Abstract
l-threonine is used in dietary supplements and nutritional products ingested by healthy consumers. The objective of this study was to determine in a randomized double blind controlled clinical trial the safety and tolerability of l-threonine used as graded doses in supplements for 4 weeks. Healthy male adults (age 42.9) ingested randomly placebo or different doses of L-threonine (0, 3, 6, 9, 12 g/day) for 4 weeks using a crossover design. At the end of supplementation period, the subjects visited the clinic for medical examination, anthropometric parameter measurements, blood sampling for biochemical tests including amino acid concentrations in plasma, measurement of blood pressure and heart rate, and dietary intake evaluation. Adverse events were recorded all along the trial. None of the anthropometric parameters measured, dietary intake and the biochemical parameters were affected by l-threonine supplementation except a non-specific minor increase in plasma aspartate amino transferase and creatine kinase which was measured in the group supplemented with 9 g l-threonine per day but not with the 12 g per day dose. Also, the concentration of L-threonine as well as the concentration of its metabolite L-2-amino butylate were found to be increased in plasma after supplementation with 6, 9, 12 g/day L-threonine. The moderate and mild adverse events were found to occur at random. All symptoms disappeared during the supplementation period despite continuous L-threonine supplementation. These results of this study indicate a no-observed-adverse-effect-level (NOAEL) value for L-threonine to be 12 g/day in healthy adult males. This study was registered at jRCT as jRCT1050230137.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00726-025-03461-6.
Keywords: Amino acid, Human study, NOAEL, Randomized controlled clinical trial, Threonine
Introduction
l-threonine was independently discovered in oat protein by Schryver SB and Buston HW and in teozein by R.A. Gortner RA and Hoffmann WF in 1925 (Wu G 2022, discoveries of natural amino acids, p2). l-threonine is an indispensable amino acid that must be obtained from the diet (Reeds 2000). l-threonine is used as a building block for the synthesis of proteins, is a precursor of the dispensable amino acid glycine, and is used as energy substrate for mitochondrial adenosine triphosphate (ATP) production (Tang et al 2021; Bird et al 1984). l-threonine is metabolized in pathways involving threonine-3-dehydrogenase (TDH), threonine aldolase (TA) and threonine dehydratase (TH). Accordingly, l-Threonine is converted into metabolites such as acetyl-CoA, succinyl-CoA and pyruvate, which play important roles in ATP production (Fig. 1) (Wu 2022, catabolism of threonine, pp 231-234; Dale 1978; Irino et al 2016), with acetyl-CoA and succinyl-CoA entering the tricarboxylic acid cycle, allowing the synthesis of reduced equivalents, and the synthesis of ATP in the mitochondrial respiratory chain. It has been shown that approximately half of dietary l-threonine is extracted by the gut on the first pass, and used for synthesis of intestinal mucosal proteins, including mucins (Schaart et al 2005; Hill et al 2022). These glycoproteins, which are rich in l-threonine are well known for their protective roles towards the intestinal epithelium (Faure et al 2002; Fogg et al 1996; Gustafsson and Johansson 2022). Adequate l-threonine supply has been shown to be critical to produce mucus in the intestine (Law et al 2007), and moderate L-threonine deficiency is associated with alterations of intestinal paracellular permeability (Hamard et al 2010). In relationship with the metabolic and physiological roles of l-threonine in the tissues and organs, notably regarding intestine, dietary supplementation with l-threonine has shown beneficial effects in the context of gastrointestinal mucosa alterations in experimental works (Stoll B 2006; An et al 2019; Faure et al 2003; Liu et al 2013). In addition, l-Threonine contributes to the regulation of melanogenesis by mucin protein MUCL1 (Kim and Choi 2022), and as a precursor for glycine synthesis, contributes to the synthesis of collagens (Li and Wu 2018; Silva et al 2020; Christner et al 1975). Among the amino acids present in body proteins, l-threonine is deeply involved in the phosphorylation and dephosphorylation of active sites within these proteins, such as protein kinase (Depaoli-Roach et al 1994). In addition, l-threonine as a nutrient is used in medical treatments aiming at maintaining or improving nutritional status, in situations such as parenteral nutrition and enteral nutrition therapy, in conjunction with other amino acids (Goudoever et al 2018; Berlana 2022; Hoover et al 1980).
Fig. 1.
Schematic view of the catabolism of L-threonine. l -Threonine is metabolized in pathways involving threonine-3-dehydrogenase (TDH), threonine aldolase (TA) and threonine dehydratase (TH). Accordingly, l -Threonine is converted into metabolites such as acetyl-CoA, succinyl-CoA and pyruvate, which play important roles in ATP production. Note: The compounds in the straight and dotted-line box indicates the substance measured in this study. *1 2-oxoaldehyde dehydrogenase, *2 2-oxobutyrate dehydrogenase, ACS acetyl-CoA synthetase, ALDH aldehyde dehydrogenase, CoA coenzyme A, ATP adenosine triphosphate, α-KG α-ketoglutaric acid, PDH pyruvate dehydrogenase, PEP phosphoenolpyruvic acid, PC Pyruvate carboxylase, PEPCK phosphoenolpyruvate carboxykinase, PK pyruvate kinase, SHMT serine hydroxymethyltransferase or glycine hydroxy-methyltransferase, SDH serine dehydratase, TCA tricarboxylic acid.
Requirement for l-threonine was reported in a joint consultation report by the World Health Organization (WHO), the Food and Agriculture Organization (FAO), and the United Nations University (UNU). The WHO/FAO/UNU consultation estimated that the mean requirement for L-threonine in healthy adults is 15 mg/kg body weight /day, thus representing 1.05 g l-threonine per day for an adult weighing 70 kg (FAO, WHO, UNU 2007). l-threonine is naturally present in several food sources, including marine products (salmon, tuba, mackerel), meat products (beef, pork, chicken), dairy products (cheese, yogurt), bean (pea, peanut, almond), grain products (soybean, tofu, miso), corn, egg, and milk. The usual consumption of L-threonine from dietary supply and supplements in the American general population 3.64–4.47 g/day (Sarkar et al 2021), a value thus largely above the mean requirement (FNB 2005). Usual consumption of L-threonine in Japanese adults is approximately the same, averaging 4.5 g/day (Ishikawa-Takada and Takimoto 2018). This average value should not mask heterogeneity in the usual consumption of l-threonine among the different subpopulations, notably in situations of protein-energy malnutrition (Crichton et al 2019). Then, in case of insufficient l-threonine intake in comparison with requirement, l-threonine supplementation may be indicated in different situations. Since the body is unable to store amino acids in large quantities, either in free form or in proteins, there is no large reserve forms of amino acid in the body in contrast with fatty acids and glucose. To the best of our knowledge, no information is available regarding the upper limit of safe intake for l-threonine in supplements in healthy adults. In particular, data on the safe intake levels of l-threonine –especially at relatively high doses –remain limited. Experimental studies in rats have determined a no-observed-adverse-effect level (NOAEL) value equal to 3627 and 3673 mg/kg body weight/day in male and female rats respectively (Aoki et al 2014). By comparing these NOAEL values with the usual consumption in rats, it appears that L-threonine is well tolerated by these animals (Blachier et al 2021). However, since extrapolation from animal studies to humans is hazardous, clinical trials are mandatory to determine the NOAEL value for l-threonine in humans. In this context, the primary objective of this study was to determine the clinical no-observed adverse effect level (NOAEL) for l-threonine given during 4 weeks in a randomized double blind controlled trial to healthy volunteers at graded doses (3, 6, 9, and 12g/day) using a clinical study design as already used in previous studies (Deutz et al 2017; McNeal et al 2018; Gheller et al 2020; Miura et al 2021; Miura et al 2023). The lowest dose of l-threonine used (i.e. 3g/day) corresponds or is below the usual consumption of L-threonine in food and supplements for the American general population (FNB 2005) and for Japanese adults (Ishikawa-Takada and Takimoto 2018), respectively, and then can be considered as safe. The limited information regarding L-threonine tolerance in humans led us to measure a broad range of criteria. In fact, there is limited information on the metabolic and physiological consequences of a temporary increase of circulating L-threonine after supplementation with this amino acid. The parameters measured in the present study included numerous blood biochemical parameters, anthropometric characteristics, blood pressure, heart rate, as well as dietary data in terms of energy and macronutrient intake. In addition, amino acid concentrations in plasma were measured at the end of the supplementation period. Adverse events were duly compiled during the whole period of supplementation and completed by medical examination.
This study thus aims to evaluate the safety of l-threonine supplementation (as opposed to dietary intake) across a range of doses (0 [placebo], 3, 6, 9, and 12 g/day) over a four-week period, using a comprehensive set of clinical laboratory parameters commonly used health checkups, as well as assessments of amino acid metabolism. Such study is expected to contribute to the safe use of amino acids in supplements by individuals for which it is indicated, in a context of globally increased consumption of dietary supplements, including those containing amino acids.
Methods
Participants
The study population consisted of 30 healthy male participants and the clinical trial was performed at the Miura Medical Clinic (Osaka, Japan) between November 2023 and July 2024. Participant recruitment, blood collection, and anthropometric and body composition measurements were conducted in the Miura Medical Clinic and managed by a project coordinator with proper training (Oneness Support Ltd., Osaka, Japan). The participants provided written, informed consent before participation. They were duly informed of the purpose of the study and of all the experimental aspects of the trial. Participants were publicly recruited in the Osaka city area (Japan). Inclusion criteria included: healthy males aged 20 to less than 60 years at the time of informed consent. Exclusion criteria were as follows: (1) continuous consumption or possible consumption of medicinal products during the trial, (2) consumption of supplements containing amino acid components from the time of participation in the study until end of trial, (3) involvement or intended involvement in other human clinical studies or involvement in other study less than 4 weeks before the present study, (4) cardiac, liver, and kidney disease, respiratory disease, history of cardiovascular disease, diabetes mellitus, intestinal disease/enteropathies, inborn errors of metabolism. Volunteers with an history of serious diseases, such as cancer or tuberculosis, were also excluded from the study, (5) as well as volunteers with a history of allergies to food or drugs, and finally (6) all other causes as determined by the appointed medical staff to be unsuitable for inclusion in the study. During the inclusion phase, all participants completed a health history and physical activity questionnaire that included current and recent medications and supplement use. The procedures in the clinical trials were in accordance with ethical standards and were approved by the Ajinomoto Co., Inc ethics review committee (2022-021) and the Miura Medical Clinic ethics review committee (R2212). This study was registered at jRCT as jRCT1050230137 (https://jrct.niph.go.jp/en-latest-detail/jRCT1050230137).
Study design
Participants in each group were randomly given either no supplement (placebo group) or 3, 6, 9 and 12 g/day of L-threonine during 4 weeks under double-blind conditions. These groups were assigned to ensure no differences in BMI, blood pressure and heart rate. l-Threonine (Ajinomoto Co., Inc., Tokyo, Japan) and corn starch (Japan corn starch Co. Ltd, Tokyo, Japan) as a placebo were encapsulated in cellulose. All capsules were then coded and allocated by an investigator who was not involved in either data collection or data analysis. The participant received 33 capsules containing l-threonine or placebo to be consumed daily over 4-weeks, with at least a 2-week washout period between each test with l-threonine dose. The participants were recommended to swallow these capsules, divided into one to three times per day. The lowest supplement dose (i.e. 3 g/day) was in the order of magnitude or below the usual consumption of l-threonine by Japanese men (Ishikawa-Takada and Takimoto 2018), thus considered as safe. Participants in each treatment arm received amino acid supplements for the whole 4-week test period in accordance with previous study design (Miura et al 2021; Miura et al 2023). The participants compliance regarding capsule consumption was calculated as percentages of the number of duly taken capsules (as noted by participants) compared to the number of capsules provided in the protocol. At the end of the 4-week intervention period, each volunteer had a clinical examination. Compensation including travel costs was provided to participants at the end of the trial.
Anthropometric measurements and dietary survey
The participants visited the clinic following overnight fasting for least 8 h for safety evaluation after the 4-week supplementation. The participants were evaluated for anthropometric parameters, undergo blood sampling, responded to questionnaire regarding their diet, and were interviewed by a clinician. The body height (BH) and body weight were measured to the nearest 0.1 cm using a fully automatic height and weight scale NGP-150L (Nitto Kagaku Co. Ltd., Nagoya, Japan). The body mass index (BMI) was calculated as the ratio between BW (Kg) and the square of the BH (m2). The blood pressure (systolic arterial pressure and diastolic blood pressure) and heart rate were measured on resting in a sitting position using digital automatic blood pressure monitor HEM-1000 (Omron Healthcare Co., Ltd., Kyoto, Japan). A dietary survey using the Sasaki Food Habit Assessment (Sasaki et al 2003; Kobayashi et al 2011) was conducted at the end of the supplementation period to assess food intake status in terms of total food energy and main macronutrient categories (proteins, lipids, and carbohydrates).
Blood sample collection and analysis
Blood chemistry, blood biochemistry and plasma amino acid analyses were conducted by LSI Medience Corporation (Tokyo, Japan) using routine clinical methodologies. For blood chemistry, venous blood sample (2 mL) was collected in a 5 mL dipotassium EDTA-2K tube (NP-EK0205; Nipro corporation, Osaka, Japan). The collected blood samples were stored at 4 ℃ after gentle inversion mixing. The treated samples were used to measure white blood cells (WBC), red blood cells (RBC) using, automated flowcytometry analyzer (XE-2100, Sysmex corporation, Hyogo, Japan), hemoglobin (HGB), hematocrit (HC), and platelet (PLT) count using the red blood cell pulse peak detection method and the electrical resistance detection method, respectively (XE-2100, Sysmex corporation, Hyogo, Japan). For blood biochemistry, venous blood samples (8.5 mL) were collected in a 10-mL neo-tube A (NP-EK0205; Nipro corporation, Osaka, Japan). The collected blood samples remained at room temperature for 30 min after 10 times gentle inversion mixing. The serum was obtained by centrifugation (3000 rpm, 20 ℃, 10 min; H-19F, Kokusan Co. Ltd., Saitama, Japan) and stored at 4 ℃. The treated samples were used for the measurement of total protein (TP), albumin (ALB), total bilirubin (T-BIL), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), aspartate aminotransferase/glutamic oxaloacetic transaminase (AST/GOT), alanine aminotransferase/glutamic pyruvic transaminase (ALT/GPT), gamma glutamyl transferase (γ-GTP), creatine kinase (CK), phospholipids (PL), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-Cho), low-density lipoprotein cholesterol (LDL-Cho), blood urea nitrogen (BUN), creatinine (CRE), uric acid (UA), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), and glucose (GLU). All blood biochemistry parameters were evaluated based on methodologies recommended by the Japanese Society of Clinical Chemistry using an automated analyzer Labospect LST008α (Hitachi High-Tech corporation, Tokyo, Japan) combined with an automatic analyzer JCA-BM8000 (JEOL Ltd., Tokyo, Japan). Albumin was analyzed by dye binding-bromocresol purple method using Labospect LST008α, and serum levels of electrolytes (Na, K, Cl and Ca) were analyzed by the electrode method using the same instrument. Blood glucose was measured enzymatically using the automatic analyzer JCA-BM9130 (JEOL Ltd., Tokyo, Japan). Estimated glomerular filtration rate (eGFR) was calculated according to the 2024 CKD practice guidelines of the Japanese Society of Nephrology. For blood amino acid analysis, venous blood samples (4 mL) were collected in a 5-mL heparin Na blood collection tubes (NP-HE0405; Nipro corporation, Osaka, Japan). Collected blood samples were mixed by 10 times gentle inversion mixing and remained for 15 min in cube cooler (CUBE-T20, Forte Grow Medical Co., Ltd., Tochigi, Japan). Plasma was obtained by centrifugation (3000 rpm, 4 ℃, 10 min; H-19FMR, Kokusan Co. Ltd., Saitama, Japan), transferred to special tube (trans-tough tube, Daisensangyo Co. Ltd., Osaka, Japan) and stored at − 80 ℃. The treated plasma samples were used to measure concentrations of amino acids (l-histidine (His), l-isoleucine (Ile), l-Lysine (Lys), l-methionine (Met), l-phenylalanine (Phe), l-threonine (Thr), l-tryptophan (Trp), l-valine (Val), L-arginine (Arg), l-aspartic acid (Asp), l-Asparagine (Asn), l-cystine (Cys), l-glutamic acid (Glu), l-glutamine (Gln), glycine (Gly), l-proline (Pro), l-serine (Ser), l-tyrosine (Tyr), and l-2-amino-butyric acid (ABA)) by an LC-MS/MS analyzer (Xevo TQ-S micro, Nihon Waters K.K., Tokyo, Japan).
Adverse events
The number of adverse events (AE) observed from the onset of the event until the participants recovered from the event was defined as the sum of single events. In all reported events, the medical doctor, as the principal investigator, made a diagnosis in order to estimate the relationship between the observed adverse effects and l-threonine supplementation under the testing conditions.
Statistical analysis
Statistical analyses were performed on the per-protocol set (PPS). Descriptive data were analyzed using SPSS version 29.0 for windows (IBM Japan Ltd., Tokyo, Japan). All results are presented as means and standard deviation (SD). The statistical significance was determined using one-factor repeated-measures analysis of variance (ANOVA) followed by post hoc analysis as Dunnett's multiple comparison test. The level of statistical significance was determined at *: P < 0.05. **: P < 0.01.
Results
Subject
As indicated in Table 1, the study included 30 participants (Age 42.9 ± 9.7 years, BW 71.6 ± 8.0 Kg and BMI 24.3 ± 2.3 Kg/m2) which were selected by screening criteria as described in the Method section. Volunteer average characteristics in placebo and L-threonine supplementation groups are given in Supplemental Table 1. In the l-threonine-supplemented group, 6 out of 30 participants were withdrawn because of deviance from the protocol or for private reasons (Supplemental Figure 1). The decision to withdraw volunteers from the study was taken by the principal investigator. The compliance of the remaining participants for capsule ingestion was overall 99.6 %. In the different experimental groups, the compliance was similar, 99.8 % for the placebo group, 99.6 % for 3 g/day supplementation group, 99.8 % for 6 g/day supplementation group, 99.1 % for 9 g/day supplementation group and 99.7 % for 12 g/day supplementation group.
Table 1.
Participants baseline characteristics in l-threonine supplementation group
| l-threonine intake group | |
|---|---|
| Age (year) | 42.9±9.7 |
| BH (cm) | 171.8 ± 5.0 |
| BW (Kg) | 71.6 ± 8.0 |
| BMI (kg/m2) | 24.2 ± 2.3 |
| SAP (mmHg) | 128.8 ± 18.7 |
| DBP (mmHg) | 78.6 ± 16.3 |
| HR (bpm) | 72.2 ±10.9 |
| Carbohydrates (g/day) | 212.4 ± 90.6 |
| Fat (g/day) | 51.6 ± 19.4 |
| Protein (g/day) | 66.1 ± 23.2 |
| Total energy (kcal/day) | 1620 ± 537 |
| TP (g/dL) | 7.5 ± 0.33 |
| ALB (g/dL) | 4.7 ± 0.21 |
| T-BIL (mg/dL) | 0.87 ± 0.26 |
| AST (U/L) | 21.9 ± 5.3 |
| ALT (U/L) | 23.8 ± 10.5 |
| LDH (U/L) | 190.0 ± 42.9 |
| ALP (U/L) | 72.5 ± 18.5 |
| γ-GTP (U/L) | 26.8 ± 15.5 |
| CK (U/L) | 154.4 ± 85.3 |
| PL (mg/dL) | 225.0 ± 37.7 |
| BUN (mg/dL) | 14.38 ± 4.01 |
| CRE (mg/dL) | 0.84 ± 0.13 |
| UA (mg/dL) | 5.81 ± 0.93 |
| GLU (mg/dL) | 89.5 ± 9.05 |
| TC (mg/dL) | 210.7 ± 35.6 |
| TG (mg/dL) | 108.5 ± 86.2 |
| HDL-Cho (mg/dL) | 63.1 ± 14.4 |
| LDL-Cho (mg/dL) | 127.1 ± 31.21 |
| Na (mEq/L) | 141.1 ± 1.23 |
| K (mEq/L) | 4.31 ± 0.32 |
| Cl (mEq/L) | 102.6 ± 1.63 |
| Ca (mEq/L) | 9.58 ± 0.23 |
| eGFR (mL/min/1.73m2) | 82.6 ± 15.0 |
| RBC (×104/µL) | 502.6 ± 35.9 |
| HGB (g/dL) | 15.1 ± 1.05 |
| HC (%) | 45.8 ± 3.36 |
| WBC (/µL) | 5623 ± 1929 |
| PLT (×104/µL) | 26.0 ± 5.3 |
| His (μmol/L) | 86.3 ± 9.1 |
| Ile (μmol/L) | 70.6 ± 16.3 |
| Leu (μmol/L) | 129.0 ± 23.4 |
| Lys (μmol/L) | 211.6 ± 31.2 |
| Met (μmol/L) | 25.3 ± 5.3 |
| Phe (μmol/L) | 64.1 ± 10.9 |
| Thr (μmol/L) | 136.4 ± 26.6 |
| Trp (μmol/L) | 65.1 ± 14.1 |
| Val (μmol/L) | 247.3 ± 41.3 |
| Ala (μmol/L) | 390.9 ± 70.6 |
| Arg (μmol/L) | 102.2 ± 16.2 |
| Asn (μmol/L) | 50.8 ± 9.1 |
| Cys (μmol/L) | 50.8 ± 5.7 |
| Glu (μmol/L) | 32.7 ± 14.2 |
| Gln (μmol/L) | 669.1 ± 76.4 |
| Gly (μmol/L) | 244.5 ± 60.6 |
| Pro (μmol/L) | 167.4 ± 53.0 |
| Ser (μmol/L) | 120.6 ±23.5 |
| Tyr (μmol/L) | 68.0 ± 11.3 |
| ABA (μmol/L) | 21.1 ± 5.2 |
The participants visited the clinic after an overnight fast for baseline screening, including anthropometric measurements, dietary surveys, blood chemistry, blood biochemistry, and plasma amino acid analyses, prior to the intervention study. Each values represent the mean and standard deviation (SD) (n = 30). Note: Aspartic acid is not detected in plasma for concentration below 5 μmol/L (RV is < 5.7 μmol/L).
ABA L-2-amino-butyric acid, ALB albumin, ALP Alkaline Phosphatase, ALT (GPT) alanine aminotransferase (Glutamic Pyruvic Transaminase), Arg L-arginine, Asn L-Asparagine, Asp L-aspartic acid, AST (GOT) aspartate aminotransferase (Glutamic Oxaloacetic Transaminase), BH body height, BW body weight, BMI body mass index, bpm beats per minute, BUN blood urea nitrogen, Ca calcium, Cl chloride, CK Creatine kinase, CRE creatinine, Cys L-cystine, DPB diastolic blood pressure, eGFR estimated glomerular filtration rate, Gln L-glutamine, GLU glucose, Glu L-glutamic acid, Gly glycine, γ-GTP γ-glutamyl Transpeptidase, glycine, HC hematocrit, HGB hemoglobin, HDL-Cho high-density lipoprotein cholesterol, His L-histidine, HR heart rate, Ile L-isoleucine, K potassium, LDL-Cho low-density lipoprotein cholesterol, LDH Lactate dehydrogenase, Lys L-Lysine, Met L-methionine, Na sodium, Phe L-phenylalanine, PLT platelet, PL phospholipids, Pro L-proline, RBC red blood cells, SAP systolic arterial pressure, Ser L-serine, Thr, L-threonine, T-BIL total bilirubin, TC total cholesterol, TP total protein, TG triglycerides, Trp L-tryptophan, Tyr L-tyrosine, UA uric acid, Val L-valine, and WBC white blood cells.
Anthropometric measurements and dietary intake estimation
The results of anthropometric measurements (BW, BMI, blood pressure and HR) and dietary intake estimation (total energy, protein, fat, and carbohydrate) conducted at the end of the supplementation period are shown in Table 2. In all the experimental groups, the results of anthropometric measurements and dietary intake estimates indicated no significant differences when compared to the placebo group, whatever the doses used (3, 6, 9, and 12 g/day).
Table 2.
Anthropometric measurements and dietary intake estimation measured at the end of the supplementation period in each experimental group
| (g/day) | n | l-threonine intake group | P | |
|---|---|---|---|---|
| BW | 0 | 22 | 71.7 ± 7.9 | – |
| (Kg) | 3 | 20 | 70.4 ± 7.7 | 0.942 |
| 6 | 22 | 71.5 ± 6.4 | 1.000 | |
| 9 | 21 | 70.7 ± 8.2 | 0.981 | |
| 12 | 20 | 71.3 ± 7.6 | 0.999 | |
| BMI | 0 | 22 | 24.3 ± 2.4 | – |
| (kg/m2) | 3 | 20 | 23.9 ± 2.3 | 0.972 |
| 6 | 22 | 24.4 ± 2.3 | 1.000 | |
| 9 | 21 | 24.3 ± 2.7 | 1.000 | |
| 12 | 20 | 24.2 ± 2.5 | 1.000 | |
| SAP | 0 | 22 | 125.6 ± 18.9 | – |
| (mmHg) | 3 | 20 | 127.1 ± 19.5 | 0.997 |
| 6 | 22 | 130.7 ± 19.1 | 0.775 | |
| 9 | 21 | 130.0 ± 20.2 | 0.854 | |
| 12 | 20 | 124.1 ± 14.1 | 0.997 | |
| DBP | 0 | 22 | 79.5 ± 13.7 | – |
| (mmHg) | 3 | 20 | 79.7 ± 16.1 | 1.000 |
| 6 | 22 | 81.7 ± 13.6 | 0.960 | |
| 9 | 21 | 81.9 ± 16.1 | 0.950 | |
| 12 | 20 | 77.4 ± 12.5 | 0.974 | |
| HR | 0 | 22 | 71.7 ± 11.0 | – |
| (bpm) | 3 | 20 | 76.9 ± 11.8 | 0.332 |
| 6 | 22 | 69.6 ± 9.5 | 0.903 | |
| 9 | 21 | 72.6 ± 8.5 | 0.997 | |
| 12 | 20 | 72.3 ± 12.0 | 0.999 | |
| Carbohydrates | 0 | 22 | 210.0 ± 78.1 | – |
| (g/day) | 3 | 20 | 233.4 ± 81.3 | 0.738 |
| 6 | 22 | 215.0 ± 75.3 | 0.999 | |
| 9 | 21 | 237.7 ± 72.3 | 0.606 | |
| 12 | 20 | 217.2 ± 85.1 | 0.995 | |
| Fat | 0 | 22 | 50.1 ± 16.3 | – |
| (g/day) | 3 | 20 | 54.8 ± 20.3 | 0.770 |
| 6 | 22 | 50.1 ± 16.7 | 1.000 | |
| 9 | 21 | 56.6 ± 13.5 | 0.506 | |
| 12 | 20 | 50.5 ± 15.2 | 1.000 | |
| Protein | 0 | 22 | 62.2 ± 19.8 | – |
| (g/day) | 3 | 20 | 69.4 ± 25.9 | 0.654 |
| 6 | 22 | 66.6 ± 21.5 | 0.903 | |
| 9 | 21 | 73.0 ± 16.4 | 0.285 | |
| 12 | 20 | 67.3 ± 22.3 | 0.854 | |
| Total energy | 0 | 22 | 1583 ± 507 | – |
| (kcal/day) | 3 | 20 | 1764 ± 594 | 0.618 |
| 6 | 22 | 1624 ± 503 | 0.997 | |
| 9 | 21 | 1811 ± 433 | 0.403 | |
| 12 | 20 | 1652 ± 518 | 0.980 |
The participants visited the clinic following overnight fasting for least 8 h for safety evaluation after the 4-week supplementation, including anthropometric measurements and dietary surveys. All results are presented as means and standard deviation (SD). The statistical significance was determined using Dunnett's multiple comparison test. The level of statistical significance was determined at *: P < 0.05. **: P < 0.01.
BW body weight, BMI body mass index, SAP systolic arterial pressure, DPB diastolic blood pressure, HR heart rate, bpm beats per minute. Values represent the mean and standard deviation (SD).
Clinical laboratory tests
The results of blood chemistry, blood biochemistry and serum levels of electrolytes measurements performed at the end of the supplementation period are shown in Table 3. In the l-threonine-supplemented group, minor but statistically significant increase in AST/GOT (P = 0.025) and CK (P = 0.036) values was observed for the 9 g/day l-threonine experimental group. The results of plasma amino acid analysis performed at the end of the supplementation period are shown in Table 4. In the l-threonine supplemented groups, statistically significant increases in plasma concentration of l-threonine were observed for the supplementation dose 6 (P = 0.020), 9 (P < 0.001) and 12 (P < 0.001) g/day. Regarding the l-threonine-derived metabolite 2-amino-butylate, its plasma concentration was increased for the supplementation doses 6 (P = 0.008), 9 (P < 0.001) and 12 (P < 0.001) g/day.
Table 3.
Blood chemistry and blood biochemistry parameter measurement at the end of the supplementation period in each experimental group
| (g/day) | n | l-threonine intake group | P | |
|---|---|---|---|---|
| TP | 0 | 22 | 7.22 ± 0.4 | – |
| RV 6.7–8.3 | 3 | 20 | 7.31 ± 0.4 | 0.833 |
| (g/dL) | 6 | 22 | 7.24 ± 0.3 | 1.000 |
| 9 | 21 | 7.26 ± 0.4 | 0.993 | |
| 12 | 20 | 7.13 ± 0.3 | 0.773 | |
| ALB | 0 | 22 | 4.52 ± 0.4 | – |
| RV 3.8–5.2 | 3 | 20 | 4.51 ± 0.3 | 1.000 |
| (g/dL) | 6 | 22 | 4.52 ± 0.3 | 1.000 |
| 9 | 21 | 4.51 ± 0.3 | 0.999 | |
| 12 | 20 | 4.43 ± 0.4 | 0.555 | |
| T-BIL | 0 | 22 | 0.809 ± 0.22 | – |
| RV 0.2–.2 | 3 | 20 | 0.900 ± 0.29 | 0.613 |
| (mg/dL) | 6 | 22 | 0.827 ± 0.26 | 0.998 |
| 9 | 21 | 0.905 ± 0.30 | 0.560 | |
| 12 | 20 | 0.860 ± 0.20 | 0.920 | |
| AST(GOT) | 0 | 22 | 20.8 ± 4.5 | – |
| RV 10–40 | 3 | 20 | 21.4 ± 6.0 | 0.998 |
| (U/L) | 6 | 22 | 21.4 ± 6.8 | 0.996 |
| 9 | 21 | 27.2 ± 13.3 | 0.025* | |
| 12 | 20 | 20.0 ± 4.0 | 0.991 | |
| ALT(GPT) | 0 | 22 | 22.9 ± 10.4 | – |
| RV 5–45 | 3 | 20 | 22.3 ± 10.8 | 0.999 |
| (U/L) | 6 | 22 | 23.0 ± 11.8 | 1.000 |
| 9 | 21 | 25.1 ± 12.5 | 0.902 | |
| 12 | 20 | 21.1 ± 8.6 | 0.956 | |
| LDH | 0 | 22 | 175.0 ± 26.3 | – |
| RV 120–240 | 3 | 20 | 187.1 ± 61.2 | 0.902 |
| (U/L) | 6 | 22 | 184.7 ± 63.8 | 0.949 |
| 9 | 21 | 197.4 ± 61.8 | 0.517 | |
| 12 | 20 | 185.0 ± 64.6 | 0.948 | |
| ALP | 0 | 22 | 67.4 ± 13.7 | – |
| RV 38–113 | 3 | 20 | 69.8 ± 14.9 | 0.958 |
| (U/L) | 6 | 22 | 67.0 ± 14.8 | 1.000 |
| 9 | 21 | 66.9 ± 15.1 | 1.000 | |
| 12 | 20 | 65.7± 12.9 | 0.986 | |
| γ-GTP | 0 | 22 | 25.5 ± 13 | – |
| RV less than 80 | 3 | 20 | 27.4 ± 15 | 0.986 |
| (U/L) | 6 | 22 | 26.4 ± 17 | 0.999 |
| 9 | 21 | 26.3 ± 15 | 0.999 | |
| 12 | 20 | 26.9 ± 17 | 0.996 | |
| CK | 0 | 22 | 154.7 ± 77.3 | – |
| RV. 60–270 | 3 | 20 | 144.2 ± 79.4 | 1.000 |
| (U/L) | 6 | 22 | 152.9 ± 81.5 | 1.000 |
| 9 | 21 | 381.3 ± 606.6 | 0.036* | |
| 12 | 20 | 165.4 ± 133.7 | 1.000 | |
| PL | 0 | 22 | 219.9 ± 0.2 | – |
| RV 150–280 | 3 | 20 | 223.4 ± 31.8 | 0.990 |
| (mg/dL) | 6 | 22 | 222.1 ± 21.6 | 0.998 |
| 9 | 21 | 226.7 ± 32.9 | 0.890 | |
| 12 | 20 | 223.8 ± 30.2 | 0.984 | |
| BUN | 0 | 22 | 13.6 ± 3.8 | – |
| RV 8.0–20 | 3 | 20 | 14.7 ± 4.5 | 0.784 |
| (mg/dL) | 6 | 22 | 15.1 ± 4.4 | 0.534 |
| 9 | 21 | 13.9 ± 3.6 | 0.997 | |
| 12 | 20 | 14.0 ± 3.4 | 0.992 | |
| CRE | 0 | 22 | 0.85 ± 0.1 | – |
| RV 0.61–1.04 | 3 | 20 | 0.89 ± 0.1 | 0.732 |
| (mg/dL) | 6 | 22 | 0.86 ± 0.1 | 0.993 |
| 9 | 21 | 0.86 ± 0.1 | 0.996 | |
| 12 | 20 | 0.81 ± 0.1 | 0.760 | |
| UA | 0 | 22 | 5.57 ± 1.1 | – |
| RV 3.8–7.0 | 3 | 20 | 5.62 ± 0.9 | 0.999 |
| (mg/dL) | 6 | 22 | 5.50 ± 1.0 | 0.997 |
| 9 | 21 | 5.53 ± 0.8 | 1.000 | |
| 12 | 20 | 5.47 ± 0.8 | 0.990 | |
| GLU | 0 | 22 | 89.8 ± 7.8 | – |
| RV 70–109 | 3 | 20 | 93.9 ± 11.1 | 0.443 |
| (mg/dL) | 6 | 22 | 89.5 ± 10.6 | 1.000 |
| 9 | 21 | 91.2 ± 9.2 | 0.970 | |
| 12 | 20 | 91.5 ± 7.6 | 0.948 | |
| TC | 0 | 22 | 205.6 ± 38.2 | – |
| RV 210–219 | 3 | 20 | 205.7 ± 33.1 | 1.000 |
| (mg/dL) | 6 | 22 | 205.6 ± 39.8 | 1.000 |
| 9 | 21 | 214.5 ± 41.2 | 0.867 | |
| 12 | 20 | 207.7 ± 38.6 | 0.999 | |
| TG | 0 | 22 | 94.0 ± 57.4 | – |
| RV 30–149 | 3 | 20 | 107.7 ± 92.7 | 0.883 |
| (mg/dL) | 6 | 22 | 99.7 ± 37.6 | 0.994 |
| 9 | 21 | 82.9 ± 44.5 | 0.936 | |
| 12 | 20 | 100.3 ± 60.7 | 0.992 | |
| HDL-Cho | 0 | 22 | 59.9 ± 16.8 | – |
| RV 40–85 | 3 | 20 | 62.5 ± 14.3 | 0.938 |
| (mg/dL) | 6 | 22 | 58.2 ± 14.7 | 0.987 |
| 9 | 21 | 64.0 ± 15.1 | 0.743 | |
| 12 | 20 | 61.6 ± 14.8 | 0.986 | |
| LDL-Cho | 0 | 22 | 125.4 ± 36.3 | – |
| RV 65–139 | 3 | 20 | 122.5 ± 31.3 | 0.997 |
| (mg/dL) | 6 | 22 | 125.6 ± 36.9 | 1.000 |
| 9 | 21 | 132.3 ± 38.5 | 0.924 | |
| 12 | 20 | 123.9 ± 36.0 | 1.000 | |
| Na | 0 | 22 | 140.6 ± 1.5 | – |
| RV 137–147 | 3 | 20 | 140.7 ± 1.4 | 1.000 |
| (mEq/L) | 6 | 22 | 140.6 ± 1.5 | 1.000 |
| 9 | 21 | 140.2 ± 1.1 | 0.852 | |
| 12 | 20 | 140.9 ± 1.2 | 0.937 | |
| K | 0 | 22 | 4.20 ± 0.3 | – |
| RV 3.5–5.0 | 3 | 20 | 4.21 ± 0.3 | 1.000 |
| (mEq/L) | 6 | 22 | 4.22 ± 0.2 | 0.996 |
| 9 | 21 | 4.23 ± 0.3 | 0.977 | |
| 12 | 20 | 4.28 ± 0.3 | 0.765 | |
| Cl | 0 | 22 | 102.9 ± 1.2 | – |
| RV 98–108 | 3 | 20 | 102.4 ± 2.2 | 0.854 |
| (mEq/L) | 6 | 22 | 102.8 ± 2.0 | 1.000 |
| 9 | 21 | 102.4 ± 2.0 | 0.874 | |
| 12 | 20 | 103.2 ± 1.6 | 0.947 | |
| Ca | 0 | 22 | 9.29 ± 0.3 | – |
| RV 8.4–10.4 | 3 | 20 | 9.35 ± 0.3 | 0.937 |
| (mg/dL) | 6 | 22 | 9.36 ± 0.2 | 0.858 |
| 9 | 21 | 9.33 ± 0.4 | 0.971 | |
| 12 | 20 | 9.24 ± 0.3 | 0.948 | |
| eGFR | 0 | 22 | 81.3 ± 14.2 | – |
| RV ≧60 | 3 | 20 | 77.6 ± 15.0 | 0.852 |
| (mL/min/1.73m2) | 6 | 22 | 80.1 ± 15.3 | 0.997 |
| 9 | 21 | 79.9 ± 14.8 | 0.994 | |
| 12 | 20 | 85.8 ± 18.4 | 0.759 | |
| RBC | 0 | 22 | 496.4 ± 36.9 | – |
| RV 430–570 | 3 | 20 | 503.9 ± 40.5 | 0.897 |
| (×104/µL) | 6 | 22 | 504.2 ± 22.1 | 0.874 |
| 9 | 21 | 500.8 ± 36.9 | 0.982 | |
| 12 | 20 | 492.1 ± 37.1 | 0.985 | |
| HGB | 0 | 22 | 15.0 ± 1.0 | – |
| RV 13.5–17.5 | 3 | 20 | 15.3 ± 1.1 | 0.829 |
| (g/dL) | 6 | 22 | 15.2 ± 0.7 | 0.906 |
| 9 | 21 | 15.0 ± 1.1 | 1.000 | |
| 12 | 20 | 14.9 ± 1.0 | 0.974 | |
| HC | 0 | 22 | 46.0 ± 3.1 | – |
| RV 39.7–52.4 | 3 | 20 | 46.9 ± 3.4 | 0.725 |
| (%) | 6 | 22 | 46.5 ± 1.8 | 0.935 |
| 9 | 21 | 46.3 ± 3.4 | 0.986 | |
| 12 | 20 | 45.3 ± 2.9 | 0.878 | |
| WBC | 0 | 22 | 5618 ± 1445 | – |
| RV 3300–9000 | 3 | 20 | 5885 ± 1882 | 0.926 |
| (/µL) | 6 | 22 | 5305 ± 1099 | 0.867 |
| 9 | 21 | 4971 ± 1199 | 0.353 | |
| 12 | 20 | 4925 ± 1123 | 0.304 | |
| PLT | 0 | 22 | 25.8 ± 7.0 | – |
| RV 14-34 | 3 | 20 | 27.9 ± 3.2 | 0.610 |
| (×104/µL) | 6 | 22 | 25.7 ± 6.8 | 1.000 |
| 9 | 21 | 25.2 ± 6.3 | 0.995 | |
| 12 | 20 | 24.7 ± 6.0 | 0.956 |
The participants visited the clinic following overnight fasting for least 8 h for safety evaluation after the 4-week supplementation, including Blood chemistry and blood biochemistry parameter measurements. All results are presented as means and standard deviation (SD). The statistical significance was determined using Dunnett's multiple comparison test. The level of statistical significance was determined at *: P < 0.05. **: P < 0.01.
ALB albumin, ALP Alkaline Phosphatase, ALT (GPT) alanine aminotransferase (Glutamic Pyruvic Transaminase), AST (GOT) aspartate aminotransferase (Glutamic Oxaloacetic Transaminase), BUN blood urea nitrogen, Ca calcium, Cl chloride, CK Creatine kinase, CRE creatinine, eGFR estimated glomerular filtration rate, GLU glucose, γ-GTP γ-glutamyl Transpeptidase, HC hematocrit, HGB hemoglobin, HDL-Cho high-density lipoprotein cholesterol, K potassium, LDL-Cho low-density lipoprotein cholesterol, LDH Lactate dehydrogenase, Na sodium, PLT platelet, PL phospholipids, RBC red blood cells, RV reference value (clinical normative values used Miura medical clinic), T-BIL total bilirubin, TC total cholesterol, TP total protein, TG triglycerides, UA uric acid, and WBC white blood cells.
Table 4.
Plasma amino acid concentration (in µmol/L) measured at the end of the supplementation period in each experimental group
| IAA (μmol/L) | (g/day) | n | Threonine intake group | P |
|---|---|---|---|---|
| His | 0 | 22 | 83.3 ± 6.5 | – |
| RV 68.0–116.6 | 3 | 20 | 85.8 ± 11.4 | 0.818 |
| 6 | 22 | 81.5 ± 9.2 | 0.910 | |
| 9 | 21 | 86.1 ± 10.5 | 0.748 | |
| 12 | 20 | 85.1 ± 8.6 | 0.927 | |
| Ile | 0 | 22 | 74.3 ± 12.7 | – |
| RV 44.9–120.3 | 3 | 20 | 72.6 ± 18.5 | 0.990 |
| 6 | 22 | 68.4 ± 12.8 | 0.566 | |
| 9 | 21 | 71.9 ± 18.5 | 0.967 | |
| 12 | 20 | 67.9 ± 17.4 | 0.509 | |
| Leu | 0 | 22 | 130.6 ± 16.8 | – |
| RV 84.4–200.2 | 3 | 20 | 129.5 ± 25.5 | 1.000 |
| 6 | 22 | 122.4 ± 19.6 | 0.508 | |
| 9 | 21 | 127.4 ± 22.0 | 0.967 | |
| 12 | 20 | 124.5 ± 20.8 | 0.764 | |
| Lys | 0 | 22 | 198.7 ± 28.5 | – |
| RV 138.6–294.2 | 3 | 20 | 210.5 ± 33.5 | 0.586 |
| 6 | 22 | 198.2 ± 36.3 | 1.00 | |
| 9 | 21 | 206.2 ± 30.1 | 0.861 | |
| 12 | 20 | 208.5 ± 32.9 | 0.728 | |
| Met | 0 | 22 | 25.4 ± 3.8 | – |
| RV 18.1–43.5 | 3 | 20 | 26.2 ± 5.5 | 0.945 |
| 6 | 22 | 24.4 ± 4.0 | 0.885 | |
| 9 | 21 | 25.8 ± 4.7 | 0.997 | |
| 12 | 20 | 26.3 ± 5.5 | 0.934 | |
| Phe | 0 | 22 | 62.4 ± 9 | – |
| RV 49.0–90.8 | 3 | 20 | 63.9 ± 11 | 0.966 |
| 6 | 22 | 58.5 ± 10 | 0.500 | |
| 9 | 21 | 62.6 ± 11 | 1.000 | |
| 12 | 20 | 60.5 ± 8 | 0.931 | |
| Thr | 0 | 22 | 127.9 ± 19.4 | – |
| RV 89.2–241.6 | 3 | 20 | 191.2 ± 76.9 | 0.261 |
| 6 | 22 | 229.7 ± 100.7 | 0.020 | |
| 9 | 21 | 307.0 ± 143.8 | <.001** | |
| 12 | 20 | 385.1 188.5 | <.001** | |
| Trp | 0 | 22 | 64.1 ± 10.2 | – |
| RV 46.7–92.0 | 3 | 20 | 64.5 ± 12.7 | 1.000 |
| 6 | 22 | 60.5 ± 9.6 | 0.676 | |
| 9 | 21 | 63.6 ± 13.3 | 1.000 | |
| 12 | 20 | 60.7 ± 10.9 | 0.742 | |
| Val | 0 | 22 | 242.2 ± 32.8 | – |
| RV 162.9–351.4 | 3 | 20 | 239.5 ± 39.4 | 0.997 |
| 6 | 22 | 235.9 ± 33.1 | 0.939 | |
| 9 | 21 | 236.8 ± 38.7 | 0.966 | |
| 12 | 20 | 235.5 ± 33.7 | 0.930 |
| DAA (μmol/L) | (g/day) | n | Threonine intake group | P |
|---|---|---|---|---|
| Ala | 0 | 22 | 380.9 ± 71.7 | – |
| RV 253.6–601.9 | 3 | 20 | 388.4 ± 61.5 | 0.991 |
| 6 | 22 | 367.7 ± 56.4 | 0.927 | |
| 9 | 21 | 359.3 ± 75.7 | 0.711 | |
| 12 | 20 | 380.2 ± 82.4 | 1.000 | |
| Arg | 0 | 22 | 95.2 ± 12.1 | – |
| RV 44.1–115.2 | 3 | 20 | 99.5 ± 19.9 | 0.788 |
| 6 | 22 | 94.7 ± 15.0 | 1.000 | |
| 9 | 21 | 100.0 ± 11.1 | 0.712 | |
| 12 | 20 | 100.6 ± 18.3 | 0.632 | |
| Asn | 0 | 22 | 47.9 ± 7.0 | – |
| RV 37.7–78.5 | 3 | 20 | 50.7 ± 10.4 | 0.673 |
| 6 | 22 | 46.4 ± 8.8 | 0.957 | |
| 9 | 21 | 51.2 ± 8.5 | 0.555 | |
| 12 | 20 | 50.2 ± 9. | 0.802 | |
| Cys | 0 | 22 | 40.8 ± 7.2 | – |
| RV 34.9-77.7 | 3 | 20 | 44.1 ± 10.0 | 0.476 |
| 6 | 22 | 42.1 ± 8.3 | 0.954 | |
| 9 | 21 | 45.1 ± 7.2 | 0.239 | |
| 12 | 20 | 42.8 ± 7.2 | 0.844 | |
| Glu | 0 | 22 | 35.0 ± 11.4 | – |
| RV 13.3–86.7 | 3 | 20 | 35.8 ± 13.2 | 0.999 |
| 6 | 22 | 35.6 ± 14.9 | 1.000 | |
| 9 | 21 | 36.9 ± 16.8 | 0.972 | |
| 12 | 20 | 35.3 ± 11.6 | 1.000 | |
| Gln | 0 | 22 | 635.2 ± 59.9 | – |
| RV 503.4–851.4 | 3 | 20 | 630.8 ± 82.6 | 0.999 |
| 6 | 22 | 617.3 ± 78.1 | 0.815 | |
| 9 | 21 | 627.2 ± 65.5 | 0.988 | |
| 12 | 20 | 609.9 ± 59.7 | 0.596 | |
| Gly | 0 | 22 | 228.8 ± 50.5 | – |
| RV 136.8–397.7 | 3 | 20 | 219.2 ± 39.0 | 0.902 |
| 6 | 22 | 214.4 ± 44.7 | 0.679 | |
| 9 | 21 | 221.5 ± 41.4 | 0.960 | |
| 12 | 20 | 221.7 ± 49.3 | 0.964 | |
| Pro | 0 | 22 | 161.1 ± 42.0 | – |
| RV 89.8–304.7 | 3 | 20 | 154.5 ± 32.1 | 0.95 |
| 6 | 22 | 168.6 ± 35.3 | 0.929 | |
| 9 | 21 | 156.1 ± 42.2 | 0.983 | |
| 12 | 20 | 163.5 ± 46.8 | 0.999 | |
| Ser | 0 | 22 | 112.6 ± 21.2 | – |
| RV 78.4–200.1 | 3 | 20 | 111.3 ± 22.3 | 0.999 |
| 6 | 22 | 109.1 ± 17.8 | 0.951 | |
| 9 | 21 | 116.2 ± 20.3 | 0.942 | |
| 12 | 20 | 113.9 ± 20.8 | 0.999 | |
| Tyr | 0 | 22 | 66.2 ± 8.9 | – |
| RV 46.7–103.6 | 3 | 20 | 67.0 ± 13.1 | 0.997 |
| 6 | 22 | 62.4 ± 11.1 | 0.620 | |
| 9 | 21 | 67.0 ± 11.4 | 0.998 | |
| 12 | 20 | 65.0 ± 10.3 | 0.991 |
| Other (μmol/L) | (g/day) | n | Threonine intake group | P |
|---|---|---|---|---|
| ABA | 0 | 22 | 20.0 ± 5.9 | – |
| RV 11.2~40.1 | 3 | 20 | 28.5 ± 11.9 | 0.304 |
| 6 | 22 | 36.1 ± 16.4 | 0.008** | |
| 9 | 21 | 48.5 ± 21.9 | <.001** | |
| 12 | 20 | 51.1 ± 23.1 | <.001** |
The participants visited the clinic following overnight fasting for least 8 h for safety evaluation after the 4-week supplementation, including plasma amino acid analyses. All results are presented as means and standard deviation (SD). The statistical significance was determined using Dunnett's multiple comparison test. The level of statistical significance was determined at *: P < 0.05. **: P < 0.01. Note: Aspartic acid is not detected in plasma for concentration below 5 μmol/L (RV is < 5.7 μmol/L).
ABA L-2-amino-butyric acid, Arg l -arginine, Asn l -Asparagine, Asp l -aspartic acid, Cys l -cystine, DAA dispensable (non-essential) amino acid, Glu l -glutamic acid, Gln l -glutamine, Gly glycine, His l -histidine, IDA indispensable (essential) amino acid, Ile l -isoleucine, Lys l -Lysine, Met l-methionine, Phe l-phenylalanine, Pro l-proline, RV reference value (clinical normative values used Miura medical clinic), Ser l-serine, Thr l -threonine, Trp l -tryptophan, Tyr l -tyrosine, and Val l-valine.
Adverse events
During the supplementation period with L-threonine, whatever the dose used, no serious adverse events were recorded. Eight moderate adverse events requiring treatment were recorded all along the supplementation period. They consist of 2 cases of stomach indefinite complaint, 1 case each for the following individual or combined symptoms: stomach pain, cold-like symptoms, lower back pain, COVID-19 infection, headache, cervicobrachial syndrome. In addition, 6 mild cases that did not require treatment were recorded. They consisted of 1 case each for the following symptoms: diarrhea, increased bowel movements, cough with runny nose, fatigue, fever and decreased appetite. These events occurred in a random manner when comparing the placebo and the l-threonine-supplemented groups. In the placebo group, 7 events were recorded, while in the 3 g/day group, 2 events were observed. In the 6 g/day group, 4 events were recorded, in the 9 g/day group,1 event was noticed, while in the 12 g/day group, no event was registered. All symptoms disappeared during the supplementation period despite continuous L-threonine and placebo supplementation. For all reported events, Thr supplementation was not found to be causally related to noncontinuous adverse events as judged from the diagnosis made by the medical doctor acting as principal investigator.
Discussion
The results of our study clearly indicate that increasing doses of l-threonine up to 12 g/day given for 4 weeks did not result in significant modifications of the dietary intake, anthropometric data, blood parameters, blood pressure, and heart rate. In this study, AST/GOT and CK were slightly but statistically significantly changed for a L-threonine intake dose of 9 g/day (Table 3). For AST, elevated levels exceeding the upper limit of the clinical reference range were observed in 5 out of 108 cases (6 g/day, n = 2; 9 g/day, n = 3), with increases ranging from 1.03 to 1.45 times the upper limit. For CK, elevated levels were found in 13 out of 108 cases (placebo, n = 3; 3 g/day, n = 2; 6 g/day, n = 2; 9 g/day, n = 4; 12 g/day, n = 2), with increases ranging from 1.08 to 9.49 times the upper limit of the clinical reference range. However, these values did not exhibit dose-dependent characteristics, and no significant increases were recorded regarding these 2 parameters using the 12 g/day, the highest dose. This, together with the widely dispersed values for AST/GOT and CK after supplementation with 9 g/day l-threonine, strongly suggest nonspecific outcome for the 9 g/day doses. The results of blood chemistry and biochemistry test indicate no signs of either metabolic dysfunctions, or liver, kidney, muscle, or bone dysfunctions. No signs of cardiovascular dysfunctions were detected as well. Glycemia, total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides were not modified by the 28 day-supplementation with any of the supplemental dose of L-threonine. Blood electrolytes and blood cell count were in addition not modified by the supplementation. Finally, dietary consumption, in terms of total energy, protein, fat, and carbohydrate intake, as well as the BMI were not modified by the dietary supplementation with l-threonine. Furthermore, and of major importance, these doses of l-threonine were not associated with any serious adverse events, the minor adverse events recorded during the 4-week supplementation being distributed at random between the placebo and experimental groups and disappearing during the supplementation period despite continuous l-threonine intake. In addition, the occurrence of adverse events in each dosage group appeared to be at random, occurring throughout both the early and late phases of the placebo or supplement intake period. All these results lead us to propose for adult males a NOAEL value for L-threonine equal to 12 g/day, thus representing the highest supplemental intake without measurable adverse effects.
To the best of our knowledge, our study is the first one to report on the NOAEL value for l-threonine used in healthy adults as supplements in medium-term randomized controlled double blind clinical trial. In previous studies, l-threonine supplementation was performed in some clinical trials in pathological situations such as upper motor syndrome (Growdon et al 1991), spinal spasticity (Lee and Patterson1993) and amyotrophic lateral sclerosis (ALS) (Tandan et al 1996). As a matter of comparison, the NOAEL value determined in the present study is above the highest supplemental doses of l-threonine tested in clinical trials for their effects. For instance, in the study by Growdon and collaborators, 6 g/day of l-threonine were given during 2 weeks to patients with familial spastic paraparesis (Growdon et al 1991), while 6 g/day of l-threonine were given to spinal spasticity patients in the study by Lee (Lee and Patterson1993). In the study by Tandan and collaborators, 4 g/day of l-threonine were given for 6 months to patients with amyotrophic lateral sclerosis (Tandan et al 1996).
The results of our study give indication on the concentrations of amino acids in the circulation as recorded at the end of the supplementation period after a one night fast. After supplementation with L-threonine, the middle to highest doses (6, 9 and 12 g/day) were associated with increased plasma concentration of l-threonine (6 g/day 179.6 %, 9 g/day 240.0 % and 12 g/day 301.1 %) and its metabolite 2-aminobutylic acid (6 g/day 180.5 %, 9 g/day 242.5 % and 12 g/day 255.5%). Following L-threonine intake, plasma levels of 2-amino-butyric acid R2 increased in parallel with plasma l-threonine concentrations (R2 = 0.550), whereas no concentration-dependent increases were observed for glycine (R2 = 0.010) or serine (R2 = 0.043) (Fig. 2). Of note, the increases of L-threonine and 2-amino-butylic acid concentrations in blood plasma, as measured in our study at the end of the supplementation period, were not associated with any measurable sign of metabolic dysfunctions or signs of organic abnormalities. In our study, the increased plasma concentration of 2-amino-butylic acid could plausibly originate from the metabolism of l-threonine to 2-oxo-butylic acid by threonine dehydratase, and then from the synthesis of 2-amino-butylic acid by aminotransferase (Fig. 1). On the other hand, the contributions of these latter enzymes for 2-amino-butylic acid synthesis remains to be explored since the concentrations of metabolites generated by the threonine aldolase (i.e. Gly and Ser) did not change in plasma. In addition, the metabolite 2-amino-3-oxobutyrate which is generated by threonine dehydrogenase was not analyzed in this study. Further work, outside of the aim of the present study, is required to test the hypothesis of the presence of three different putative enzyme activities for the catabolism of L-threonine in human tissues and organs.
Fig. 2.
Correlation between L-threonine and its metabolites (ABA, Gly and Ser) concentration in blood. These figures indicate correlation between L-threonine and ABA A, Gly B and Ser C. Liner regression and R2 square calculated by Microsoft 365 version 16.96 (n=105). Abbreviation: ABA L-2-Amino butylate, Gly Glycine, R2 Coefficient of Determination, Ser l-Serine.
The present study has inherent limitations. Firstly, the clinical trial included only male volunteers. Experiments with rodents have revealed that for l-threonine, the NOAEL values are similar between males and females (Aoki et al 2014). Although experiments with animal models give useful preliminary information, clinical trials with female volunteers are necessary to confirm that the NOAELs for l-threonine are similar in men and women. Secondly, the chemical and biochemical tests were performed only at the end of the study. Although our experimental design allowed us to avoid repeated blood samplings, it gives only a view on the situation at the end of the supplementation period. With these reservations in mind, our randomized double blind controlled study allows to determine the NOAEL for supplementation with L-threonine in healthy men.
Finally, our study results of l-threonine NOAEL of 12 g/day contribute to complete previous reports which determined the NOAELs for different amino acids. The reported NOAELs from previous studies are related to the indispensable amino acids: histidine (Gheller et al 2020), methionine (Deutz et al 2017), phenylalanine (Miura et al 2021), tryptophan (Hiratsuka et al 2013); to the conditionally indispensable or dispensable amino acids: arginine (McNeal et al 2018), glycine (Inagawa et al 2006), serine (Miura et al 2021); and to the non-proteinogenic amino acids ornithine (Miura et al 2023), and citrulline (Miura et al 2023). These findings are important for optimizing the safe intake of amino acids in healthy humans.
In conclusion, this study determined the no-observed-adverse-effect-level (NOAEL) of L-threonine supplementation (4 weeks) in healthy adult males, to be 12 g/day, the highest dose. These findings are important for optimizing the safe intake of indispensable amino acids in healthy humans and should be useful for future utilization of L-threonine supplementation in individuals for which it is indicated.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
The authors are grateful to Dr. F Blachier for helpful discussion on the results of this study. We also thank S Nakano, M Terashima, and T Terashima for support on experimental design and statistical analysis.
Abbreviations
- ANOVA
Analysis of variance
- BHDQ
Brief-type self-administered diet history questionnaire
- DAA
Dispensable amino acid/non-essential amino acid
- FAO
The Food and Agriculture Organization
- jRCT
Japan Registry of clinical Trials
- IAA
Indispensable amino acid/essential amino acid
- NOAEL
Non observed adverse effect level
- PPS
Per-protocol set
- SD
Standard deviation
- Thr
Threonine
- UNU
The United Nations University
- WHO
World Health Organization
Author contributions
The authors’ responsibilities were as follows - HM, NM, MN: designed research; NM: conducted research; HM, MN: provided amino acids; NM: analyzed data and performed statistical analysis; HM, MN: not contribute to statistical analysis using raw data and diagnoses of adverse effects; HM, RE: wrote the article; HM, NM: had primary responsibility for final content; and all authors: read and approved the final manuscript.
Funding
Funding provided by Ajinomoto Co., Inc., Tokyo, Japan.
Data availability
Data from the study in a summarized format can be made available upon reasonable request.
Declarations
Conflict of interest
HM, MN are employed by Ajinomoto Co., Inc., Tokyo, Japan. NM, RE declares no conflict of interest.
Ethical approval
The procedures in the clinical trials were in accordance with ethical standards and were approved by the Ajinomoto Co., Inc ethics review committee (2022-021) and the Miura Medical Clinic ethics review committee (R2212).
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- An M, Kang EA, Han YM, Kim YS, Hong YG, Hah BS, Hong SP, Hahm KB (2019) Dietary threonine prevented stress-related mucosal diseases in rat. J Physiol Pharmacol 70(3):467–478. 10.26402/jpp.2019.3.14 [DOI] [PubMed] [Google Scholar]
- Aoki M, Ishida S, Fukuzumi H, Morishita K (2014) A 13-week feeding toxicity study of l-threonine in rats with a recovery period of 5 weeks. Fund Toxicol Sci 1(2):49–62. 10.2131/fts.1.49 [Google Scholar]
- Berlana D (2022) Parenteral nutrition overview. Nutrients 14(21):4480. 10.3390/nu14214480 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bird MI, Nunn PB, Lord LA (1984) Formation of glycine and aminoacetone from L-threonine by rat liver mitochondria. BBA-Gen Subj 802(2):29–36. 10.1016/0304-4165(84)90166-1 [DOI] [PubMed] [Google Scholar]
- Blachier F, Blais A, Elango R, Saito K, Shimomura Y, Matsumoto H et al (2021) Tolerable amounts of amino acids for human supplementation: summary and lessons, from published peer-reviewed studies. Amino Acids 53:1313–1328. 10.1007/s00726-021-03054-z [DOI] [PMC free article] [PubMed] [Google Scholar]
- Christner P, Carpousis A, Harsch M, Rosenbloom J (1975) Inhibition of the assembly and secretion of procollagen by incorporation of a threonine analogue, hydroxynorvaline. J Biol Chem 250(19):7623–7630. 10.1016/S0021-9258(19)40861-2 [PubMed] [Google Scholar]
- Crichton M, Craven D, Mackay H, Marx W, De van der Schueren M, Marshall S (2019) A systematic review, meta-analysis and meta-regression of the prevalence of protein-energy malnutrition: associations with geographical region and sex. Age Ageing 48(1):38–48. 10.1093/ageing/afy144 [DOI] [PubMed] [Google Scholar]
- Dale RA (1978) Catabolism of threonine in mammals by coupling of l-threonine 3-dehydrogenase with 2-amino-3-oxobutyrate-CoA ligase. BBA-Gen Subjects 544(3):496–503. 10.1016/0304-4165(78)90324-0 [DOI] [PubMed] [Google Scholar]
- Depaoli-Roach AA, Park IK, Cerovsky V, Csortos C, Durbin SD, Kuntz MJ et al (1994) Serine/threonine protein phosphatases in the control of cell function. Adv Enz Regul 34:199–224. 10.1016/0065-2571(94)90017-5 [DOI] [PubMed] [Google Scholar]
- Deutz NEP, Simbo SY, Ligthart-Melis GC, Cynober L, Smriga M, Engelen MP (2017) Tolerance to increased supplemented dietary intakes of methionine in healthy older adults. Am J Clin Nutr 106:675–683. 10.3945/ajcn.117.152520 [DOI] [PubMed] [Google Scholar]
- FAO, WHO, UNU, (2007) Expert consultation on amino acid requirements in human nutrition Amino acid requirements of adults. Protein and amino acid requirements in human nutrition. WHO technical report series. WHO press, Geneva, Switzerland, pp 138–139 [Google Scholar]
- Faure M, Moennoz D, Montigon F, Fay LB, Breuille D, Finot PA, Ballèvre O, Boza J (2002) Development of a rapid and convenient method to purify mucins and determine their in vivo synthesis in rats. Anal Biochem 307:244–251. 10.1016/s0003-2697(02)00048-9 [DOI] [PubMed] [Google Scholar]
- Faure M, Moennoz D, Montigon F, Mettraux C, Mercier S, Schiffrin EJ et al (2003) Mucin production and composition is altered in dextran sulfate sodium-induced colitis in rats. Digest Dis Sci 48:1366–1373. 10.1023/a:1024175629909 [DOI] [PubMed] [Google Scholar]
- Fogg FJ, Hutton DA, Jumel K, Pearson JP, Harding SE, Allen A (1996) Characterization of pig colonic mucins. Biochem J 316(3):937–942. 10.1042/bj3160937 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Food and Nutrition Board (FNB) Institute of Medicine (IOM) (2005) Dietary reference intake for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids. The National Academies Press, Washington DC, pp 682–691 [Google Scholar]
- Gheller ME, Vermeylen F, Handzlik MK, Gheller BJ, Bender E, Met-allo C et al (2020) Tolerance to graded dosages of histidine supplementation in healthy human adults. Am J Clin Nutr 112:1358–1367. 10.1093/ajcn/nqaa210 [DOI] [PubMed] [Google Scholar]
- Goudoever JB, Carnielli V, Darmaun D, Pipaon MS (2018) ESPGHAN/ESPEN/ESPR/CSPEN guidelines on pediatric parenteral nutrition: amino acids. Clin Nutr 37(6):2315–2323. 10.1016/j.clnu.2018.06.945 [DOI] [PubMed] [Google Scholar]
- Growdon JH, Nader TM, Wurtman RJ (1991) L-Threonine in the treatment of spasticity. Clin neuropharmacol 14(5):403–412. 10.1097/00002826-199110000-00003 [DOI] [PubMed] [Google Scholar]
- Gustafsson JK, Johansson MEV (2022) The role of goblet cells and mucus in intestinal homeostasis. Nat Rev Gastro Hepat 19(12):785–803. 10.1038/s41575-022-00675-x [DOI] [PubMed] [Google Scholar]
- Hamard A, Mazurais D, Boudry G, Huërou-Luron IL, Sève B, Floc’h NL (2010) A moderate threonine deficiency affects gene expression profile, paracellular permeability and glucose absorption capacity in the ileum of piglets. J Nutr Biochem 21(10):914–21. 10.1016/j.jnutbio.2009.07.004 [DOI] [PubMed] [Google Scholar]
- Hill DB, Button B, Rubinstein M, Boucher RC (2022) Physiology and pathophysiology of human airway mucus. Physiol Rev 102(4):1757–1836. 10.1152/physrev.00004.2021 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hiratsuka C, Fukuwatari T, Sano M, Saito K, Sasaki S, Shibata K (2013) Supplementing healthy women with up to 5.0 g/d of L-tryptophan has no adverse effects. J Nutr 143(6):859–866. 10.3945/jn.112.173823 [DOI] [PubMed] [Google Scholar]
- Hoover HC, Ryan JA, Anderson EJ, Fischer JE (1980) Nutritional benefits of immediate postoperative jejunal feeding of an elemental diet. AM J Surg 139(1):153–159. 10.1016/0002-9610(80)90245-7 [DOI] [PubMed] [Google Scholar]
- Inagawa K, Kawai N, Ono K, Sukegawa E, Tsubuku S, Takahashi M (2006) Assessment of acute adverse events of glycine ingestion at a high dose in human volunteers. J Urban Living Health Assoc 50(1):27–32. 10.11468/SEIKATSUEISEI.50.27 [Google Scholar]
- Irino Y, Toh R, Nagao M, Mori T, Honjo T, Hirata K et al (2016) 2-Aminobutyric acid modulates glutathione homeostasis in the myocardium. Sci Rep 6:36749. 10.1038/srep36749 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ishikawa-Takada K, Takimoto H (2018) Current protein and amino acid intakes among Japanese people: analysis of the 2012 national health and nutrition survey. Geriatr Gerontol Int 18(5):723–731. 10.1111/ggi.13239 [DOI] [PubMed] [Google Scholar]
- Kim J, Choi H (2022) The mucin protein MUCL1 regulates melanogenesis and melanoma genes in a manner dependent on threonine content. Brit J Dermatol 186(3):532–543. 10.1111/bjd.20761 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kobayashi S, Murakami K, Sasaki S, Okubo S, Hirota N, Notsu A et al (2011) Comparison of relative validity of food group intakes estimated by comprehensive and brief-type self-administered diet history questionnaires against 16 d dietary records in Japanese adults. Public Health Nutr 14(7):1200–1211. 10.1017/S1368980011000504 [DOI] [PubMed] [Google Scholar]
- Law GK, Bertolo RF, Adjiri-Awere A, Pencharz PB, Ball RO (2007) Adequate oral threonine is critical for mucin production and gut function in neonatal piglets. Am J Physiol-Gastr L 292:G1293–G1301. 10.1152/ajpgi.00221.2006 [DOI] [PubMed] [Google Scholar]
- Lee A, Patterson V (1993) A double-blind study of l-threonine in patients with spinal spasticity. Acta Neurol Scand 88(5):334–338. 10.1111/j.1600-0404.1993.tb05353.x [DOI] [PubMed] [Google Scholar]
- Li P, Wu G (2018) Roles of dietary glycine, proline, and hydroxyproline in collagen synthesis and animal growth. Amino Acids 50:29–38. 10.1007/s00726-017-2490-6 [DOI] [PubMed] [Google Scholar]
- Liu X, Beaumont M, Walker F, Chaumontet C, Andriamihaja M, Matsumoto H et al (2013) Beneficial effects of an amino acid mixture on colonic mucosal healing in rats. Inflamm Bowel Dis 19(13):2895–2905. 10.1097/01.MIB.0000435849.17263.c5 [DOI] [PubMed] [Google Scholar]
- McNeal CJ, Meininger CJ, Wilborn CD, Tekwe CD, Wu G (2018) Safety of dietary supplementation with arginine in adult humans. Amino Acids 50:1215–1229. 10.1007/s00726-018-2594-7 [DOI] [PubMed] [Google Scholar]
- Miura N, Matsumoto H, Cynober L, Stover PJ, Elango R, Kadowaki M et al (2021) Subchronic tolerance trials of graded oral supplementation with phenylalanine or serine in healthy adults. Nnutrients 13(6):1976. 10.3390/nu13061976 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miura N, Morishita K, Yasuda T, Akiduki S, Matsumoto H (2023) Subchronic tolerance trials of graded oral supplementation with ornithine hydrochloride or citrulline in healthy adults. Amino Acids 55:299–311. 10.1007/s00726-022-03227-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reeds P (2000) Dispensable and indispensable amino acids for humans. J Nutr 130(7):1835S-1840S. 10.1093/jn/130.7.1835S [DOI] [PubMed] [Google Scholar]
- Sarkar TR, McNeal CJ, Meininger CJ, Niu Y, Mallick BK, Carroll RJ, Wu G (2021) Dietary intakes of amino acids and other nutrients by adult humans. Adv Exp Med Biol 1332:211–227. 10.1007/978-3-030-74180-8_12 [DOI] [PubMed] [Google Scholar]
- Sasaki S, Takahashi T, Iitoi Y, Iwase Y, Kobayashi M, Ishihara J et al (2003) Food and nutrient intakes assessed with dietary records for the validation study of a self-administered food frequency questionnaire in JPHC study cohort I. J Epidemiol 13:S23–S50. 10.2188/jea.13.1sup_23 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schaart MW, Schierbeek H, van der School SRD, Stoll B, Burrin DG, van Goudoever JB et al (2005) Threonine utilization is high in the intestine of piglets. J Nutr 135(4):765–770. 10.1093/jn/135.4.765 [DOI] [PubMed] [Google Scholar]
- Silva KE, Huber LE, Mansilla WD, Shoveller AK, Htoo JK, Cant JP et al (2020) The effect of reduced dietary glycine and serine and supplemental threonine on growth performance, protein deposition in carcass and viscera, and skin collagen abundance of nursery pigs fed low crude protein diets. J Anim Sci 98(5):skaa157. 10.1093/jas/skaa157 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stoll B (2006) Intestinal uptake and metabolism of threonine: nutritional impact. Adv Pork Prod 17:257–263 [Google Scholar]
- Tandan R, Bromberg MB, Forshew D, Fries TJ, Badger GJ, Nau K et al (1996) A controlled trial of amino acid therapy in amyotrophic lateral sclerosis: I. Clinical, functional, and maximum isometric torque data. Neurology 47(5):1220–1226. 10.1212/wnl.47.5.1220 [DOI] [PubMed] [Google Scholar]
- Tang Q, Tan P, Ma N, Ma X (2021) Physiological functions of threonine in animals: beyond nutrition metabolism. Nutrients 13(8):2592. 10.3390/nu13082592 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wu G (2022) Amino acids: biochemistry and nutrition, 2nd edn. CRC Press Taylor and Francis Group LLC, Boca Raton, USA [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Data from the study in a summarized format can be made available upon reasonable request.