Alanine-fortified tomatoes relieve the acute alcohol-induced adverse effects in healthy men: a randomized cross-over study

This study was proposed and approved by the Ethics Committee of Huma R & D Co. Ltd. Informed consent was obtained from 78 healthy Japanese men older than 20 years who participated in baseline screening. Inclusion criteria for the clinical study were as follows: (i) aldehyde dehydrogenase 2 (ALDH2) genotype: ALDH2*1/*1; (ii) healthy males, free of chronic illness such as liver, kidney, heart, gastrointestinal, lung, endocrine, metabolic diseases, and mental disorders; (iii) the ability to drink 1.2 g/kg alcohol as a Japanese distilled spirit (Shochu) and conduct abstinence according to the protocol. ALDH2*1/*2 or ALDH2*2/*2 subjects were excluded as they have a genetic intolerance to drinking higher quantities of alcohol [16].

Both the commercially available tomato paste and dl-alanine were kindly provided by Kagome Co., Ltd (Tokyo, Japan) and Ajinomoto Healthy Supply, Inc. (Tokyo, Japan) respectively. Tomato paste (2,000 g), dl-alanine (250 g) and tap water (2,750 g) were mixed well and the mixture was prepared as freeze-dried products that yielded a total of 880 g. The dry AFT product was stored in a desiccator attached to a vacuum aspirator at room temperature for use in Experiments 1 and 2. A commercially available table sugar was used as a control meal and shochu (20 % alcohol, v/v) was utilized as the alcohol in all experiments.

A non-blind, single-center, randomized cross-over design was used for this study. The study flowchart is shown in Fig. 1. Subjects were scheduled to receive sugar (as a control) and AFT on two separate test days, 4 weeks apart in a randomized order. Prior to each Experiment (1 or 2), participants were instructed not to consume alcohol for two days prior. The timeline of the experiments (1 or 2) is shown in Fig. 2. On the day of the experiments, subjects were provided lunch at 12:00 pm in the Sekino Clinical Pharmacology Clinic (Tokyo, Japan). They had a free time until the start of the experiment at the institution. All of the subjects ingested a commercially available micronutrients-fortified food as meal (716 kcal, BALANCEUP, Asahi food & healthcare Co., Ltd., Japan), shochu (1.2 g alcohol/kg body weight), and the mixed dry product of AFT (40 g, 150 kcal). The AFT was equivalent to approximately 400 g of raw tomato with 10.4 g alanine or sugar (38 g, 152 kcal) used as the control. All subjects refrained from eating foods except for the supper provided and the test meals. However, they were allowed to freely consume water throughout the trial. The volume of any consumed water and the excreted urine were recorded during the experiment. The energy and nutrient contents of the supper and each test meal are shown in Table 1. The subjects ingested the supper and the alcoholic drink containing the test meal within 1 h. Peripheral blood specimens from the subjects were collected from the cubital vein at 18:00, 21:00, 00:00, and 6:00 (awakening time), as well as before and after the ingestion of each meal to determine the blood acetaldehyde, acetate, and plasma glucose levels. Serum alanine concentrations measured from each sample obtained at 18:00, 00:00, and 6:00. Breath alcohol concentrations (BrAC) were monitored using AlcoQuant® 6020 plus (EnviteC, Germany) as a breath alcohol analyzer at 18:00, 19:00, 20:00, 21:00, 22:00, 23:00, 00:00, and 6:00. Urine specimens were also collected from 12:00 to 18:00, 18:00 to 21:00, 21:00 to 00:00, and 00:00 to 6:00. The volume and pH was recorded for each of the four urine samples. The subjective sensations following alcohol consumption were evaluated based on the questionnaire data. The subjects were required to answer 14 questionnaires at the same time that the BrAC was determined. After a wash out period of 4 weeks, the cross-over trial was performed using the same protocol.

The blood acetaldehyde concentration was measured using a 2, 4-dinitrophenylhydrazine derivative method for acetaldehyde by ultraviolet detection (365 nm) using a high-performance liquid chromatography (HPLC) technique used in a previous report [17]. The blood acetate level was determined using HPLC (Shimadzu organic acid determination system; Shimadzu, Japan) fitted with an ion-exclusion column and a conductivity detector [18]. Following collection, the blood samples (0.5 mL) were mixed with 1.0 mL of 0.5 N perchloric acid and the supernatants were prepared by centrifugation. The serum alanine concentration was analyzed using an automated precolumn derivatization amino acid analytical method based on HPLC/electrospray ionization mass spectrometry (UF-Amino Station system; Shimadzu, Japan) as previously described [19]. The levels of plasma glucose and vasopressin; blood vitamin B1; serum vitamin B6, folic acid and vitamin C; and urinary pH were measured by a local laboratory for clinical examination (SRL Inc., Tokyo, Japan).

It is well established that alcohol produces both stimulant and sedative effects [20‐22]. Martin et al reported the biphasic effects of alcohol on a scale (a seven-item stimulant subscale and a seven-item sedative subscale), which held promise as a self-reporting measure of the impact of alcohol [20]. The stimulant items included the descriptions of elated, energized, excited, stimulated, talkative, up, and vigorous. The sedative items included difficulty concentrating, down, heavy head, inactive, sedated, slow thoughts, and sluggish. Each subject reported on each the 14 items as a subjective sensation on a 10 cm visual analog scale (VAS). The distance (cm) from the left edge of the line to the mark placed by the subject was measured at each time point. This measurement was then used in subsequent analyses as the sensation score from 0 (not at all) to 10 (extremely) that best described their present feelings. The stimulant or sedative score added the score of each of the seven items, and the scores were expressed as the median, quartile as well as the lowest and highest data in box plots. Each of the seven items of the sedative subscale performed at 6:00 was box plotted in a similar way.

All statistical analyses were performed using Dr. SPSS II software (SPSS Inc.). Each item between the AFT and control group was compared using a paired t-test. Temporal changes of each item were analyzed using a paired t-test or repeated ANOVA (if there were more than three points). These data were presented as mean ± SD. The acceptable level of significance was 5 and 1 % for each analysis. The VAS scores on subjective sensations between two conditions were evaluated using a Wilcoxon signed-rank test. A value of p < 0.05 was considered to be significant.