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European Journal of Clinical (1998) 52, 425±431 ß 1998 Stockton Press. All rights reserved 0954±3007/98 $12.00 http://www.stockton-press.co.uk/ejcn

A controlled investigation of the effects of or placebo on subjective and objective measures of fatigue

A Cunliffe, OA Obeid and J Powell-Tuck

Department of , St Bartholomew's and Royal London School of Medicine and Dentistry, Queen Mary and West®eld College, London E1 2AD

Objective: To examine the effect of L-tryptophan administration on subjective and objective measures of fatigue in healthy volunteers. Subjects: Six healthy volunteers (4M:2F) were recruited from staff and students at the College. Setting: Department of Human Nutrition, St. Bartholomews and the Royal London School of Medicine and Dentistry. Design: Subjects were tested for central and peripheral fatigue using a visual analogue scale, ¯icker fusion frequency, grip strength, reaction time and wrist ergometry. In addition, plasma free tryptophan concentrations and Trp:LNAA ratio were determined. Measurements were made before, and at 1, 2, 3 and 4 h after drinking one of two test drinks. The drinks were of either caffeine free Coca-Cola (placebo) or caffeine free diet Coca- Cola plus L-tryptophan (30 mg/kg: active drink). Each of the six subjects was tested after placebo and active drink with a one week washout period between test days. Results: Subjective fatigue was signi®cantly increased following tryptophan compared to placebo (P < 0.002), and objective measures of central fatigue were signi®cantly increased by tryptophan compared to placebo (¯icker fusion frequency: P < 0.001; reaction time P < 0.001). No signi®cant changes in grip strength were found following tryptophan compared to placebo, but a signi®cant increase in ergometric work output was seen (P < 0.001). Conclusion: Tryptophan ingestion in the quantity 30 mg/kg leads to increased subjective and central fatigue. Increases in work output observed following tryptophan may be as a result of a reduced perception of discomfort during ergometry. Sponsorship: Pharmacia Ltd. Descriptors: tryptophan; fatigue; amino acids

Introduction in our previous investigations, be differentially affected. To further investigate and clarify the functional outcomes of We have previously investigated the effect of dietary changes in plasma free amino acids, the present investiga- macronutrient manipulation on measures of fatigue and tion aimed to assess the effect on fatigue and plasma amino plasma amino acids (Cunliffe et al, 1997a). We have acids of the ingestion of L-tryptophan itself. shown that we can demonstrate increases in fatigue as the working day progresses (Cunliffe et al, 1997b) and in relation to meals of varying composition (Cunliffe et al, 1997a). Central and peripheral fatigue measurements were Methods not always coincident with each other. A diet induced rise Six healthy volunteers (students and staff aged 24±35 y; in the ratio of free plasma tryptophan to competitor large M:F 4:2) were recruited from within the college with a mean neutral amino acids (Trp:LNAA) has been implicated in the age of 30.5 ( Æ 3.9) y and mean BMI of 23.5 ( Æ 1.6) kg/m2. genesis of central fatigue (Fernstrom, 1994). Rises in this The following methods were used for measuring fatigue; (1) ratio allow more tryptophan to enter the brain and a Visual analogue scale (13 item)Ðmeasuring subjective consequent increase in 5-HT synthesis to occur. impressions of fatigue; (2) Flicker Fusion Frequency This study was designed to investigate the effect of a (FFF)Ðobjective measure of central fatigueÐthe ¯icker placebo, and a simple metabolic intervention expected to rate was started at 200 Hz and reduced gradually until the increase fatigue. Tryptophan administration (2 g) has pre- subject reported seeing ¯icker. Increasing fatigue is accom- viously been shown to induce drowsiness in healthy volun- panied by reductions in FFF score, namely, perception of teers (Thorleifsdottir et al, 1989) and when given in the ¯icker is detected at a lower number of cycles per second quantity 50 mg/kg increase reaction time relative to tyro- when fatigued; (3) Simple Visual Reaction TimeÐ sine (Lieberman et al, 1985). Much of the work on objective measure of central fatigueÐslowing of reaction tryptophan has employed self ratings of mood and vigour time indicating increased central fatigue. Reaction time to a and it was therefore decided to test tryptophan against a colour change on a computer screen was measured in placebo with our newly developed range of tests for fatigue seconds/1000; (4) Grip StrengthÐobjective measure of to assess if the aspects of fatigue being measured would, as peripheral fatigue (brief maximal voluntary contraction) Correspondence: Dr A Cunliffe. using a hand dynamometerÐgrip strength was measured Received 30 January 1998; reviewed 3 March 1998; accepted 9 March in kg force; (5) Wrist ergometryÐobjective measure 1998 of peripheral fatigue (endurance)Ðsubjects raised and A placebo controlled investigation A Cunliffe et al 426 lowered a ®xed weight (1.5 kg females, 2.0 kg males) in mance. Between tests subjects worked as normal. For the time with a metronome (69 beats per minute) until they subjects in this study, this represented light administrative could no longer continue. Total work done was calculated in work or study. Joules. Ethical approval for all the test procedures on healthy A more detailed description of these techniques has been volunteers was granted by the East London and City Health given in our previous investigation (Cunliffe et al, 1997a). Authority. A 5 ml blood sample was also taken from each subject before receiving the drink and then at each test interval. Data analysis Unmarked bottles and drinking vessels were used and the Data for fatigue tests are expressed for each hourly test as subjects were unaware of which drink was being given. the percentage changes from initial pre-test drink readings 200 ml of the diet cola used contains less than 1 kcal onwards through the test period and presented graphically. energy. Blood samples were analysed for plasma free Error bars show standard error of the mean. The general amino acids using HPLC (Cunliffe et al, 1997a). linear model of the analysis of variance (2 way analysis of Subjects were fasted overnight before testing and variance) was carried out to determine the effect of supple- instructed to take only water on the morning of the test. mentation on the different tests (time and diet were used as Any subject who had not complied with the fast or who had factors in the model). A one way analysis of variance was had less than 5 h sleep was excluded. Testing began performed to assess the effect of time on each measure between 9.30 am and 10.00 am in all cases. Each subject individually. The actual baseline readings and units are also was tested for fatigue, before and then at hourly intervals given for reference. Data for the plasma amino acids is for 4 h after being given either the placebo drink (200 ml tabulated giving plasma free tryptophan levels, tryptophan caffeine free diet Coca-Cola) or the active drink (200 ml to competitor large neutral ratios (Trp:LNAA) caffeine free diet Coca-Cola plus L-tryptophan (SHS, and summed levels of the competitor large neutral amino Liverpool, England) in the quantity 30 mg/kg body acids ( S , , , , isoleu- weight). After a one week (minimum) washout period, cine). each subject was re-tested before and at hourly intervals for 4 h after having received either the active drink (if previously having had placebo) or placebo (if previously Results having had active drink). A 70 kg subject given 30 mg/kg Figure 1 shows that following tryptophan, mean subjective tryptophan received 2.1 g tryptophan, representing fatigue scores increased over the ®rst 3 h of the test by 8.4 kcals. We considered it very unlikely that this amount about 100% from pre-test drink values and in the ®nal hour of energy intake would measurably affect subject perfor- increased markedly by more than another 100%. Following

Figure 1 Mean percentage change from baseline (pre-drink) for subjective fatigue measured by VAS: Vertical bars show standard error. The baseline before the Tryptophan test drink was 2.13 Æ 0.77 (cm) and before the Placebo test drink was 1.45 Æ 0.34 (cm). * 2-way ANOVA Tryptophan vs Placebo P < 0.002. A placebo controlled investigation A Cunliffe et al 427

Figure 2 Mean percentage change from baseline (pre-drink) for Flicker Fusion Frequency: Vertical bars show standard error. The baseline before the Tryptophan test drink was 62.73 Æ 3.62 (Hz) and before the Placebo test drink was 59.95 Æ 3.50 (Hz). * 2-way ANOVA Tryptophan vs Placebo, P < 0.001. placebo on the other hand no appreciable increases in an overall mean decrease of nearly 10% was seen following subjective fatigue score were observed until the ®nal hour placebo. Post tryptophan, an increase in ergometric work of the test when the mean increase was 44% from baseline. output of just over 6% above baseline was seen at 1 h A signi®cant difference was found (P < 0.002) with respect though by the second test this was reduced to a 4% increase to test drink and a signi®cant effect of time was found and then maintained at about this level till the ®nal test. overall (P < 0.02). Following placebo, work output dropped to 7 17.5% by The results from ¯icker fusion frequency (Figure 2) 2 h and was improved only to 7 12% below baseline by showing mean decreases in ¯icker fusion frequency the ®nal test. No signi®cant effect for passage of time alone scores in subjects taking tryptophan indicate that central was found for the grip strength or wrist ergometry tests. fatigue also increased signi®cantly as measured by this Data from analysis of blood samples shows that at objective test (P < 0.001), compared to those taking pla- baseline, no signi®cant differences were found in plasma cebo. Decreases in FFF score (Figure 2) following the free tryptophan, Trp:LNAA, or LNAAs. Following trypto- tryptophan test drink and increases in FFF score following phan ingestion there was a signi®cant increase in plasma the placebo test drink were evident at the ®rst test interval free tryptophan compared to placebo (Table 1: P < 0.002), (post tryptophan reduced by 4.5% and post placebo the placebo drink leading to a decrease in plasma levels of increased by 4.2%) and maintained at 4 h. A similar this amino acid. During the test, the Trp:LNAA ratio was divergence was noted in reaction times (Figure 3), another signi®cantly raised in the plasma samples from those measure of central arousal, which were signi®cantly subjects given the drink containing tryptophan (Table 1: increased (slowed) following tryptophan (P < 0.001) P < 0.002) compared to placebo. With respect to LNAAs when compared to placebo, which produced mean no signi®cant difference was found between tryptophan and decreases in reaction time (faster reactions) at each test placebo or as a result of time alone, indicating that rises in interval. The greatest differential between effects post the Trp:LNAA observed post tryptophan were as a result of tryptophan and post placebo was found at the ®rst hourly increasing plasma levels of free tryptophan test for ¯icker fusion frequency and at the third hour for The increase in plasma free tryptophan peaked at 2 h reaction time. No signi®cant effect of time alone was found (219 mM), declining to 50 mM at 4 h. The greatest percentage for either objective test of central fatigue. increase in the Trp:LNAA was seen at 2 h from baseline No signi®cant differences were found between test and peaked at over 129% increase from baseline by 2 h drinks in the grip strength test of brief maximal voluntary from baseline. By 4 h however following tryptophan inges- contraction (Figure 4). Work output during wrist ergometry tion the Trp:LNAA was actually reduced to 19% below however (Figure 5) was signi®cantly greater following baseline, almost exactly the same percentage change as the tryptophan (P < 0.001) compared to placebo and main- mean value post placebo at 4 h. The placebo itself led to tained above baseline throughout the test period. While decreases in the Trp:LNAA at each time interval, the this only represented an overall mean increase of about 4%, lowest point being 7 50% of baseline at 3 h, with a A placebo controlled investigation A Cunliffe et al 428

Figure 3 Mean percentage change from baseline (pre-drink) for Reaction Times: Vertical bars show standard error. The baseline before the Tryptophan test drink was 209 Æ 4.5 (ms) and before the Placebo test drink was 228 Æ 6.5 (ms). * 2-way ANOVA Tryptophan vs Placebo P < 0.001.

Figure 4 Mean percentage change from baseline (pre-drink) for Grip Strength: Vertical bars show standard error. The baseline before the Tryptophan test drink was 46.4 Æ 5.2 (kg) and before the Placebo test drink was 44.0 Æ 4.7 (kg). A placebo controlled investigation A Cunliffe et al 429

Figure 5 Mean percentage change from baseline (pre-drink) for Ergometry: Vertical bars show standard error. The baseline before the Tryptophan test drink was 487.6 Æ 37.0 (J) and before the Placebo test drink was 558.6 Æ 56.2 (J). * 2-way ANOVA Tryptophan vs Placebo P < 0.001.

Table 1 Table gives mean plasma free tryptophan (mMol), SLNAA (tyrosine, phenylalanine, valine, leucine, ) (mMol), and the Trp:LNAA for tryptophan vs placebo at times 0±4 h

Group

Tryptophana Placebo Tryptophan Placebo Tryptophanb Placebo Trp (mM) Trp (mM) LNAA (mM) LNAA (mM) Trp:LNAA Trp:LNAA

Time (h) Mean s.e.m. Mean s.e.m. Mean s.e.m. Mean s.e.m. Mean s.e.m. Mean s.e.m.

0 67.6 27.7 95.7 33.3 572 109 649 211 0.128 0.05 0.081 0.03 1 139 42.3 18.8 8.4 652 87.8 488 50.0 0.237 0.07 0.043 0.02 2 219 48.7 13.4 5.4 656 110 456 68.6 0.312 0.06 0.044 0.02 3 168 67.9 13.5 6.3 617 98.0 479 40.0 0.234 0.06 0.033 0.02 4 50.4 14.0 20.5 7.2 628 201 424 36.0 0.109 0.05 0.062 0.02 a 2-way ANOVA Tryptophan vs Placebo P < 0.002. b 2-way ANOVA Tryptophan vs Placebo P < 0.002. mean overall decrease of 7 28% from baseline compared performed signi®cantly better during the ergometry test for to a mean overall increase of 50% post tryptophan. endurance following tryptophan than following placebo. This may be a result of increased subject compliance or tolerance to discomfort due to the tryptophan administra- tion as has been implicated in both the determi- Discussion nation of pain threshold and the behavioural response to The results from this study con®rm the ®ndings of other painful stimuli (Seltzer et al, 1981). researchers (Thorleifsdottir et al, 1989; Yuwiler et al, With respect to voluntary muscle function it is interest- 1981) that tryptophan administration can lead to increases ing to note that at 2 h post test drink, mean increases in in subjective sensations of fatigue. The results from the work done during ergometry were near peak level follow- ¯icker fusion frequency and reaction time tests, objective ing tryptophan and at their nadir following placebo (point measures of central fatigue, also support the notion that of the largest differential between test drinks; > 21%). The tryptophan can have centrally fatiguing effects that can be greatest divergence in grip strength performance following objectively assessed. While no signi®cant difference in the test drinks was also seen at 2 h though this time in the effects of the test drinks was noted in grip strength, subjects opposite direction (post tryptophan grip strength A placebo controlled investigation A Cunliffe et al 430 decreased). This divergent effect on voluntary muscle Wilson & Maughan, 1992 administered Paroxetin, a ser- function occurred at the same time as the largest increase otonin re-uptake inhibitor. Time of day of exercise is also in the Trp:LNAA. It seems therefore that if an increased infrequently reported in such studies. availability of tryptophan to the brain is affecting perfor- It seems that tryptophan administration is centrally and mance, it has different effects on function, revealed accord- objectively fatiguing but that this is associated with an ing to the test applied. increased tolerance to discomfort. Given that no increases A mean increase in plasma free tryptophan occurred in grip strength performance were noted following trypto- following tryptophan ingestion. This would be expected as phan it seems that changes to the discomfort threshold would the increase in the Trp:LNAA that was observed during wrist ergometry were probably responsible for the following the ingestion of pure tryptophan, leading to the signi®cant increases in work output observed. This is introduction of the amino acid into the bloodstream follow- consistent with the idea that endogenous opioid-induced ing absorption. While an increase in free tryptophan was analgesia depends on an increase in the uptake of trypto- seen following tryptophan ingestion, a mean decrease phan to the brain (Kelly & Franklin, 1985), a greater occurred following placebo, consistent with the metabolic amount of free tryptophan being available would then utilisation of the amino acid already present in plasma enhance this effect. following a and energy free meal. The Trp:LNAA peaked at 2 hours (130% increase) following the tryptophan test drink and was actually reduced by 4 h. This increase is Conclusions pronounced when compared to the 19% rise in the Increases in Trp:LNAA were associated with increases in Trp:LNAA brought about by the ingestion of 1672 kJ subjective and objective measures of central fatigue similar pure in our previous study (Cunliffe et al, though more pronounced than those seen following pure 1997a). Given that under normal conditions tryptophan carbohydrate ingestion (Cunliffe et al, 1997a). This implies hydroxylase (rate limiting enzyme in the tryptophan to that central fatigue may be in¯uenced by changes in the 5-HT synthetic pathway), is unsaturated, (Growdon & plasma Trp:LNAA and 5-HT synthesis though this mechan- Wurtman, 1979), the large increase in the Trp:LNAA ism cannot explain increases in central fatigue observed seen in this study would allow for increased central 5-HT following fat ingestion (Wells et al, 1995; Cunliffe et al, synthesis and as a consequence in increased brain trypto- 1997a) which does not increase this ratio. phan uptake. The increases in subjective and central fatigue measured were maintained at 4 h post ingestion, suggesting that while 5-HT synthesis may be increased as a result of References the raised Trp:LNAA that the actual release of the neuro- transmitter may persist for some time after substrate for its Cunliffe A, Obeid O & Powell-Tuck J (1997a): Post-prandial changes in synthesis is depleted. 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