Eighteen healthy students (nine men and nine women aged between 18 and 30 years) at Maastricht University participated in the study. Exclusion criteria for participation were chronic and current illness; history of psychiatric or medical illness; medication use; metabolic, hormonal, or intestinal diseases; irregular diets or deviant eating habits; excessive use of alcohol (>2 U/day), cigarettes, coffee, and/or drugs; aversion for sugar-rich products; pregnancy; and any discomfort with blood drawing procedures as assessed by health and lifestyle questionnaires. Subjects participating in the experiment had a body mass index in the normal range (BMI in kg/m2 between 20 and 25), and female subjects were matched for contraception. Women participated during their mid–late follicular phase (day 4–10), while women using contraception participated when they actually used the contraception pill. All subjects that participated in the experiment were nonsmokers and nondrug users and were not allowed to drink alcohol starting 2 days before the experiment until the end of the experiment. The study was approved by the Medical Ethics Committee (MEC 06-3-029) of the Academic Hospital Maastricht (Maastricht, The Netherlands), and the procedures followed were in accordance with the Helsinki Declaration of 1975 as revised in 1983. All subjects gave their signed informed consent to participate in the experiment prior to their inclusion in the experiment and received a financial compensation for their participation.
Approximately 600 students at Maastricht University received written information about the study and an invitation to participate. In addition, students who were interested to participate were requested to complete and return a questionnaire package concerning general information (health, smoking and drinking habits, caffeine consumption, weight and height, and use of psychoactive drugs) and several questionnaires concerning psychopathology (mood disorders, family history of depression, medical complaints, etc.). From the subjects showing interest in taking part in the experiment, 18 healthy subjects were selected for the experiment. Subsequently, subjects taking part in the experiment were invited at the laboratory to receive information about the study and to become familiar with the environment and experimental procedures.
During five experimental morning sessions, subjects visited the laboratory to monitor plasma TRP/LNAA concentrations and mood following intake of a drink containing different TRP/LNAA ratios (see the “Diets” section). The order of presentation of dietary conditions was counterbalanced, and the five experimental days were separated by a 1-week period. Before and 15, 30, 60, 90 120, 180, and 210 min after ingestion, blood samples were taken to measure the dose-dependent effect (response curve) of the different tryptophan or protein sources on plasma amino acid concentrations and the TRP/LNAA ratio. In addition, mood was measured before, 60 min, and 210 min after intake (Fig. 1).
On each experimental morning, six subjects arrived at the laboratory at 8:30 a.m. Subjects had been instructed to fast overnight; only water or tea without sugar was permitted. In addition, subjects were not allowed to use any kind of drugs before and during the experiment (see selection criteria) or to drink alcohol the day before their participation and arrival at the laboratory. After arrival, subjects were allowed to rest for a while before an indwelling catheter was inserted in their nonpreferred forearm. Then all subjects were brought into a laboratory room containing sitting areas with separated computer systems. Each subject was separately seated in front of his or her personal computer screen and instructed about the experiment. Subsequently, subjects were exposed to a computerized version of the profile of mood state (POMS) (for detailed description, see below). After the first test session, subjects received a test drink containing different TRP/LNAA ratios. Before, 60 min, and 210 min after intake, respectively, a first, second, and third mood measure was conducted. Before and 15, 30, 60, 90 120, 180, and 210 min after intake, blood samples were taken to measure the dietary effects on plasma amino acid concentrations. Between measurements, all subjects stayed in the laboratory room to read or work on their own private computer.
On each experimental morning, a 312-ml drink was consumed containing different tryptophan (TRP) or LNAA concentrations. The reference condition included 20 g casein protein (PLC: DSM, Delft, The Netherlands) with 0.4 g TRP and 10 g LNAA, whereas the test conditions included 15 g intact alpha-lactalbumin (ALAC) whey protein (BioPURE, Davisco Foods International, USA) with 0.8 g TRP and 9.4 g LNAA, protein hydrolysate or HPROT (PeptoBalance™: DSM, Delft, The Netherlands) with 0.8 g TRP and 4 g LNAA, 0.8 g pure tryptophan (TRP: Orthica, Almere, The Netherlands) and 1.2 g synthetic peptide (SYN: Ser–TRP, DSM, Delft; Molecular formula C14H17N3O4) containing 0.8 g tryptophan (Tables 1 and 2).
All drinks are prepared by mixing the powder with 0.10 g sweetener (acesulfame) and were filled up by plain water in order to reach a 312-ml drink. Research assistants blind to the dietary conditions conducted the administration of the different drinks.
Profile of mood states
Changes in mood are measured using a computerized Dutch shortened version of the POMS questionnaire (Wald and Mellenbergh 1990) as a visual analogue scale ranging from “strongly disagree” to “strongly agree.” The POMS was calculated by the total mood scores on five different subscales, ranging from anger, depression, fatigue, and tension that refer to a negative mood state to vigor concerning a positive mood.
Blood samples were collected in duplicate in 5 ml vacutainer tubes containing sodium heparin and centrifuged at 5,000 rpm for 5 min at 4°C. Subsequently, the supernatants were directly stored at −80°C until analysis. Before storage, the supernatant (100 μl) was mixed with 4 mg sulfasalicyl acid. Plasma amino acid analysis was conducted with high-pressure liquid chromatography (HPLC), making use of a 2- to 3-μm Bischof Spherisorb ODS II column. The plasma tryptophan ratio was calculated by dividing the plasma tryptophan concentration (in μmol/L) by the sum of the other large neutral amino acids, i.e., valine, isoleucine, leucine, tyrosine, and phenylalanine.
Experimental design and statistical analysis
The main research questions formulated in the introduction were analyzed by means of repeated-measures multivariate and univariate analyses of variance (MANOVA and ANOVA) by using the general linear model (GLM; SPSS 12.0 for Windows) with one within-subjects factors “condition” (five different conditions: PLC, ALAC, HPROT, TRP, SYN) on the several dependent measures from mood and plasma amino acids. Although we already counterbalanced for order of condition, this variable was preliminarily taken as a between-subjects factor. Yet, because order of intervention did not contribute to any of the results, final analyses were performed with only condition as a within-subjects factor. For the effect of diet manipulation on blood samples, repeated-measures MANOVA was performed with first- and second-order polynomial contrasts. Only significant results revealed by these procedures were further examined by univariate tests. Huynh–Feldt or Greenhouse–Geisser corrected P values, their corresponding epsilons, as well as the original, i.e., uncorrected, degrees of freedom are reported when the sphericity assumption was not met. The study, including validation of the group size, was designed to detect a large effect size (μ
2 0.20) for a power of 0.80 at alpha = 0.05 (requiring a group size of at least 17 subjects). All statistics were evaluated at a significance level of 5%. Data are reported as the means±SD.