who score highly on this dimension are known to display strong emotional reactions to stress [37]. Consequently, the baseline emotional state of an individual might influence the efficacy of PS in altering feeling states during exercise.

The present invention has been described above with reference to the examples. However, the invention is not limited to the examples. As will be apparent to the person skilled in the art, the present invention extends to all variations which fall within the spirit and scope of the following claims. REFERENCES

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Claims

1. A phosphoglyceride (as defined in the description) for use in improving heart rate recovery.

2. Phosphatidylserine for use in improving heart rate recovery.

3. A pharmaceutical composition comprising a phosphoglyceride (as defined in the description) in combination with a pharmaceutically acceptable carrier, for use in improving heart rate recovery.

4. A pharmaceutical composition comprising a phosphoglyceride (as defined in the description) and docosahexaenoic acid for combined, simultaneous or sequential administration.

5. The pharmaceutical composition of claim 3 or claim 4, wherein the phosphoglyceride is phosphatidyl serine.

6. Use of a phosphoglyceride (as defined in the description) in the manufacture of a medicament for improving heart rate recovery.

7. The use of claim 6, wherein the phosphoglyceride is phosphatidyl serine.

8. A method for improving heart rate recovery comprising treating a subject with a phosphoglyceride (as defined in the description).

9. The method of claim 8, wherein the phosphoglyceride is phosphatidyl serine.

10. The method of claim 8 or claim 9, which further comprises treating the subject simultaneously or sequentially with docosahexaenoic acid.

11. The method of claim 10, wherein the subject is treated with from 5 to 25 mg/kg/day of the docosahexaenoic acid.

12. The method of any one of claims 8 to 10, wherein the subject is treated with from 1.5 to 12 mg/kg/day of the phosphoglyceride.

13. A phosphoglyceride (as defined in the description) for use in improving exercise capacity.

14. Phosphatidylserine for use in improving exercise capacity.

15. A pharmaceutical composition comprising a phosphoglyceride (as defined in the description) in combination with a pharmaceutically acceptable carrier, for use in improving exercise capacity.

16. The pharmaceutical composition of claim 15, wherein the phosphoglyceride is phosphatidyl serine.

17. Use of a phosphoglyceride (as defined in the description) in the manufacture of a medicament for improving exercise capacity.

18. The use of claim 17, wherein the phosphoglyceride is phosphatidyl serine.

19. A method for improving exercise capacity comprising treating a subject with a phosphoglyceride (as defined in the description).

20. The method of claim 19, wherein the phosphoglyceride is phosphatidyl serine.

21. The method of claim 19 or claim 20, which further comprises treating the subject simultaneously or sequentially with docosahexaenoic acid.

22. The method of claim 21, wherein the subject is treated with from 5 to 25 mg/kg/day of the docosahexaenoic acid.

23. The method of any one of claims 19 to 21, wherein the subject is treated with from 1.5 to 12 mg/kg/day of the phosphoglyceride.

24. The method of any one of claims 19 to 21, wherein the subject is treated with from 500 to 1000 mg/day of the phosphoglyceride.