Abstract
Branched-chain amino acids (
Exhaustive exercise oxidizes the branched-chain amino acids (BCAAs;
Because there is an inadequate amount of preclinical safety information available on BCAAs,
the purpose of this study was to evaluate the subchronic toxicity of diet-incorporated
MATERIALS AND METHODS
Animals and Feeding Protocols—Study 1 (Ile)
Seventy-five 4-week-old male and 75 female Sprague-Dawley rats (Charles River Japan,
Tokyo, Japan) were housed individually in conventional stainless steel hanging cages
(Lead Engineering, Tokyo, Japan), and provided with a standard diet (CRF-1; Oriental
Yeast, Tokyo, Japan) and water ad libitum in an animal room with controlled
temperature (22°C ± 2 °C), relative humidity (55% ± 10%) and illumination (12 h
illumination per day, from 7:00
Animals and Feeding Protocols—Studies 2 (Leu) and 3 (Val)
Another 150 Sprague-Dawley rats of both genders were purchased (Charles River Japan), divided into two equally sized groups, and used for testing of Leu (Lot. no. 301FKB7) and Val (301EL12) as described above. Both Leu and Val were provided by Ajinomoto Co., Inc. (Tokyo, Japan) and mixed into a standard diet at the above-described doses (1.25%, 2.50%, and 5.00% w/w). The study was conducted in compliance with the Good Laboratory Practice Standards for Safety Studies on Drugs (Notification N. 313, March 31, 1982) and Guidelines for Toxicity Studies Required for Applications fro Approval to Manufacture (Import) Drugs (Ordinance N.1, Article N.24, September 11, 1989).
General Examination
All rats were observed twice daily (morning and afternoon) during the 13-week-long
testing period, and once daily (morning) during the 5-week-long recovery period. The
rats were weighted twice a week at a specified time (9:00
Ophthalmologic Examination
Using an ophthalmoscope, all animals were examined prior to the start of study. Animals with ocular abnormalities of external appearance of the eyes, the anterior part, the vitreous body, and the fundus oculi of the eyes were documented and not included in the study. The ophthalmologic examination was repeated in week 13 of administration on six randomly selected rats from each group, and on all rats at the end of the recovery period.
Urinalysis
Urine was collected and analyzed from all rats in weeks 5 and 13 of the administration period, and in week 5 of the recovery. Collection occurred during a 4-h period while the rats were housed in metabolic cages and provided with water, but deprived of food. Immediately thereafter, rats were given food and further additional 20-h urine samples were collected, during which both food and water were provided. The following parameters were evaluated only from the 4-h samples: pH, protein, ketone body, glucose, occult blood, bilirubin, urobilinogen (all parameters measured by Uriflet 7A; Kyoto Daiichi Kagaku, Kyoto, Japan), urine color, and sedimentation (microscopic examination). The following parameters were evaluated only from the 24-h samples: volume of urine (volumetry), specific gravity (refractometry), and electrolyte concentration.
Hematology and Blood Chemistry
Hematological examination was conducted from the blood samples collected on the day following the final administration (week 13), and at the end of recovery period from the rats deprived of food overnight prior to blood sample collection. Blood samples were collected from the abdominal aorta by laparotomy under ether anesthesia into blood collecting tubes (SB-41; Toa Medical Electronics, Tokyo, Japan) containing an anticoaugulant (EDTA-2K). Following parameters were measured, red blood cell count (RBC) (electronic counting method using Coulter 8 Item Automatic Blood Cell Analyzer T890; Japan Scientific Instrument, Tokyo, Japan), mean corpuscular volume (MCV) (electronic counting method using automatic coagulometer ACL100), hemoglobin (Hb) (cyanmethemoglobin method using automatic coagulometer ACL100) to reticulocyte ratio (Brecher method), platelet and white blood cell count (electronic counting method using automatic coagulometer ACL100), differential leucocyte count (microscopic method using May-Giemsa staining), prothrombin and activated partial thromboplastin time (PT and APTT) (clot method using automatic analyzer Monarch), and fibrinogen (thromboplastin method using automatic analyzer Monarch). Hematocrit and mean corpuscular hemoglobin were calculated from the above-measured parameters. Additional plasma parameters (GOT, GPT, and lactate dehydrogenase [LDH]) were obtained from blood samples collected from the abdominal aorta into tubes containing heparin. The plasma was acquired by centrifugation (3000 rpm, 10 min). The serum parameters (total cholesterol, triglycerides, phospholipids, total bilirubin, blood glucose, urea nitrogen, creatine, uric acid, sodium, potassium, chloride, calcium, inorganic phosphorus, and total protein) were obtained from blood samples that were allowed to stand for 30 to 60 min, and thereafter centrifuged at 3000 rpm (10 min).
Pathology and Histopathology
Femoral bone marrow samples were collected at autopsy from all rats and May-Giemsa–stained specimen were prepared and examined microscopically. The rats were sacrificed by exsanguinations from the abdominal aorta and observed for any external malformations. Then, the organs and tissues in the cephalic, thoratic, and abdominal cavities were examined macroscopically. The brain, pituitary, salivary, and thyroid glands, heart, lungs (including bronchia), liver, spleen, kidneys, adrenals, testes, prostate, ovaries, and uterus were excised and weighted. The relative organ weights were calculated from the animals’ fasting body weights. All the organs listed above, plus spinal cord, sciatic nerve, thoratic aorta, trachea, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, thymus, mesenteric lymph nodes, cervical lymph nodes, epididymides, seminal vesicles, vagina, mammary glands, skin, eyes, optic nerve, Harderian glands, sternum (bone marrow), femur (bone marrow), femoral muscle, and gross lesions were excised and fixed in phosphate-buffered formalin solution. After paraffin embedding, the excised organs and tissues were prepared for microscopic examination by sectioning and staining with hematoxylin and eosin. Representative samples were photographed.
Statistical Analysis
Data were analyzed for homogeneity of variance using Bartlett’s test. Homogenous data observed at the level of 5% (w/w) were analyzed using the parametric one-way analysis of variance (ANOVA), and the significance of differences was assessed using Scheffe’s method to compare the values between the control group and each amino acid–administered group. Heterogeneous data converted to rank-sum were analyzed using the Kruskal-Wallis nonparametric test. Any significant differences observed were further evaluated using the method of distribution free multiple comparison (Gad and Weil 1982). Means ± standard deviations (SD) are shown.
RESULTS
No deaths and no signs related to administration of the amino acids (Iso, Leu, Val) were observed during the administration period. Excoriation of the dorsal neck skin was found in two females ingesting 5.0% Ile-supplemented diet, swelling of the hindlimbs was found in one female in the control group, mal-occlusion of the incisors was observed in two males (control group, 1.25% Leuingesting group), and grypsosis of the upper jaw was observed in one female in the 2.5% Val-ingesting group. All above changes were incidental. No clinical signs were observed during recovery period.
Effects of Ile
Body weights in male and female rats in each concentration group were comparable to those recorded in controls, and no significant difference was observed (Table 1). The diet consumption of both males and females in 1.25% and 2.5% concentration groups were identical to those found in controls. The diet consumption of the males in the 5.0% concentration group was slightly lower than that of the control group during the first 3 weeks of administration; however, significant differences in daily intake were infrequently observed. The diet consumption during the rest of the administration period and during the recovery period was comparable among the groups. The average intake of Ile over the course of the study is shown in Table 2. No administration-related changes in water intake were recorded.
Ophthalmology
There were no changes that were thought to be treatment-related. In the examination at the end of the administration period, choroids atrophy was observed in one animal of each gender in the 2.5% concentration group.
Urinalysis—Week 5
No intergroup differences were observed in the quantitative parameters. In the qualitative parameters, a tendency toward an increase was observed in the number of positive incidence of urine protein (above 30 to 70 mg/dl) in the 2.5% and 5.0% concentration groups (controls, 6 rats; 2.5% group, 10 rats; 5.0% group, 16 rats), but the changes were mostly within the range observed in controls. Additionally, a tendency toward an increase in the incidence of urine sediment of phosphate salt (above slight level) was observed in the 2.5% and 5.0% groups (controls, 6 rats; 2.5% group, 9 rats; 5.0% group, 14 rats).
Urinalysis—Week 13
In the quantitative parameters, a significant decrease in the excreted sodium (controls, 2.38 ± 0.46 mEq/24 h; 5.0% group, 1.71±0.49 mEq/24 h), potassium (controls, 3.80±0.71 mEq/24 h; 5.0% group, 3.06 ± 0.71 mEq/24 h), and chloride (controls, 2.82 ± 0.50 mEq/24 h; 5.0% group, 2.07 ± 0.52 mEq/24 h) was seen in the 5.0% group, as compared to controls. Among the qualitative parameters observed in the 5.0% concentration group (males), a tendency toward an increase was seen in the number of positive incidence of urine protein (above 30 to 70 mg/dl) and positive urine glucose (controls, 1 case; 5.0% group, 7 cases) and urobilinogen (controls, 4 cases; 5.0% group, 9 cases). However, all variations were within the range observed in the control group.
Hematology and Blood Chemistry
There were no Ile-related changes in any of the hematological parameters when measured at the end of the administration period. A significant reduction of the activated thromboplastin time was observed in males in the 2.5% group (control, 16.0 ± 0.9 s; 2.5% group, 14.5 ± 1.6 s). In females of the 5.0% concentration group, significant increases in GOT (control, 57.5 ± 6.1 U/L; 5.0% group, 75.4 ± 21.0 U/L) and GPT (control, 31.5 ± 5.6 U/L; 5.0% group, 49.2 ± 21.5 U/L) activities were found. No other changes between each administration group and the control group were observed.
The changes seen at the end of the administration period were no longer observed at the end of the recovery period. Only incidental slight changes in the lymphocyte and neutrophil ratio were seen in males of the 5.0% concentration group. A significant decrease in total cholesterol (controls, 113.8 ± 19.5 mg/dl: 5.0% group, 79.8 ± 16.6 mg/dl) and phospholipids (controls, 170.5 ± 26.9 mg/dl; 5.0% group, 131.3 ± 21.8 mg/dl) was found in males of the 5.0% concentration group, but as these changes were not observed at the end of the administration period, they were considered to be accidental ones.
Pathology and Histopathology
There were no significant treatment-related pathologies; minor changes were extremely few and dose independent. At the end of the administration period, there were no treatment-related changes in absolute (Tables 3A, 3B) or relative organ weights. A significant increase in the absolute weight of the salivary gland (unilateral) was observed in the males of the 5.0% concentration group at the end of recovery period, but the change was judged incidental, as it was not observed at the end of the administration period.
Histopathological alterations at the end of the administration and recovery periods were not related to the treatment. For the purpose of demonstration of incidence and severity, the findings are listed below. Focal hemorrhage of lungs was found at the end of the administration period in three males in the control group, two males in the 1.25% concentration group, and one male in each of the 2.5% and 5.0% concentration groups. Erosion in the glandular layer of the stomach wall was recorded in one female in each of the control and 2.5% and 5.0% concentration groups. Only single case of the erosion was found in a control animal at the end of the recovery period. Ileal granulomas in Peyer’s patches were found at the end of administration period in one female each of the 2.5% and 5.0% concentration groups. At the end of the administration period, cellular infiltration in the lamina propria mucosae of the cecum was recorded in one female in the control group, three males and one female in the 1.25% group, three females in the 2.5% concentration group, and one male and two females in the 5.0% concentration group. Mild cell necrosis of the hepatocytes (one female in the 5.0% concentration group) and microgranulomas (one male and two females in the control group, two males and three females in each of the 1.25% and 2.5% concentration groups and one male and two females in the 5.0% concentration group) were recorded at the end of the administration period. Only microgranulomas pathology was found at the end of the recovery period (one male in the control group and one male and three females in the 5.0% concentration group). Fibrosis in the pancreatic islets was observed in two males in the 2.5% concentration group and one male in the 5.0% concentration group at the end of the administration period. At the end of the recovery period, only a single case of this pathology was found (control group). Ectopic thymic tissue at the end of the administration period characterized two males and one female in the 2.5% concentration group and two females in the 5.0% concentration group. Arteritis of kidneys was found in one male in the 5.0% group and cyst was recorded in two females in the 5.0% concentration group. Atrophy of the somniferous tubules of the testis was observed in one animal in the 2.5% concentration group, in which edematous changes in the testis were seen macroscopically. Interstitial cellular infiltration was observed in one rat in the 5.0% concentration group. Calcific granulomas in the conjunctiva of the eye were found in one male rat from the 2.5% concentration group at the end of the administration period. Hemorrhage in the vitreous body of the eye was recorded in the eye of a single male rat from the 5.0% concentration group at the end of the recovery period. Degeneration of femoral muscle fibers was observed in one male rat of the 5.0% concentration group. Finally, arthritis characterized by indurations of the intertarsal joints of the hind limbs was seen in one female in the control group. No abnormalities were observed in the organs and tissues other than those listed above.
Effects of Val
There was no significant difference in body weight between males in each concentration group and females in the 1.25% and 2.5% concentration groups, and the controls. The body weights in females in the 5.0% concentration group were lower than those of the control group after week 7 of administration, and showed significantly low values almost constantly until the end of the administration period (Table 1). However, the body weights were comparable among all four groups during the recovery period. In females of the 5.0% concentration group, a significantly increased value for body weight gain was observed during the recovery period. The total diet consumption of males in all groups, and females in 1.25% concentration group, were identical to those found in controls. In females in the 2.5% concentration group, decreased diet consumption was observed sporadically throughout the administration period. In males and females in the 5.0% concentration group, a significantly decreased diet consumption was observed on day 3, and continuously thereafter (consumption volume was approximately 92% of that found in controls). The average intake of Val over the course of the study is shown in Table 2. No administration-related changes in water intake were recorded, besides slightly increased water intake in females fed 5.0% diet on a single day (day 7 of the administration). In females in the 1.25% concentration group, a significantly high intake of water consumption was noted on a single day (day 20 of the administration).
Ophthalmology
No abnormalities in any of the tested rats were recorded.
Urinalysis—Week 5
No intergroup differences were observed in the quantitative and qualitative parameters.
Urinalysis—Week 13
There were no changes that were thought to be treatment related. In males in the 1.25% concentration group, a significant increase in the specific gravity of urine (controls, 1.051 ± 0.01; 1.25% group, 1.063 ± 0.01) was noted, but the change was minor and dose-independent.
Hematology and Blood Chemistry
Examinations at the end of both the administration and the recovery period did not reveal any significant administration-related changes in hematological parameters and blood chemistry. In the examination conducted at the end of the recovery period, a significant increase in the γ-globulin fraction ratio was noted in females in the 5.0% concentration group (controls, 6.8% ± 0.9%; 5.0% group, 8.2% ± 0.9%). As the change was not observed at the end of the administration period, it was not considered to be a specific one.
Pathology and Histopathology
A small number of pathologies in three organs (liver, kidney, and stomach) at the end of the administration period were judged to be incidental. Absolute organ weights recorded at the end of the administration period are depicted in Tables 3A (males) and 3B (females).
Infrequent histopathology findings were observed at the end of the administration period in the following organs: the cerebrum, heart, stomach, cecum, liver, pancreas, kidneys, prostate, and the eye. None of these changes was administration related.
Effects of Leu
Treatment-related malfunctions were not observed in any of the concentration groups at the end of the administration or the recovery period. In one male in the 1.25% concentration group, malocclusion was seen from days 38 to 84 of administration, but the finding was not dose dependent and was considered to be an incidental one. Body weights of males and females in all tested concentration groups during the administration and recovery periods were comparable to those recorded in controls (Table 1). The diet consumption of males fed 2.5% diet increased singularly on days 45 and 59 of the administration. No other significant changes were observed in the total diet consumption at the end of the administration period. Similarly, no group differences in diet intake were observed during the recovery period, and no water intake changes were found throughout the study. The average intake of Leu in both male and female rats is shown in Table 2.
Ophthalmology
Opacity in the anterior portion (cornea) of the left eye was observed in one male in each of the 1.25% and 5.0% concentration groups, and abnormal running of vessels (retina) was found in one male in the control group at the end of the administration period.
Urinalysis—Week 5 and Week 13
No intergroup differences were observed in the quantitative and qualitative parameters.
Hematology and Blood Chemistry
Examinations at the end of the administration period did not reveal any significant administration-related changes in hematological parameters and blood chemistry. A significant decrease in myeloblasts was found in females in the 1.25% concentration group (controls, 0.2% ± 0.1%; 1.25% group, 0.1% ± 0.1%), and a significantly high value of M/E ratio (controls, 1.2% ± 0.3%; 2.5% group, 1.5% ± 0.2%) was seen in females in the 2.5% concentration group. Those changes were not reproduced at the end of the recovery period.
At the end of the recovery period, a significant increase in the level of inorganic phosphorus (controls, 5.9 ± 0.4 mg/dl; 5.0% group, 6.7 ± 0.3 nmg/dl) and albumin ratio (controls, 43.3% ± 1.4%; 5.0% group, 45.3% ± 1.6%) and a significantly low value of α1-globulin ratio (controls, 22.2% ± 2.8%; 5.0% group, 18.9% ± 2.1%) were noted in males in the 5.0% concentration group. In females of this concentration group, significant increases in the levels of creatine (controls, 0.59 ± 0.04 mg/dl; 5.0% group, 0.64 ± 0.03 mg/dl) and total protein (controls, 6.5% ± 0.11 g/dl; 5.0% group, 7.1 ± 0.5 g/dl) were observed. As none of the above changes was seen at the end of the administration period, they were thought to be incidental.
Pathology and Histopathology
A small number of abnormalities in two organs (kidney and stomach) at the end of the administration period were judged to be incidental. At the end of the administration period, absolute organ weights recorded are depicted in Tables 3A (males) and 3B (females). A significant increase in the adrenal gland weight was recorded in the 2.5% concentration group; however, the increase was recoverable and dose independent. At the end of the recovery period, there were significant increases in the absolute weight of the pituitary (controls, 15.1 ± 1.8 mg; 5.0% group 17.9 ± 1.1 mg) in females in the 5.0% concentration groups. Because these changes were not observed at the end of the administration period, they were considered incidental.
DISCUSSION
No deaths were observed throughout the administration and recovery periods. The effects on body weight gain and diet intake differed among the three amino acid tested. No effect of Leu on diet consumption was noticed, whereas Ile at 5.0% (w/w) slightly decreased diet consumption during the first 3 weeks of administration in female rats. As the decrease was not accompanied by body weight reduction, it was not considered to be specifically related to Ile administration. Val at 5.0% (w/w) partly, but significantly, reduced diet consumption volume in the female rats from the week 7 onwards. A significant drop in body weight accompanied this reduction. The above changes were recoverable, because no group differences were found at the end of the 5-week-long recovery period. It is not clear why the three BCAAs had different effects on diet consumption and body weight gain. The bulk of BCAA metabolism begins in the muscle; other organs are significantly less involved, with only Leu being significantly processed also in the liver by a mitochondrial pathway (Paul and Adibi 1984). The existence of multiple metabolic centers for Leu might indicate that a high dietary load of Leu is more efficiently metabolized than Ile, and especially Val. In addition, Val and Ile serve as stronger gluconeogenic agents than Leu (Jungas, Halperin, and Brosnan 1992), indicating a weaker Leu-induced satiety signaling. Yet, it is important to note that all presently observed body weight and diet consumption changes were recoverable and relatively weak.
The random findings seen in water intake and ophthalmology at the end of the administration period were not considered toxicologically relevant to effects of Ile, Val, and Leu, due to the absence of consistency, and because of the speedy recovery. Importantly, none of the observed changes was outside of the range seen in controls. Only a single organ weight change was recorded in the females of the 2.5% Leu group; however, this change was regarded as unrelated to the Leu diet, because it was dose independent and disappeared after the 5 weeks of the recovery.
Ile mixed into a standard diet at the highest tested dose (5.0% w/w) affected the urine electrolytes, protein, ketone bodies, urine glucose and urobilinogen, yet the changes remained mostly within the range observed in controls. These results differ from the observations made by Kawabe and colleagues (1996), who reported significantly elevated urine volume and pH in F344 rats fed for 13 weeks on 8.0% Ile diet. Both the use of F344 rat strain and high dose of Ile (8.0% w/w) could be the main reasons for the above effects. BCAA supplementation to humans that contained Ile at 2.85 g/person (Yamamoto et al. 2000) did not affect the urine parameters.
Evaluations of blood chemistry and hematology documented no toxicology-relevant, dose-dependent, and unrecoverable effects of Val and Leu, and only minor increases of plasma GPT and GOP were found in the highest Ile concentration group, indicating a slight elevation of hepatic enzyme activities or minor hyperlipidemia during the high-dose loading with Ile. The changes disappeared within the recovery period.
We estimate the no-observed-adverse-effect level (NOAEL) for Ile at 2.5% for both genders (male, 1.57 ± 0.06 g/kg/day; females, 1.65 ± 0.10 g/kg/day), Val at 5.0% for males (3.23 ± 0.14 g/kg/day) and 2.5% for females (1.85 ± 0.06 g/kg/day), and Leu at 5.0% for both genders (males, 3.33 ± 0.10 g/kg/day: females, 3.84 ± 0.26 g/kg/day). The results of this study demonstrate the absence of any significant toxicological findings with BCAAs at a dose equal or lower than 2.5% (w/w) given orally in a diet during 13 weeks of continuous exposure.