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Toxicological evaluation of l -proline in a 90-day feeding study with Fischer 344 rats
Regulatory Toxicology and Pharmacology 58 (2010) 114–120
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Toxicological evaluation of L-proline in a 90-day feeding study with Fischer 344 rats Y. Tada *, N. Yano, H. Takahashi, K. Yuzawa, H. Ando, Y. Kubo, A. Nagasawa, N. Ohashi, A. Ogata, D. Nakae Department of Environmental Health and Toxicology, Tokyo Metropolitan Institute of Public Health, Japan
a r t i c l e
i n f o
Article history: Received 1 March 2010 Available online 4 May 2010 Keywords: L-proline Toxicological evaluation Subchronic Feeding study Fischer 344 rat
a b s t r a c t L-proline (L-Pro) is a non-essential amino acid, and has become widely used as supplements and health foods, recently. A subchronic oral toxicity study of L-Pro was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.625%, 1.25%, 2.5% and 5.0% of L-Pro for 90 days. No treatment-related clinical signs and mortality were noted. We observed no clear treatment-related effects with regard to body weight, food intake or urinalysis data. The average daily water intakes of the treated female groups were significantly increased compared to the controls. The hematology (red blood cell parameter) and serum biochemistry (glucose, blood urea nitrogen, creatinine or uric acid) of the treated male and/or female groups were lower than those of the control groups. However, these changes were lacked dose-dependence, and no abnormalities were found in corresponding pathological findings. In conclusion, the no-observed-adverse-effect-level (NOAEL) for L-Pro was determined to be a dietary dose of 5.0% (2772.9 mg/kg body weight/day for males and 3009.3 mg/kg body weight/day for females) under the present experimental conditions. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction L-Pro ((S)-2-pyrrolidinecarboxylic acid) [CAS No. 147-85-3] is a non-essential amino acid that can be formed from and converted to glutamic acid. It is incorporated into tissue proteins and can then be hydroxylated to form hydroxyproline. Both proline and hydroxyproline are found in large quantities in collagen (Dietary Reference Intakes, 2005). L-Pro has recently become widely used as an ingredient of supplements, health foods and cosmetics. Under these circumstances, potential risks of L-Pro must be well assessed and appropriately managed. There are, however, few reports available regarding the toxicity of L-Pro. In the reviews of Harper et al. (1970) in regard to the effects of disproportionate intake levels of amino acids, 5% of L-Pro has small depressions of growth and food intake of rats fed low-protein diets. The acute toxicity data of L-Pro, including 50% lethal dose (LD50) of L-Pro, is not recorded in Registry of Toxic Effects of Chemical Substances (RTECS, 2009). The only study in humans on effects of the long-term oral administration of proline was a clinical study on the efficacy of proline (isomer not specified) to alter the progression of the gyrate atrophy of the choroid and retina (Haya-
* Corresponding author. Address: Department of Environmental Health and Toxicology, Tokyo Metropolitan Institute of Public Health, 3-24-1 Hyakunin’cho, Shin’juku, Tokyo 169-0073, Japan. Fax: +81 3 3368 4060. E-mail address: [email protected] (Y. Tada). 0273-2300/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2010.04.011
saka et al., 1985). Four patients (aged 4–32 years) were treated with doses of supplementary proline (2–10 g/day) and vitamin B6 (120–600 mg/day) between for 2–5 years. No overt adverse effects were reported (Hayasaka et al., 1985). On the other hand, it is known that L-Pro has a specific function or induces toxicity in central nervous system (Takemoto and Semba, 2006; Delwing et al., 2003; Nadler et al., 1988). In the literature, D-proline (D-Pro; 50 mg/kg body weight/day) is toxic as causing liver and kidney lesions (Kampel et al., 1990). The information available concerning the potential risks of L-Pro is thus limited, and considering the human exposure situation, the risks need to be assessed in an urgent but careful manner by well-established protocols. This study was therefore conducted to examine influence of L-Pro when administered to male and female Fischer 344 rats for 90 days in the diet. In Japan, the Ministry of Health, Labour and Welfare (MHLW) has responsibility for risk assessment and management of food additives. In 1995, MHLW amended the Food Sanitation Law for food safety/hygiene, and the designation system was applied to all additives including the natural food additives. Because the natural food additives had already been marketed or had been used for the date of the amendment of the Food Sanitation Law in 1995, they were put on the List of Existing Food Additives (2007). However, the risks of all existing food additives have not been assessed. MHLW gave the research fund to deal with this issue, and this study was conducted as a part of this effort.
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observed twice daily, and clinical signs and mortality (if any) were recorded. Body weight and food and water intakes were monitored weekly.
2. Materials and methods 2.1. Ethical considerations This study was performed in accordance with the Guidelines for Designation of Food Additives and for Revision of Standards for Use of Food Additives released by the MHLW (Standards and Evaluation Division Department of Food Safety, 1996). The experimental protocol was approved prior to its execution by our in-house committee, which monitored every step during the experimentation for its scientific and ethical propriety, with strict obedience to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, Japanese Government Animal Protection and Management Law, Japanese Government Notification on Feeding and Safekeeping of Animals and Guidelines for Animal Experimentation (Japanese Association for Laboratory Animal Science, 1987). 2.2. Test chemical and diet preparation L-Pro (Lot No. LSR7-8-45; purity >99.8%) was supplied by Ajinomoto Co., Inc. (Kanagawa, Japan). It was admixed into a modified AIN-93G powder diet (Oriental Yeast Co., Ltd., Tokyo, Japan) to produce concentrations of 0% (control), 0.625%, 1.25%, 2.5% or 5.0% every 4 weeks (three time preparation for experimentation period) ; the diet composition is shown in Table 1. The diet was kept at 4 °C and was given to the rat every week. The L-Pro content in all experimental diets was analyzed at their preparation, and the actual values were 6.57 ± 0.48, 13.30 ± 0.39, 27.18 ± 0.71 and 54.04 ± 1.34 g/kg diet for the 0.625%, 1.25%, 2.5% and 5.0% doses, respectively. After keeping the 5.0% diet for 7 days at room temperature (22–24 °C), or 30 days at 4 °C, the contents of L-Pro were found to remain fairly stable, with values of 54.20 g/kg diet, or 56.29 g/kg diet, respectively.
2.4. Animal sacrifice and assessments At the end of the experimentation period of 90 days, all rats were deprived of food (but not water) overnight, and fresh urine samples were obtained for urinalysis of urobilinogen, occult blood, bilirubin, ketone, glucose, protein, pH and nitrous acid using test papers (N-multisticks, Siemens Medical Solutions Diagnostics Ltd., Tokyo, Japan). All rats were then lightly anesthetized and sacrificed by exsanguination after collecting blood samples via the abdominal aorta. Using the blood samples and subsequently prepared sera, hematological and serological examinations were performed. Hematological examination was carried out using an automatic analyzer (Sysmex KX-21NV; Sysmex Co., Hyogo, Japan) for the red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit level (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count (WBC) and platelet count (PLT). Differential counts of leukocytes were made by a light microscopic observation of smeared specimens stained with a routine May-Grünwald-Giemsa protocol. Serum biochemistry determination was performed with an automatic analyzer (TBA120FR; Toshiba Medical Systems Co., Tokyo, Japan) for the levels of total protein (TP), albumin (ALB), albumin/globulin ratio (A/G), glucose (GLU), total cholesterol (T-CHO), triglyceride (TG), total bilirubin (T-BIL), blood urea nitrogen (BUN), creatinine (CRE), uric acid (UA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), sodium (Na), potassium (K), chlorine (Cl) and calcium (Ca). At terminal sacrifice, complete
350 2.3. Animals and treatments
300
Ingredient
(g/kg dry matter)
b-Cornstarch Casein (vitamin free) Soybean oil (no additives) Cellulose powder Mineral mix (AIN-93G) Vitamin mix (AIN-93G)
629.486–X 200.000 70.000 50.000 35.000 10.000 3.000
L-proline
200 150 100 50 0 200
0
1
2
3
4
5
2
3
4
5
6
7
8
9
10
11 12
6
7
8
9
10
11 12
Female
150 100
Table 1 Composition of a modified AIN-93G diet containing L-proline.
L-cystine Choline bitartrate (41.1% choline) Tert-butylhydroquinone
250
Body weight (g)
A total of 53 male and 53 female specific pathogen-free Fischer 344 (F344/DuCrlCrlj) rats were purchased from Charles River Japan, Inc. (Kanagawa, Japan) at 5 weeks of age, and acclimatized to the control diet for 1 week before the start of the experiment. The rats were housed individually in stainless steel cages; kept under controlled conditions of temperature (23 ± 2 °C), relative humidity (55 ± 10%) and ventilation (more than 10 times/hour) with a 12-h light/dark cycle; and allowed free access to food and drinking water throughout both the acclimation and experimentation periods. After confirming normal health status at the end of the acclimation period, 50 rats of each sex were selected for use (3 rats of each sex being omitted), randomly allocated to 5 groups each consisting of 10 rats and given the control or experimental diets for 90 days. During the experimentation period, the rats were
Male
2.500 0.014 X
50 0
0
1
Weeks Fig. 1. Weekly changes in average body weights of Fischer 344 rats (10 animals for each group) given L-proline at dietary levels of 0% (control, open circles), 0.625% (closed triangles), 1.25% (closed circles), 2.5% (asterisks) or 5.0% (closed squares) for 90 days.
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necropsies were performed on all animals. For each animal, the body weight was determined, and gross observations were made. The brain, thyroids (with parathyroids), heart, spleen, liver, adrenal glands, kidneys, testes, ovaries and uterus were then excised, and their absolute and relative weights were determined. These organs as well as the pituitary gland, eyes, harderian glands, thymus, nasal cavity, trachea, lungs (including bronchi, fixed by inflation with fixative), salivary glands, tongue, esophagus, stomach, duodenum, jejunum, ileum, caecum, rectum, pancreas, urinary bladder, skin with mammary gland, skeletal muscle, epididymides, seminal vesicles, prostate, preputials, oviducts, vagina, lymph nodes (submandibular and mesenteric), thoracic aorta, sciatic nerve, spinal cords
(cervical, mid-thoracic and lumbar), bones (femur and sternum) with bone marrows, Zymbal’s glands and all gross lesions of each animal were fixed in 10% neutral buffered formalin. Paraffinembedded sections were then routinely prepared. All organs were histopathologically examined using these sections stained via a routine hematoxylin and eosin (HE) protocol. 2.5. Statistical analysis For numerical data such as body weight and hematological data, statistical evaluation of equality of means between the control and treated group values was assessed by Bartlett’s test. Homogeneity
Table 2 Average daily food, water and chemical intakes in Fischer 344 rats given L-proline for 90 days. Item
Dietary levels of L-proline (%) 0 (control)
0.625
1.25
2.5
5.0
Male Effective number of rats Food intake (g/kg body weight/day) Water intake (mL/kg body weight/day) Chemical intake (mg/kg body weight/day)
10 57.6 ± 16.3a 58.1 ± 14.7 –
10 56.4 ± 16.7 60.5 ± 15.5 352.4 ± 104.6
10 56.8 ± 16.5 60.3 ± 15.4 710.3 ± 205.7
10 56.8 ± 16.1 57.8 ± 15.3 1419.2 ± 401.8
10 55.5 ± 14.7 57.9 ± 15.1 2772.9 ± 733.6
Female Effective number of rats Food intake (g/kg body weight/day) Water intake (mL/kg body weight/day) Chemical intake (mg/kg body weight/day)
10 61.5 ± 14.4 73.5 ± 16.6 –
10 62.4 ± 14.9 79.7 ± 16.9* 389.8 ± 93.2
10 62.7 ± 15.6 76.0 ± 16.4 784.3 ± 194.5
10 62.2 ± 15.5 78.6 ± 15.5* 1554.2 ± 387.4
10 60.2 ± 11.9 81.3 ± 17.2* 3009.3 ± 595.8
a *
Values are the means ± standard deviations. Significantly different from the corresponding control values (p < 0.05, Dunnett’s test).
Table 3 Hematology in Fischer 344 rats given L-proline for 90 days. Item
a
0 (control)
0.625
1.25
2.5
5.0
Male Effective number of rats RBC ( 104/lL) HGB (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL) WBC ( 102/lL) Lymphocyte ( 102/lL) Neutrophil ( 102/lL) Eosinophil ( 102/lL) Basophil ( 102/lL) Monocyte ( 102/lL) Others ( 102/lL) PLT ( 104/lL)
10a 909.9 ± 15.4b 15.3 ± 0.3 46.3 ± 1.0 50.9 ± 0.3 16.8 ± 0.1 33.1 ± 0.3 40.8 ± 7.6 27.1 ± 3.6 12.7 ± 5.9 0.3 ± 0.3 0.0 ± 0.0 0.6 ± 0.6 0.0 ± 0.0 55.5 ± 9.7
8 895.9 ± 22.7 14.9 ± 0.3* 45.3 ± 1.1 50.6 ± 0.4 16.7 ± 0.3 33.0 ± 0.4 36.4 ± 10.9 26.4 ± 8.1 9.3 ± 3.7 0.3 ± 0.3 0.0 ± 0.0 0.5 ± 0.4 0.0 ± 0.0 54.1 ± 12.8
9 889.6 ± 17.6 14.9 ± 0.3* 45.2 ± 0.9* 50.8 ± 0.4 16.8 ± 0.2 33.0 ± 0.3 40.3 ± 5.7 27.7 ± 4.0 11.9 ± 4.9 0.3 ± 0.3 0.0 ± 0.0 0.5 ± 0.5 0.0 ± 0.0 59.6 ± 4.0
9 904.6 ± 15.1 15.1 ± 0.3 45.9 ± 0.7 50.8 ± 0.4 16.8 ± 0.2 33.0 ± 0.2 44.3 ± 11.3 28.3 ± 6.6 15.0 ± 7.1 0.4 ± 0.3 0.0 ± 0.0 0.7 ± 1.1 0.0 ± 0.0 53.1 ± 17.3
10 922.6 ± 18.3 15.4 ± 0.2 46.9 ± 0.9 50.9 ± 0.4 16.7 ± 0.2 32.8 ± 0.2 51.0 ± 8.6 33.9 ± 9.3 16.2 ± 3.6 0.3 ± 0.4 0.0 ± 0.0 0.5 ± 0.4 0.0 ± 0.0 55.1 ± 9.7
Female Effective number of rats RBC ( 104/lL) HGB (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL) WBC ( 102/mL) Lymphocyte ( 102/lL) Neutrophil ( 102/lL) Eosinophil ( 102/lL) Basophil ( 102/lL) Monocyte ( 102/lL) Others ( 102/lL) PLT ( 104/lL)
10 907.4 ± 20.7 16.1 ± 0.4 47.6 ± 1.2 52.4 ± 0.3 17.8 ± 0.1 33.9 ± 0.2 27.9 ± 5.3 23.2 ± 5.2 4.4 ± 2.7 0.2 ± 0.2 0.0 ± 0.0 0.2 ± 0.2 0.0 ± 0.0 62.8 ± 4.2
10 889.8 ± 32.3 15.7 ± 0.6 46.8 ± 1.8 52.6 ± 0.3 17.6 ± 0.2 33.5 ± 0.3 30.4 ± 5.8 24.3 ± 4.7 5.4 ± 1.9 0.3 ± 0.2 0.0 ± 0.0 0.3 ± 0.3 0.0 ± 0.0 61.1 ± 3.2
10 894.8 ± 18.7 15.7 ± 0.3* 46.7 ± 0.9 52.2 ± 0.2 17.5 ± 0.1* 33.6 ± 0.2 27.1 ± 3.8 21.9 ± 3.4 4.7 ± 1.7 0.3 ± 0.3 0.0 ± 0.0 0.2 ± 0.2 0.0 ± 0.0 62.1 ± 4.1
10 897.7 ± 15.2 15.8 ± 0.2 47.0 ± 0.7 52.4 ± 0.3 17.6 ± 0.2 33.6 ± 0.4 26.3 ± 3.2 19.2 ± 3.6 6.4 ± 2.4 0.3 ± 0.4 0.0 ± 0.0 0.3 ± 0.4 0.0 ± 0.0 59.7 ± 11.2
10 913.8 ± 25.1 16.0 ± 0.4 47.7 ± 1.2 52.2 ± 0.3 17.5 ± 0.3* 33.5 ± 0.5 33.9 ± 9.3 27.2 ± 9.6 6.3 ± 2.7 0.2 ± 0.2 0.0 ± 0.0 0.2 ± 0.2 0.0 ± 0.0 62.6 ± 4.5
Effective numbers smaller than 10 occurred due to the failure of taking samples. Values are the means ± standard deviations. Significantly different from the corresponding control values (p < 0.05, Dunnett’s test).
b *
Dietary levels of L-proline (%)
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of variance was then analyzed by a one-way analysis of variance, and differences between the control and treated group values were evaluated by Dunnett’s test. If the Bartlett’s test was significant, the data were subjected to the Kruskal–Wallis test and the Dunnett’s type rank sum test (Gad and Weil, 1994). For contingent data such as incidences of histopathological lesions and urinalysis, differences between the control and treated group values were evaluated by the Fisher’s exact probability test (Gad and Weil, 1994). Statistical processing was conducted using StatLight software (Yukms Ltd., Tokyo, Japan). In all cases, statistical significance was set at p-value less than 0.05.
and treated groups (for both sexes). The average daily water intakes of the 0.625%, 2.5% and 5.0% females were significantly increased compared to the controls (Table 2). The average L-Pro intakes of each group were listed in Table 2. 3.2. Urinalysis Comparison of the results of urinalysis between the control and treated groups revealed no treatment-related changes in the analyzed parameters. 3.3. Hematology and serum biochemistry
3. Results 3.1. General findings No rats died or became moribund until the end of the experiment. All treated rats showed no abnormal signs for general appearance, attitude (such as excitement or inanimation), behavior (such as exploration or grooming) or nervous system (such as tremor or convulsion) compared to the control rats during the study. There were no significant differences in average body weights (Fig. 1) or average daily food intakes (Table 2) between the control
In the male rats, the HGB of the 0.625% and 1.25% groups and HCT of the 1.25% group were significantly lower than those of the control groups (Table 3). In the female rats, the HGB of the 1.25% group and MCH of the 1.25% and 5.0% groups were significantly lower than those of the control groups. The morphological findings and differential counts of leukocytes showed no significant effects in any of the treated groups. Serum biochemistry demonstrated that the values for GLU of the 0.625% or greater males, BUN of the 0.625%, 1.25% and 5.0% females, CRE of the 5.0% males and females, UA of the 2.5% or greater males, and 0.625% and 5.0% females, and K of the 5.0% males were
Table 4 Serum biochemistry in Fischer 344 rats given L-proline for 90 days. Item
a *
Dietary levels of L-proline (%) 0 (control)
0.625
1.25
2.5
5.0
Male Effective number of rats TP (g/dL) ALB (g/dL) A/G GLU (mg/dL) T-CHO (mg/dL) TG (mg/dL) T-BIL (mg/dL) BUN (mg/dL) CRE (mg/dL) UA (mg/dL) AST (U/L) ALT (U/L) GGT (U/L) ALP (U/L) Na (mmol/L) K (mmol/L) Cl (mmol/L) Ca (mg/dL)
10 6.82 ± 0.09a 4.45 ± 0.05 1.88 ± 0.05 147.9 ± 10.1 76.8 ± 7.2 88.7 ± 42.2 0.01 ± 0.01 15.2 ± 0.8 0.28 ± 0.02 1.21 ± 0.15 88.9 ± 12.5 47.4 ± 7.6 0.00 ± 0.67 403.4 ± 28.8 142.1 ± 1.3 4.75 ± 0.17 100.5 ± 1.3 10.6 ± 0.1
9 6.86 ± 0.18 4.47 ± 0.09 1.87 ± 0.05 137.2 ± 6.7* 79.0 ± 6.2 97.7 ± 21.3 0.01 ± 0.01 15.5 ± 1.5 0.27 ± 0.02 1.18 ± 0.26 88.7 ± 11.5 47.1 ± 7.1 0.00 ± 0.67 379.3 ± 15.0 141.8 ± 2.2 4.87 ± 0.58 101.1 ± 1.7 10.7 ± 0.1
10 6.89 ± 0.13 4.50 ± 0.06 1.89 ± 0.08 137.3 ± 8.1* 80.0 ± 8.7 88.7 ± 28.6 0.02 ± 0.01 15.6 ± 1.6 0.27 ± 0.02 1.13 ± 0.16 87.4 ± 11.5 43.8 ± 5.5 0.00 ± 0.47 385.2 ± 25.1 142.8 ± 2.2 4.63 ± 0.27 101.5 ± 1.4 10.8 ± 0.2
10 6.79 ± 0.19 4.43 ± 0.10 1.88 ± 0.05 132.1 ± 10.3* 75.7 ± 9.8 80.1 ± 40.5 0.02 ± 0.01 14.9 ± 1.5 0.26 ± 0.02 0.99 ± 0.18* 97.1 ± 22.9 47.7 ± 11.0 0.00 ± 0.47 383.3 ± 29.9 143.6 ± 1.9 4.57 ± 0.33 101.7 ± 1.5 10.7 ± 0.1
10 6.83 ± 0.20 4.50 ± 0.10 1.94 ± 0.06 137.1 ± 7.1* 81.4 ± 7.4 91.7 ± 31.2 0.02 ± 0.01 15.1 ± 1.6 0.25 ± 0.02* 0.98 ± 0.15* 93.1 ± 6.4 46.0 ± 4.8 0.40 ± 0.70 391.8 ± 25.3 143.9 ± 1.8 4.43 ± 0.16* 100.9 ± 1.9 10.8 ± 0.2*
Female Effective number of rats TP (g/dL) ALB (g/dL) A/G GLU (mg/dL) T-CHO (mg/dL) TG (mg/dL) T-BIL (mg/dL) BUN (mg/dL) CRE (mg/dL) UA (mg/dL) AST (U/L) ALT (U/L) GGT (U/L) ALP (U/L) Na (mmol/L) K (mmol/L) Cl (mmol/L) Ca (mg/dL)
10 6.62 ± 0.13 4.37 ± 0.09 1.94 ± 0.06 109.3 ± 28.4 72.6 ± 8.3 18.3 ± 7.4 0.02 ± 0.00 19.2 ± 1.0 0.31 ± 0.03 1.47 ± 0.41 80.0 ± 3.1 33.4 ± 2.7 0.80 ± 0.42 307.0 ± 25.5 145.2 ± 1.3 4.30 ± 0.28 107.7 ± 1.4 10.5 ± 0.2
10 6.71 ± 0.25 4.40 ± 0.14 1.91 ± 0.07 104.2 ± 14.7 73.5 ± 6.3 21.7 ± 9.6 0.01 ± 0.01 17.0 ± 1.1* 0.29 ± 0.03 1.12 ± 0.17* 77.5 ± 5.5 32.9 ± 4.4 0.50 ± 0.53 318.3 ± 39.9 145.8 ± 2.0 4.50 ± 0.34 106.9 ± 2.6 10.5 ± 0.3
10 6.69 ± 0.18 4.39 ± 0.11 1.91 ± 0.05 101.6 ± 17.1 73.1 ± 6.4 15.9 ± 4.1 0.02 ± 0.01 17.3 ± 1.3* 0.29 ± 0.02 1.16 ± 0.13 77.0 ± 2.9 31.7 ± 2.8 0.70 ± 0.48 297.4 ± 35.8 145.0 ± 1.3 4.16 ± 0.16 108.0 ± 1.8 10.5 ± 0.2
10 6.56 ± 0.22 4.37 ± 0.10 2.00 ± 0.09 97.3 ± 14.6 73.0 ± 3.4 13.6 ± 2.7 0.01 ± 0.01 18.8 ± 1.4 0.29 ± 0.03 1.15 ± 0.18 81.0 ± 8.8 32.6 ± 3.8 0.70 ± 0.48 286.4 ± 21.1 146.2 ± 0.5 4.11 ± 0.15 108.4 ± 1.2 10.4 ± 0.3
10 6.60 ± 0.19 4.36 ± 0.12 1.94 ± 0.07 97.3 ± 13.6 69.7 ± 8.3 12.8 ± 2.0 0.01 ± 0.00 17.3 ± 1.2* 0.28 ± 0.02* 1.04 ± 0.10* 85.2 ± 22.3 34.2 ± 2.8 0.60 ± 0.70 297.6 ± 22.9 148.1 ± 1.6* 4.19 ± 0.30 108.8 ± 1.9 10.6 ± 0.2
Values are the means ± standard deviations. Significantly different from the corresponding control values (p < 0.05, Dunnett’s test).
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significantly lower than those of the control groups (Table 4). Ca of the 5.0% males and Na of the 5.0% females were significantly higher than those of the control groups (Table 4). 3.4. Pathology Final body weight of the 0.625% females was significantly higher than that of the control group (Table 5). In the male rats, the relative spleen weights of the 2.5% or greater groups and relative kidney weight of the 5.0% group were significantly higher than those of the control groups (Table 5). No treatment-related macroscopic changes were observed in any organs of either sex. In the histopathological study, there were no treatment-related changes in any organs (for both sexes), whereas sporadic spontaneous lesions were observed in the control and treated animals. In the spleen or the kidney, relative weights were significantly higher, there were no treatment-related histopathological changes compared to the control rats (Fig. 2).
4. Discussion In the present 90-day feeding study of L-Pro in Fischer 344 rats, no deaths, treatment-related body weight changes or clinical signs were observed in either sex. Sauberlich (1961) reported the growth-depressing effect of amino acids when fed in low-protein diets to weanling rats. In their report, with 5% of L-Pro, the growth of rats was depressed slightly. The addition of 3% of L-Pro in 14.3% casein basal diet had smaller and inconsistent effects on growth rate with male weanling rats (Abernathy and Miller, 1965). Muramatsu et al. (1971) demonstrated no or slight growth depression in Donryu male growing rats fed a 10% casein diet containing 5% L-Pro for 3 weeks. On the other hand, female Sprague–Dawley rats given L-Pro in drinking water for 1 month (mean dose 50 mg/kg body weight/day) did not exhibit any adverse effects (Kampel et al., 1990). In this study, no growth or food-depressing effects were recognized in proline-treated rats. Water intakes were higher in the female treated groups (0.625%, 2.5% and 5.0%), but these were
Table 5 Absolute and relative organ weights in Fischer 344 rats given L-proline for 90 days. Item
Dietary levels of L-proline (%) 0 (control)
0.625
1.25
2.5
5.0
Male Effective number of rats Final body weight (g)
10 304.9 ± 13.4b
10 308.1 ± 13.5
10 307.3 ± 12.6
10 296.2 ± 22.8
10 296.6 ± 16.9
Absolute organ weight (mg) Brain Thyroidsa Heart Spleen Liver Adrenals Kidneys Testes
2007.7 ± 26.2 14.3 ± 1.2 848.8 ± 41.4 594.9 ± 38.1 6767.2 ± 382.1 38.5 ± 1.8 1654.6 ± 71.0 3044.2 ± 93.5
2000.2 ± 55.1 14.9 ± 1.9 856.4 ± 46.4 608.1 ± 21.5 6954.3 ± 595.1 37.8 ± 4.3 1715.2 ± 86.1 3024.9 ± 109.0
2009.3 ± 25.9 14.5 ± 1.6 865.4 ± 52.9 618.6 ± 27.9 6973.8 ± 327.0 39.0 ± 2.0 1683.5 ± 72.9 3051.5 ± 111.3
1981.2 ± 43.0 14.1 ± 1.61.2 841.1 ± 55.3 610.7 ± 50.4 6551.9 ± 805.0 37.8 ± 3.6 1648.0 ± 108.7 2932.7 ± 250.8
2001.0 ± 20.5 14.4 ± 1.3 856.4 ± 47.5 625.0 ± 43.0 6898.4 ± 676.2 40.0 ± 2.7 1713.5 ± 96.2 3002.0 ± 118.0
Relative organ weight (mg/100 g body weight) Brain 659.6 ± 31.2 Thyroids 4.7 ± 0.3 Heart 278.6 ± 12.1 Spleen 195.1 ± 9.6 Liver 2218.9 ± 59.5 Adrenals 12.6 ± 0.7 Kidneys 543.2 ± 26.1 Testes 999.5 ± 38.4
649.9 ± 24.6 4.8 ± 0.5 278.0 ± 10.8 197.5 ± 5.9 2254.1 ± 109.4 12.3 ± 1.1 556.6 ± 11.8 982.6 ± 39.2
654.8 ± 27.8 4.7 ± 0.5 281.5 ± 10.7 201.4 ± 6.6 2269.8 ± 70.1 12.7 ± 0.7 548.1 ± 18.7 994.1 ± 45.0
671.6 ± 41.5 4.8 ± 0.4 284.3 ± 9.6 206.4 ± 11.7* 2206.6 ± 132.5 12.8 ± 1.1 557.0 ± 16.0 991.9 ± 78.5
676.3 ± 34.2 4.8 ± 0.3 288.9 ± 9.5 210.6 ± 6.3* 2321.5 ± 110.2 13.5 ± 1.3 578.1 ± 22.5* 1014.3 ± 57.3
Female Effective number of rats Final body weight (g)
10 153.6 ± 7.8
10 166.5 ± 10.0*
10 164.2 ± 10.7
10 157.1 ± 11.4
10 156.9 ± 12.8
Absolute organ weight (mg) Brain Thyroids Heart Spleen Liver Adrenals Kidneys Ovaries Uterus
1835.0 ± 43.8 9.4 ± 1.1 494.9 ± 33.0 332.6 ± 19.9 3191.7 ± 220.2 40.3 ± 3.0 923.5 ± 46.1 46.3 ± 4.1 334.7 ± 41.6
1858.5 ± 30.3 11.2 ± 2.0 526.6 ± 34.6 363.7 ± 27.5* 3419.5 ± 240.9* 42.0 ± 3.1 982.6 ± 62.2* 50.8 ± 5.6 413.0 ± 64.6*
1847.0 ± 44.3 10.1 ± 1.3 528.9 ± 34.7 360.8 ± 25.9 3390.7 ± 165.1 40.9 ± 3.2 983.6 ± 40.9* 50.9 ± 5.5 384.7 ± 59.6
1836.6 ± 25.8 9.2 ± 0.9 507.5 ± 34.2 349.4 ± 27.1 3197.4 ± 198.1 40.8 ± 2.3 950.8 ± 38.1 47.6 ± 4.6 378.0 ± 62.1
1853.2 ± 23.8 10.5 ± 0.6* 527.1 ± 29.9 352.0 ± 24.2 3246.4 ± 164.4 40.5 ± 1.9 955.0 ± 44.3 48.3 ± 3.7 368.2 ± 62.2
1119.8 ± 63.6 6.7 ± 1.4 316.5 ± 14.2 218.4 ± 6.8 2054.1 ± 74.2 25.3 ± 1.5 590.5 ± 20.8 30.5 ± 2.6 248.2 ± 36.4
1128.1 ± 59.7 6.2 ± 0.7 322.6 ± 19.4 219.8 ± 9.1 2068.5 ± 88.3 25.0 ± 1.7 600.2 ± 27.1 31.1 ± 3.4 234.8 ± 36.8
1174.2 ± 79.7 5.9 ± 0.8 323.6 ± 18.2 222.7 ± 13.0 2037.9 ± 71.8 26.0 ± 1.4 606.8 ± 27.4 30.3 ± 2.2 240.7 ± 35.8
1187.4 ± 88.9 6.7 ± 0.5 337.4 ± 26.5 224.7 ± 11.4 2074.5 ± 93.4 25.9 ± 2.2 610.6 ± 33.3 30.9 ± 3.0 234.5 ± 32.2
Relative organ weight (mg/100 g body weight) Brain 1197.3 ± 59.1 Thyroids 6.1 ± 0.6 Heart 322.4 ± 16.2 Spleen 217.0 ± 15.7 Liver 2079.9 ± 131.6 Adrenals 26.3 ± 2.4 Kidneys 602.4 ± 37.0 Ovaries 30.1 ± 2.0 Uterus 218.5 ± 30.7 a
Thyroids were weighed after the fixation. Values are the means ± standard deviations. Significantly different from the corresponding control values (p < 0.05, Dunnett’s test).
b *
Y. Tada et al. / Regulatory Toxicology and Pharmacology 58 (2010) 114–120
119
Fig. 2. Representative histology of the spleen (A and B) and kidney (C and D) of male rats given 0 (control: A and C) and 5.0% (B and D) of L-proline in the diet for 90 days. There were no treatment-related histopathological changes. HE, 100.
slight changes and lacked dose-dependence. These were probably due to the taste of diets containing L-Pro and thus without toxicological significance. In the present L-Pro-administered groups, statistically significant changes were detected in some hematological parameters, such as HGB (low in the 0.625% males and 1.25% both sexes), HCT (low in the 1.25% males) and MCH (low in the 1.25% and 5.0% females). However, these hematological changes were slight, and lacked dose-dependence, and no abnormalities were observed in the microscopically assessed red blood cell figures in the blood smear or on histopathological examination of the hematopoietic organs. The observed hematological changes can therefore be considered incidental and not treatment-related. The serum GLU (all treated males), BUN (0.625%, 1.25% and 5.0% females), CRE (5.0% both sexes) and UA (0.625% females, 2.5% males and 5.0% both sexes) levels were significantly lower than those of the control groups. However, the values in L-Pro-administered groups were within the range of the historic control values (Mitruka and Rawnsley, 1981). Moreover, no corresponding pathological findings were observed. It is thus conceivable that the changes are toxicologically insignificant, even if they were treatment-related. The relative spleen (2.5% or greater males) and kidney (5.0% male) weights were significantly higher than those of the control groups. The histological findings of the spleen (2.5% or greater males) showed no abnormalities such as extramedullary hematopoiesis, lymphoid hyperplasia, pigmentation, fibrosis, red pulp hyperplasia or atrophy of lymphoid follicles. The hematological study revealed no significant differences between the control and the 2.5% or greater males. In the kidney weight, the increased changes were slight and lacked dose-dependence, and no abnor-
malities were observed in the urinalysis and histopathology. Moreover, the absolute spleen and kidney weights showed no significant differences between the control and the treated groups. It is suggested that the organ weight changes are toxicologically insignificant, even if they were treatment-related. In the literature, L-Pro administration in the drinking water (50 mg/kg body weight) for 1 month did not result in any histological changes in liver and kidney, whereas D-Pro (50 mg/kg body weight) treated rats had severe pathological changes of periportal fibrosis and necrosis of liver cells, and severe renal tubular dystrophy and necrosis (Kampel et al., 1990). In this study, L-Pro produced no treatment-related functional or morphological liver or kidney injury in male and female rats. It is thus likely that the liver and kidney toxicity of proline depends on its optical isomerism, namely, D-isomer-specific. Hyperprolinemia is an inherited metabolic disorder, and is a condition which occurs when the amino acid proline is not broken down properly by the enzymes, proline oxidase or pyrroline-5-carboxylate dehydrogense, causing a build up of proline in the body. Genetically hyperprolinemic PRO/Re mice have 7.1 times higher concentration of proline in the brain than control CD-1 mice and 10.8 times higher concentration of proline in plasma (Baxter et al., 1985). Nadler et al. (1988) described that intrahippocampal injections of L-Pro non-selectively destroyed pyramidal and granule cells. L-Pro destroyed far more hippocampal neurons than D-Pro, in keeping with its greater neuroexcitatory potency. Its excitotoxic action may be related to the neurological and cognitive deficits associated with hyperprolinemia (Nadler et al., 1988). Delwing et al. (2003) investigated the in vivo and in vitro effects of proline on some parameters of oxidative stress in rat cerebral cortex. Ten-day-old Wistar rats received one subcutaneous
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injection of proline (12.8 lmol/g body weight), while control rats received saline in the same volumes. The results indicate that proline induces oxidative stress in the brain, which may be related, at least in part, to the neurological dysfunction observed in hyperprolinemia. In this study, even in the 5% L-Pro group, no clinical signs or symptoms suggesting neurotoxicity of L-Pro (such as excitement, inanimation, tremor, convulsion) were observed, and the pathological studies showed no histopathological changes in central nervous system of proline-treated rats. In conclusion, the no-observed-adverse-effect-level (NOAEL) for L-Pro was determined to be at least a dietary dose of 5.0% (2772.9 mg/kg body weight/day for males and 3009.3 mg/kg body weight/day for females) under the present experimental conditions.
Acknowledgments The authors thank Dr. Hideki Wanibuchi (Department of Pathology, Osaka City University Medical School, Osaka, Japan) for helpful suggestions. This work was supported in part by a Health and Labour Sciences Research Grant (awarded to Dai Nakae) from the MHLW.
References Abernathy, R.P., Miller, J., 1965. Effects of imbalances or antagonisms among nonessential amino acids on growth and nitrogen utilization by rats. J. Nutr. 86, 231–235. Baxter, C.F., Baldwin, R.A., Davis, J.L., Flood, J.F., 1985. High proline levels in the brains of mice as related to specific learning deficits. Pharmacol. Biochem. Behav. 22, 1053–1059.
Delwing, D., Bavaresco, C.S., Wannmacher, C.M., Wajner, M., Dutra-Filho, C.S., Wyse, A.T., 2003. Proline induces oxidative stress in cerebral cortex of rats. Int. J. Dev. Neurosci. 21, 105–110. Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids (Macronutrients), 2005. Proline. Available from: (accessed 20.02.2010). Gad, S.C., Weil, C.S., 1994. Statistics for toxicologist. In: Hayes, A.W. (Ed.), Principles and Methods of Toxicology, third ed. Raven Press, New York, pp. 221–274. Harper, A.E., Benevenga, N.J., Wohlhueter, R.M., 1970. Effects of ingestion of disproportionate amounts of amino acids. Physiol. Rev. 50, 428–558. Hayasaka, S., Saito, T., Nakajima, H., Takahashi, O., Mizuno, K., Tada, K., 1985. Clinical trials of vitamin B6 and proline supplementation for gyrate atrophy of the choroid and retina. Br. J. Ophthalmol. 69, 283–290. Japanese Association for Laboratory Animal Science, 1987. Guideline for animal experimentation. Exp. Anim. 23, 285–288 (in Japanese). Kampel, D., Kupferschmidt, R., Lubec, G., 1990. Toxicity of D-proline. In: Lubec, G., Rosenthal, G.A. (Eds.), Amino Acids: Chemistry, Biology, and Medicine. ESCOM, Leiden, The Netherlands, pp. 1164–1171. List of Existing Food Additives, 2007. Available from: (accessed 20.02.2010). Mitruka, B.M., Rawnsley, H.M., 1981. Clinical biochemical reference values. In: Clinical Biochemical and Hematological Reference Values in Normal Experimental Animals and Normal Humans, second ed. Masson Publishing, New York, pp. 153–314. Muramatsu, K., Odagiri, H., Morishita, S., Takeuchi, H., 1971. Effect of excess levels of individual amino acids on growth of rats fed casein diets. J. Nutr. 101, 1117– 1126. Nadler, J.V., Wang, A., Hakim, A., 1988. Toxicity of L-proline toward rat hippocampal neurons. Brain Res. 456, 168–172. RTECS, 2009. Registry of Toxic Effects of Chemical Substances, Croner-i CHEMBANK. Sauberlich, H.E., 1961. Studies on the toxicity and antagonism of amino acids for weanling rats. J. Nutr. 75, 61–72. Standards and Evaluation Division Department of Food Safety, Eika No. 29, 1996. Available from: (accessed 20.02.2010). Takemoto, Y., Semba, R., 2006. Immunohistochemical evidence for the localization of neurons containing the putative transmitter L-proline in rat brain. Brain Res. 16 (1073–1074), 311–315.
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Toxicological evaluation of L-proline in a 90-day feeding study with Fischer 344 rats Y. Tada *, N. Yano, H. Takahashi, K. Yuzawa, H. Ando, Y. Kubo, A. Nagasawa, N. Ohashi, A. Ogata, D. Nakae Department of Environmental Health and Toxicology, Tokyo Metropolitan Institute of Public Health, Japan
a r t i c l e
i n f o
Article history: Received 1 March 2010 Available online 4 May 2010 Keywords: L-proline Toxicological evaluation Subchronic Feeding study Fischer 344 rat
a b s t r a c t L-proline (L-Pro) is a non-essential amino acid, and has become widely used as supplements and health foods, recently. A subchronic oral toxicity study of L-Pro was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.625%, 1.25%, 2.5% and 5.0% of L-Pro for 90 days. No treatment-related clinical signs and mortality were noted. We observed no clear treatment-related effects with regard to body weight, food intake or urinalysis data. The average daily water intakes of the treated female groups were significantly increased compared to the controls. The hematology (red blood cell parameter) and serum biochemistry (glucose, blood urea nitrogen, creatinine or uric acid) of the treated male and/or female groups were lower than those of the control groups. However, these changes were lacked dose-dependence, and no abnormalities were found in corresponding pathological findings. In conclusion, the no-observed-adverse-effect-level (NOAEL) for L-Pro was determined to be a dietary dose of 5.0% (2772.9 mg/kg body weight/day for males and 3009.3 mg/kg body weight/day for females) under the present experimental conditions. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction L-Pro ((S)-2-pyrrolidinecarboxylic acid) [CAS No. 147-85-3] is a non-essential amino acid that can be formed from and converted to glutamic acid. It is incorporated into tissue proteins and can then be hydroxylated to form hydroxyproline. Both proline and hydroxyproline are found in large quantities in collagen (Dietary Reference Intakes, 2005). L-Pro has recently become widely used as an ingredient of supplements, health foods and cosmetics. Under these circumstances, potential risks of L-Pro must be well assessed and appropriately managed. There are, however, few reports available regarding the toxicity of L-Pro. In the reviews of Harper et al. (1970) in regard to the effects of disproportionate intake levels of amino acids, 5% of L-Pro has small depressions of growth and food intake of rats fed low-protein diets. The acute toxicity data of L-Pro, including 50% lethal dose (LD50) of L-Pro, is not recorded in Registry of Toxic Effects of Chemical Substances (RTECS, 2009). The only study in humans on effects of the long-term oral administration of proline was a clinical study on the efficacy of proline (isomer not specified) to alter the progression of the gyrate atrophy of the choroid and retina (Haya-
* Corresponding author. Address: Department of Environmental Health and Toxicology, Tokyo Metropolitan Institute of Public Health, 3-24-1 Hyakunin’cho, Shin’juku, Tokyo 169-0073, Japan. Fax: +81 3 3368 4060. E-mail address: [email protected] (Y. Tada). 0273-2300/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2010.04.011
saka et al., 1985). Four patients (aged 4–32 years) were treated with doses of supplementary proline (2–10 g/day) and vitamin B6 (120–600 mg/day) between for 2–5 years. No overt adverse effects were reported (Hayasaka et al., 1985). On the other hand, it is known that L-Pro has a specific function or induces toxicity in central nervous system (Takemoto and Semba, 2006; Delwing et al., 2003; Nadler et al., 1988). In the literature, D-proline (D-Pro; 50 mg/kg body weight/day) is toxic as causing liver and kidney lesions (Kampel et al., 1990). The information available concerning the potential risks of L-Pro is thus limited, and considering the human exposure situation, the risks need to be assessed in an urgent but careful manner by well-established protocols. This study was therefore conducted to examine influence of L-Pro when administered to male and female Fischer 344 rats for 90 days in the diet. In Japan, the Ministry of Health, Labour and Welfare (MHLW) has responsibility for risk assessment and management of food additives. In 1995, MHLW amended the Food Sanitation Law for food safety/hygiene, and the designation system was applied to all additives including the natural food additives. Because the natural food additives had already been marketed or had been used for the date of the amendment of the Food Sanitation Law in 1995, they were put on the List of Existing Food Additives (2007). However, the risks of all existing food additives have not been assessed. MHLW gave the research fund to deal with this issue, and this study was conducted as a part of this effort.
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observed twice daily, and clinical signs and mortality (if any) were recorded. Body weight and food and water intakes were monitored weekly.
2. Materials and methods 2.1. Ethical considerations This study was performed in accordance with the Guidelines for Designation of Food Additives and for Revision of Standards for Use of Food Additives released by the MHLW (Standards and Evaluation Division Department of Food Safety, 1996). The experimental protocol was approved prior to its execution by our in-house committee, which monitored every step during the experimentation for its scientific and ethical propriety, with strict obedience to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, Japanese Government Animal Protection and Management Law, Japanese Government Notification on Feeding and Safekeeping of Animals and Guidelines for Animal Experimentation (Japanese Association for Laboratory Animal Science, 1987). 2.2. Test chemical and diet preparation L-Pro (Lot No. LSR7-8-45; purity >99.8%) was supplied by Ajinomoto Co., Inc. (Kanagawa, Japan). It was admixed into a modified AIN-93G powder diet (Oriental Yeast Co., Ltd., Tokyo, Japan) to produce concentrations of 0% (control), 0.625%, 1.25%, 2.5% or 5.0% every 4 weeks (three time preparation for experimentation period) ; the diet composition is shown in Table 1. The diet was kept at 4 °C and was given to the rat every week. The L-Pro content in all experimental diets was analyzed at their preparation, and the actual values were 6.57 ± 0.48, 13.30 ± 0.39, 27.18 ± 0.71 and 54.04 ± 1.34 g/kg diet for the 0.625%, 1.25%, 2.5% and 5.0% doses, respectively. After keeping the 5.0% diet for 7 days at room temperature (22–24 °C), or 30 days at 4 °C, the contents of L-Pro were found to remain fairly stable, with values of 54.20 g/kg diet, or 56.29 g/kg diet, respectively.
2.4. Animal sacrifice and assessments At the end of the experimentation period of 90 days, all rats were deprived of food (but not water) overnight, and fresh urine samples were obtained for urinalysis of urobilinogen, occult blood, bilirubin, ketone, glucose, protein, pH and nitrous acid using test papers (N-multisticks, Siemens Medical Solutions Diagnostics Ltd., Tokyo, Japan). All rats were then lightly anesthetized and sacrificed by exsanguination after collecting blood samples via the abdominal aorta. Using the blood samples and subsequently prepared sera, hematological and serological examinations were performed. Hematological examination was carried out using an automatic analyzer (Sysmex KX-21NV; Sysmex Co., Hyogo, Japan) for the red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit level (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count (WBC) and platelet count (PLT). Differential counts of leukocytes were made by a light microscopic observation of smeared specimens stained with a routine May-Grünwald-Giemsa protocol. Serum biochemistry determination was performed with an automatic analyzer (TBA120FR; Toshiba Medical Systems Co., Tokyo, Japan) for the levels of total protein (TP), albumin (ALB), albumin/globulin ratio (A/G), glucose (GLU), total cholesterol (T-CHO), triglyceride (TG), total bilirubin (T-BIL), blood urea nitrogen (BUN), creatinine (CRE), uric acid (UA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), sodium (Na), potassium (K), chlorine (Cl) and calcium (Ca). At terminal sacrifice, complete
350 2.3. Animals and treatments
300
Ingredient
(g/kg dry matter)
b-Cornstarch Casein (vitamin free) Soybean oil (no additives) Cellulose powder Mineral mix (AIN-93G) Vitamin mix (AIN-93G)
629.486–X 200.000 70.000 50.000 35.000 10.000 3.000
L-proline
200 150 100 50 0 200
0
1
2
3
4
5
2
3
4
5
6
7
8
9
10
11 12
6
7
8
9
10
11 12
Female
150 100
Table 1 Composition of a modified AIN-93G diet containing L-proline.
L-cystine Choline bitartrate (41.1% choline) Tert-butylhydroquinone
250
Body weight (g)
A total of 53 male and 53 female specific pathogen-free Fischer 344 (F344/DuCrlCrlj) rats were purchased from Charles River Japan, Inc. (Kanagawa, Japan) at 5 weeks of age, and acclimatized to the control diet for 1 week before the start of the experiment. The rats were housed individually in stainless steel cages; kept under controlled conditions of temperature (23 ± 2 °C), relative humidity (55 ± 10%) and ventilation (more than 10 times/hour) with a 12-h light/dark cycle; and allowed free access to food and drinking water throughout both the acclimation and experimentation periods. After confirming normal health status at the end of the acclimation period, 50 rats of each sex were selected for use (3 rats of each sex being omitted), randomly allocated to 5 groups each consisting of 10 rats and given the control or experimental diets for 90 days. During the experimentation period, the rats were
Male
2.500 0.014 X
50 0
0
1
Weeks Fig. 1. Weekly changes in average body weights of Fischer 344 rats (10 animals for each group) given L-proline at dietary levels of 0% (control, open circles), 0.625% (closed triangles), 1.25% (closed circles), 2.5% (asterisks) or 5.0% (closed squares) for 90 days.
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necropsies were performed on all animals. For each animal, the body weight was determined, and gross observations were made. The brain, thyroids (with parathyroids), heart, spleen, liver, adrenal glands, kidneys, testes, ovaries and uterus were then excised, and their absolute and relative weights were determined. These organs as well as the pituitary gland, eyes, harderian glands, thymus, nasal cavity, trachea, lungs (including bronchi, fixed by inflation with fixative), salivary glands, tongue, esophagus, stomach, duodenum, jejunum, ileum, caecum, rectum, pancreas, urinary bladder, skin with mammary gland, skeletal muscle, epididymides, seminal vesicles, prostate, preputials, oviducts, vagina, lymph nodes (submandibular and mesenteric), thoracic aorta, sciatic nerve, spinal cords
(cervical, mid-thoracic and lumbar), bones (femur and sternum) with bone marrows, Zymbal’s glands and all gross lesions of each animal were fixed in 10% neutral buffered formalin. Paraffinembedded sections were then routinely prepared. All organs were histopathologically examined using these sections stained via a routine hematoxylin and eosin (HE) protocol. 2.5. Statistical analysis For numerical data such as body weight and hematological data, statistical evaluation of equality of means between the control and treated group values was assessed by Bartlett’s test. Homogeneity
Table 2 Average daily food, water and chemical intakes in Fischer 344 rats given L-proline for 90 days. Item
Dietary levels of L-proline (%) 0 (control)
0.625
1.25
2.5
5.0
Male Effective number of rats Food intake (g/kg body weight/day) Water intake (mL/kg body weight/day) Chemical intake (mg/kg body weight/day)
10 57.6 ± 16.3a 58.1 ± 14.7 –
10 56.4 ± 16.7 60.5 ± 15.5 352.4 ± 104.6
10 56.8 ± 16.5 60.3 ± 15.4 710.3 ± 205.7
10 56.8 ± 16.1 57.8 ± 15.3 1419.2 ± 401.8
10 55.5 ± 14.7 57.9 ± 15.1 2772.9 ± 733.6
Female Effective number of rats Food intake (g/kg body weight/day) Water intake (mL/kg body weight/day) Chemical intake (mg/kg body weight/day)
10 61.5 ± 14.4 73.5 ± 16.6 –
10 62.4 ± 14.9 79.7 ± 16.9* 389.8 ± 93.2
10 62.7 ± 15.6 76.0 ± 16.4 784.3 ± 194.5
10 62.2 ± 15.5 78.6 ± 15.5* 1554.2 ± 387.4
10 60.2 ± 11.9 81.3 ± 17.2* 3009.3 ± 595.8
a *
Values are the means ± standard deviations. Significantly different from the corresponding control values (p < 0.05, Dunnett’s test).
Table 3 Hematology in Fischer 344 rats given L-proline for 90 days. Item
a
0 (control)
0.625
1.25
2.5
5.0
Male Effective number of rats RBC ( 104/lL) HGB (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL) WBC ( 102/lL) Lymphocyte ( 102/lL) Neutrophil ( 102/lL) Eosinophil ( 102/lL) Basophil ( 102/lL) Monocyte ( 102/lL) Others ( 102/lL) PLT ( 104/lL)
10a 909.9 ± 15.4b 15.3 ± 0.3 46.3 ± 1.0 50.9 ± 0.3 16.8 ± 0.1 33.1 ± 0.3 40.8 ± 7.6 27.1 ± 3.6 12.7 ± 5.9 0.3 ± 0.3 0.0 ± 0.0 0.6 ± 0.6 0.0 ± 0.0 55.5 ± 9.7
8 895.9 ± 22.7 14.9 ± 0.3* 45.3 ± 1.1 50.6 ± 0.4 16.7 ± 0.3 33.0 ± 0.4 36.4 ± 10.9 26.4 ± 8.1 9.3 ± 3.7 0.3 ± 0.3 0.0 ± 0.0 0.5 ± 0.4 0.0 ± 0.0 54.1 ± 12.8
9 889.6 ± 17.6 14.9 ± 0.3* 45.2 ± 0.9* 50.8 ± 0.4 16.8 ± 0.2 33.0 ± 0.3 40.3 ± 5.7 27.7 ± 4.0 11.9 ± 4.9 0.3 ± 0.3 0.0 ± 0.0 0.5 ± 0.5 0.0 ± 0.0 59.6 ± 4.0
9 904.6 ± 15.1 15.1 ± 0.3 45.9 ± 0.7 50.8 ± 0.4 16.8 ± 0.2 33.0 ± 0.2 44.3 ± 11.3 28.3 ± 6.6 15.0 ± 7.1 0.4 ± 0.3 0.0 ± 0.0 0.7 ± 1.1 0.0 ± 0.0 53.1 ± 17.3
10 922.6 ± 18.3 15.4 ± 0.2 46.9 ± 0.9 50.9 ± 0.4 16.7 ± 0.2 32.8 ± 0.2 51.0 ± 8.6 33.9 ± 9.3 16.2 ± 3.6 0.3 ± 0.4 0.0 ± 0.0 0.5 ± 0.4 0.0 ± 0.0 55.1 ± 9.7
Female Effective number of rats RBC ( 104/lL) HGB (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL) WBC ( 102/mL) Lymphocyte ( 102/lL) Neutrophil ( 102/lL) Eosinophil ( 102/lL) Basophil ( 102/lL) Monocyte ( 102/lL) Others ( 102/lL) PLT ( 104/lL)
10 907.4 ± 20.7 16.1 ± 0.4 47.6 ± 1.2 52.4 ± 0.3 17.8 ± 0.1 33.9 ± 0.2 27.9 ± 5.3 23.2 ± 5.2 4.4 ± 2.7 0.2 ± 0.2 0.0 ± 0.0 0.2 ± 0.2 0.0 ± 0.0 62.8 ± 4.2
10 889.8 ± 32.3 15.7 ± 0.6 46.8 ± 1.8 52.6 ± 0.3 17.6 ± 0.2 33.5 ± 0.3 30.4 ± 5.8 24.3 ± 4.7 5.4 ± 1.9 0.3 ± 0.2 0.0 ± 0.0 0.3 ± 0.3 0.0 ± 0.0 61.1 ± 3.2
10 894.8 ± 18.7 15.7 ± 0.3* 46.7 ± 0.9 52.2 ± 0.2 17.5 ± 0.1* 33.6 ± 0.2 27.1 ± 3.8 21.9 ± 3.4 4.7 ± 1.7 0.3 ± 0.3 0.0 ± 0.0 0.2 ± 0.2 0.0 ± 0.0 62.1 ± 4.1
10 897.7 ± 15.2 15.8 ± 0.2 47.0 ± 0.7 52.4 ± 0.3 17.6 ± 0.2 33.6 ± 0.4 26.3 ± 3.2 19.2 ± 3.6 6.4 ± 2.4 0.3 ± 0.4 0.0 ± 0.0 0.3 ± 0.4 0.0 ± 0.0 59.7 ± 11.2
10 913.8 ± 25.1 16.0 ± 0.4 47.7 ± 1.2 52.2 ± 0.3 17.5 ± 0.3* 33.5 ± 0.5 33.9 ± 9.3 27.2 ± 9.6 6.3 ± 2.7 0.2 ± 0.2 0.0 ± 0.0 0.2 ± 0.2 0.0 ± 0.0 62.6 ± 4.5
Effective numbers smaller than 10 occurred due to the failure of taking samples. Values are the means ± standard deviations. Significantly different from the corresponding control values (p < 0.05, Dunnett’s test).
b *
Dietary levels of L-proline (%)
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of variance was then analyzed by a one-way analysis of variance, and differences between the control and treated group values were evaluated by Dunnett’s test. If the Bartlett’s test was significant, the data were subjected to the Kruskal–Wallis test and the Dunnett’s type rank sum test (Gad and Weil, 1994). For contingent data such as incidences of histopathological lesions and urinalysis, differences between the control and treated group values were evaluated by the Fisher’s exact probability test (Gad and Weil, 1994). Statistical processing was conducted using StatLight software (Yukms Ltd., Tokyo, Japan). In all cases, statistical significance was set at p-value less than 0.05.
and treated groups (for both sexes). The average daily water intakes of the 0.625%, 2.5% and 5.0% females were significantly increased compared to the controls (Table 2). The average L-Pro intakes of each group were listed in Table 2. 3.2. Urinalysis Comparison of the results of urinalysis between the control and treated groups revealed no treatment-related changes in the analyzed parameters. 3.3. Hematology and serum biochemistry
3. Results 3.1. General findings No rats died or became moribund until the end of the experiment. All treated rats showed no abnormal signs for general appearance, attitude (such as excitement or inanimation), behavior (such as exploration or grooming) or nervous system (such as tremor or convulsion) compared to the control rats during the study. There were no significant differences in average body weights (Fig. 1) or average daily food intakes (Table 2) between the control
In the male rats, the HGB of the 0.625% and 1.25% groups and HCT of the 1.25% group were significantly lower than those of the control groups (Table 3). In the female rats, the HGB of the 1.25% group and MCH of the 1.25% and 5.0% groups were significantly lower than those of the control groups. The morphological findings and differential counts of leukocytes showed no significant effects in any of the treated groups. Serum biochemistry demonstrated that the values for GLU of the 0.625% or greater males, BUN of the 0.625%, 1.25% and 5.0% females, CRE of the 5.0% males and females, UA of the 2.5% or greater males, and 0.625% and 5.0% females, and K of the 5.0% males were
Table 4 Serum biochemistry in Fischer 344 rats given L-proline for 90 days. Item
a *
Dietary levels of L-proline (%) 0 (control)
0.625
1.25
2.5
5.0
Male Effective number of rats TP (g/dL) ALB (g/dL) A/G GLU (mg/dL) T-CHO (mg/dL) TG (mg/dL) T-BIL (mg/dL) BUN (mg/dL) CRE (mg/dL) UA (mg/dL) AST (U/L) ALT (U/L) GGT (U/L) ALP (U/L) Na (mmol/L) K (mmol/L) Cl (mmol/L) Ca (mg/dL)
10 6.82 ± 0.09a 4.45 ± 0.05 1.88 ± 0.05 147.9 ± 10.1 76.8 ± 7.2 88.7 ± 42.2 0.01 ± 0.01 15.2 ± 0.8 0.28 ± 0.02 1.21 ± 0.15 88.9 ± 12.5 47.4 ± 7.6 0.00 ± 0.67 403.4 ± 28.8 142.1 ± 1.3 4.75 ± 0.17 100.5 ± 1.3 10.6 ± 0.1
9 6.86 ± 0.18 4.47 ± 0.09 1.87 ± 0.05 137.2 ± 6.7* 79.0 ± 6.2 97.7 ± 21.3 0.01 ± 0.01 15.5 ± 1.5 0.27 ± 0.02 1.18 ± 0.26 88.7 ± 11.5 47.1 ± 7.1 0.00 ± 0.67 379.3 ± 15.0 141.8 ± 2.2 4.87 ± 0.58 101.1 ± 1.7 10.7 ± 0.1
10 6.89 ± 0.13 4.50 ± 0.06 1.89 ± 0.08 137.3 ± 8.1* 80.0 ± 8.7 88.7 ± 28.6 0.02 ± 0.01 15.6 ± 1.6 0.27 ± 0.02 1.13 ± 0.16 87.4 ± 11.5 43.8 ± 5.5 0.00 ± 0.47 385.2 ± 25.1 142.8 ± 2.2 4.63 ± 0.27 101.5 ± 1.4 10.8 ± 0.2
10 6.79 ± 0.19 4.43 ± 0.10 1.88 ± 0.05 132.1 ± 10.3* 75.7 ± 9.8 80.1 ± 40.5 0.02 ± 0.01 14.9 ± 1.5 0.26 ± 0.02 0.99 ± 0.18* 97.1 ± 22.9 47.7 ± 11.0 0.00 ± 0.47 383.3 ± 29.9 143.6 ± 1.9 4.57 ± 0.33 101.7 ± 1.5 10.7 ± 0.1
10 6.83 ± 0.20 4.50 ± 0.10 1.94 ± 0.06 137.1 ± 7.1* 81.4 ± 7.4 91.7 ± 31.2 0.02 ± 0.01 15.1 ± 1.6 0.25 ± 0.02* 0.98 ± 0.15* 93.1 ± 6.4 46.0 ± 4.8 0.40 ± 0.70 391.8 ± 25.3 143.9 ± 1.8 4.43 ± 0.16* 100.9 ± 1.9 10.8 ± 0.2*
Female Effective number of rats TP (g/dL) ALB (g/dL) A/G GLU (mg/dL) T-CHO (mg/dL) TG (mg/dL) T-BIL (mg/dL) BUN (mg/dL) CRE (mg/dL) UA (mg/dL) AST (U/L) ALT (U/L) GGT (U/L) ALP (U/L) Na (mmol/L) K (mmol/L) Cl (mmol/L) Ca (mg/dL)
10 6.62 ± 0.13 4.37 ± 0.09 1.94 ± 0.06 109.3 ± 28.4 72.6 ± 8.3 18.3 ± 7.4 0.02 ± 0.00 19.2 ± 1.0 0.31 ± 0.03 1.47 ± 0.41 80.0 ± 3.1 33.4 ± 2.7 0.80 ± 0.42 307.0 ± 25.5 145.2 ± 1.3 4.30 ± 0.28 107.7 ± 1.4 10.5 ± 0.2
10 6.71 ± 0.25 4.40 ± 0.14 1.91 ± 0.07 104.2 ± 14.7 73.5 ± 6.3 21.7 ± 9.6 0.01 ± 0.01 17.0 ± 1.1* 0.29 ± 0.03 1.12 ± 0.17* 77.5 ± 5.5 32.9 ± 4.4 0.50 ± 0.53 318.3 ± 39.9 145.8 ± 2.0 4.50 ± 0.34 106.9 ± 2.6 10.5 ± 0.3
10 6.69 ± 0.18 4.39 ± 0.11 1.91 ± 0.05 101.6 ± 17.1 73.1 ± 6.4 15.9 ± 4.1 0.02 ± 0.01 17.3 ± 1.3* 0.29 ± 0.02 1.16 ± 0.13 77.0 ± 2.9 31.7 ± 2.8 0.70 ± 0.48 297.4 ± 35.8 145.0 ± 1.3 4.16 ± 0.16 108.0 ± 1.8 10.5 ± 0.2
10 6.56 ± 0.22 4.37 ± 0.10 2.00 ± 0.09 97.3 ± 14.6 73.0 ± 3.4 13.6 ± 2.7 0.01 ± 0.01 18.8 ± 1.4 0.29 ± 0.03 1.15 ± 0.18 81.0 ± 8.8 32.6 ± 3.8 0.70 ± 0.48 286.4 ± 21.1 146.2 ± 0.5 4.11 ± 0.15 108.4 ± 1.2 10.4 ± 0.3
10 6.60 ± 0.19 4.36 ± 0.12 1.94 ± 0.07 97.3 ± 13.6 69.7 ± 8.3 12.8 ± 2.0 0.01 ± 0.00 17.3 ± 1.2* 0.28 ± 0.02* 1.04 ± 0.10* 85.2 ± 22.3 34.2 ± 2.8 0.60 ± 0.70 297.6 ± 22.9 148.1 ± 1.6* 4.19 ± 0.30 108.8 ± 1.9 10.6 ± 0.2
Values are the means ± standard deviations. Significantly different from the corresponding control values (p < 0.05, Dunnett’s test).
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significantly lower than those of the control groups (Table 4). Ca of the 5.0% males and Na of the 5.0% females were significantly higher than those of the control groups (Table 4). 3.4. Pathology Final body weight of the 0.625% females was significantly higher than that of the control group (Table 5). In the male rats, the relative spleen weights of the 2.5% or greater groups and relative kidney weight of the 5.0% group were significantly higher than those of the control groups (Table 5). No treatment-related macroscopic changes were observed in any organs of either sex. In the histopathological study, there were no treatment-related changes in any organs (for both sexes), whereas sporadic spontaneous lesions were observed in the control and treated animals. In the spleen or the kidney, relative weights were significantly higher, there were no treatment-related histopathological changes compared to the control rats (Fig. 2).
4. Discussion In the present 90-day feeding study of L-Pro in Fischer 344 rats, no deaths, treatment-related body weight changes or clinical signs were observed in either sex. Sauberlich (1961) reported the growth-depressing effect of amino acids when fed in low-protein diets to weanling rats. In their report, with 5% of L-Pro, the growth of rats was depressed slightly. The addition of 3% of L-Pro in 14.3% casein basal diet had smaller and inconsistent effects on growth rate with male weanling rats (Abernathy and Miller, 1965). Muramatsu et al. (1971) demonstrated no or slight growth depression in Donryu male growing rats fed a 10% casein diet containing 5% L-Pro for 3 weeks. On the other hand, female Sprague–Dawley rats given L-Pro in drinking water for 1 month (mean dose 50 mg/kg body weight/day) did not exhibit any adverse effects (Kampel et al., 1990). In this study, no growth or food-depressing effects were recognized in proline-treated rats. Water intakes were higher in the female treated groups (0.625%, 2.5% and 5.0%), but these were
Table 5 Absolute and relative organ weights in Fischer 344 rats given L-proline for 90 days. Item
Dietary levels of L-proline (%) 0 (control)
0.625
1.25
2.5
5.0
Male Effective number of rats Final body weight (g)
10 304.9 ± 13.4b
10 308.1 ± 13.5
10 307.3 ± 12.6
10 296.2 ± 22.8
10 296.6 ± 16.9
Absolute organ weight (mg) Brain Thyroidsa Heart Spleen Liver Adrenals Kidneys Testes
2007.7 ± 26.2 14.3 ± 1.2 848.8 ± 41.4 594.9 ± 38.1 6767.2 ± 382.1 38.5 ± 1.8 1654.6 ± 71.0 3044.2 ± 93.5
2000.2 ± 55.1 14.9 ± 1.9 856.4 ± 46.4 608.1 ± 21.5 6954.3 ± 595.1 37.8 ± 4.3 1715.2 ± 86.1 3024.9 ± 109.0
2009.3 ± 25.9 14.5 ± 1.6 865.4 ± 52.9 618.6 ± 27.9 6973.8 ± 327.0 39.0 ± 2.0 1683.5 ± 72.9 3051.5 ± 111.3
1981.2 ± 43.0 14.1 ± 1.61.2 841.1 ± 55.3 610.7 ± 50.4 6551.9 ± 805.0 37.8 ± 3.6 1648.0 ± 108.7 2932.7 ± 250.8
2001.0 ± 20.5 14.4 ± 1.3 856.4 ± 47.5 625.0 ± 43.0 6898.4 ± 676.2 40.0 ± 2.7 1713.5 ± 96.2 3002.0 ± 118.0
Relative organ weight (mg/100 g body weight) Brain 659.6 ± 31.2 Thyroids 4.7 ± 0.3 Heart 278.6 ± 12.1 Spleen 195.1 ± 9.6 Liver 2218.9 ± 59.5 Adrenals 12.6 ± 0.7 Kidneys 543.2 ± 26.1 Testes 999.5 ± 38.4
649.9 ± 24.6 4.8 ± 0.5 278.0 ± 10.8 197.5 ± 5.9 2254.1 ± 109.4 12.3 ± 1.1 556.6 ± 11.8 982.6 ± 39.2
654.8 ± 27.8 4.7 ± 0.5 281.5 ± 10.7 201.4 ± 6.6 2269.8 ± 70.1 12.7 ± 0.7 548.1 ± 18.7 994.1 ± 45.0
671.6 ± 41.5 4.8 ± 0.4 284.3 ± 9.6 206.4 ± 11.7* 2206.6 ± 132.5 12.8 ± 1.1 557.0 ± 16.0 991.9 ± 78.5
676.3 ± 34.2 4.8 ± 0.3 288.9 ± 9.5 210.6 ± 6.3* 2321.5 ± 110.2 13.5 ± 1.3 578.1 ± 22.5* 1014.3 ± 57.3
Female Effective number of rats Final body weight (g)
10 153.6 ± 7.8
10 166.5 ± 10.0*
10 164.2 ± 10.7
10 157.1 ± 11.4
10 156.9 ± 12.8
Absolute organ weight (mg) Brain Thyroids Heart Spleen Liver Adrenals Kidneys Ovaries Uterus
1835.0 ± 43.8 9.4 ± 1.1 494.9 ± 33.0 332.6 ± 19.9 3191.7 ± 220.2 40.3 ± 3.0 923.5 ± 46.1 46.3 ± 4.1 334.7 ± 41.6
1858.5 ± 30.3 11.2 ± 2.0 526.6 ± 34.6 363.7 ± 27.5* 3419.5 ± 240.9* 42.0 ± 3.1 982.6 ± 62.2* 50.8 ± 5.6 413.0 ± 64.6*
1847.0 ± 44.3 10.1 ± 1.3 528.9 ± 34.7 360.8 ± 25.9 3390.7 ± 165.1 40.9 ± 3.2 983.6 ± 40.9* 50.9 ± 5.5 384.7 ± 59.6
1836.6 ± 25.8 9.2 ± 0.9 507.5 ± 34.2 349.4 ± 27.1 3197.4 ± 198.1 40.8 ± 2.3 950.8 ± 38.1 47.6 ± 4.6 378.0 ± 62.1
1853.2 ± 23.8 10.5 ± 0.6* 527.1 ± 29.9 352.0 ± 24.2 3246.4 ± 164.4 40.5 ± 1.9 955.0 ± 44.3 48.3 ± 3.7 368.2 ± 62.2
1119.8 ± 63.6 6.7 ± 1.4 316.5 ± 14.2 218.4 ± 6.8 2054.1 ± 74.2 25.3 ± 1.5 590.5 ± 20.8 30.5 ± 2.6 248.2 ± 36.4
1128.1 ± 59.7 6.2 ± 0.7 322.6 ± 19.4 219.8 ± 9.1 2068.5 ± 88.3 25.0 ± 1.7 600.2 ± 27.1 31.1 ± 3.4 234.8 ± 36.8
1174.2 ± 79.7 5.9 ± 0.8 323.6 ± 18.2 222.7 ± 13.0 2037.9 ± 71.8 26.0 ± 1.4 606.8 ± 27.4 30.3 ± 2.2 240.7 ± 35.8
1187.4 ± 88.9 6.7 ± 0.5 337.4 ± 26.5 224.7 ± 11.4 2074.5 ± 93.4 25.9 ± 2.2 610.6 ± 33.3 30.9 ± 3.0 234.5 ± 32.2
Relative organ weight (mg/100 g body weight) Brain 1197.3 ± 59.1 Thyroids 6.1 ± 0.6 Heart 322.4 ± 16.2 Spleen 217.0 ± 15.7 Liver 2079.9 ± 131.6 Adrenals 26.3 ± 2.4 Kidneys 602.4 ± 37.0 Ovaries 30.1 ± 2.0 Uterus 218.5 ± 30.7 a
Thyroids were weighed after the fixation. Values are the means ± standard deviations. Significantly different from the corresponding control values (p < 0.05, Dunnett’s test).
b *
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Fig. 2. Representative histology of the spleen (A and B) and kidney (C and D) of male rats given 0 (control: A and C) and 5.0% (B and D) of L-proline in the diet for 90 days. There were no treatment-related histopathological changes. HE, 100.
slight changes and lacked dose-dependence. These were probably due to the taste of diets containing L-Pro and thus without toxicological significance. In the present L-Pro-administered groups, statistically significant changes were detected in some hematological parameters, such as HGB (low in the 0.625% males and 1.25% both sexes), HCT (low in the 1.25% males) and MCH (low in the 1.25% and 5.0% females). However, these hematological changes were slight, and lacked dose-dependence, and no abnormalities were observed in the microscopically assessed red blood cell figures in the blood smear or on histopathological examination of the hematopoietic organs. The observed hematological changes can therefore be considered incidental and not treatment-related. The serum GLU (all treated males), BUN (0.625%, 1.25% and 5.0% females), CRE (5.0% both sexes) and UA (0.625% females, 2.5% males and 5.0% both sexes) levels were significantly lower than those of the control groups. However, the values in L-Pro-administered groups were within the range of the historic control values (Mitruka and Rawnsley, 1981). Moreover, no corresponding pathological findings were observed. It is thus conceivable that the changes are toxicologically insignificant, even if they were treatment-related. The relative spleen (2.5% or greater males) and kidney (5.0% male) weights were significantly higher than those of the control groups. The histological findings of the spleen (2.5% or greater males) showed no abnormalities such as extramedullary hematopoiesis, lymphoid hyperplasia, pigmentation, fibrosis, red pulp hyperplasia or atrophy of lymphoid follicles. The hematological study revealed no significant differences between the control and the 2.5% or greater males. In the kidney weight, the increased changes were slight and lacked dose-dependence, and no abnor-
malities were observed in the urinalysis and histopathology. Moreover, the absolute spleen and kidney weights showed no significant differences between the control and the treated groups. It is suggested that the organ weight changes are toxicologically insignificant, even if they were treatment-related. In the literature, L-Pro administration in the drinking water (50 mg/kg body weight) for 1 month did not result in any histological changes in liver and kidney, whereas D-Pro (50 mg/kg body weight) treated rats had severe pathological changes of periportal fibrosis and necrosis of liver cells, and severe renal tubular dystrophy and necrosis (Kampel et al., 1990). In this study, L-Pro produced no treatment-related functional or morphological liver or kidney injury in male and female rats. It is thus likely that the liver and kidney toxicity of proline depends on its optical isomerism, namely, D-isomer-specific. Hyperprolinemia is an inherited metabolic disorder, and is a condition which occurs when the amino acid proline is not broken down properly by the enzymes, proline oxidase or pyrroline-5-carboxylate dehydrogense, causing a build up of proline in the body. Genetically hyperprolinemic PRO/Re mice have 7.1 times higher concentration of proline in the brain than control CD-1 mice and 10.8 times higher concentration of proline in plasma (Baxter et al., 1985). Nadler et al. (1988) described that intrahippocampal injections of L-Pro non-selectively destroyed pyramidal and granule cells. L-Pro destroyed far more hippocampal neurons than D-Pro, in keeping with its greater neuroexcitatory potency. Its excitotoxic action may be related to the neurological and cognitive deficits associated with hyperprolinemia (Nadler et al., 1988). Delwing et al. (2003) investigated the in vivo and in vitro effects of proline on some parameters of oxidative stress in rat cerebral cortex. Ten-day-old Wistar rats received one subcutaneous
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injection of proline (12.8 lmol/g body weight), while control rats received saline in the same volumes. The results indicate that proline induces oxidative stress in the brain, which may be related, at least in part, to the neurological dysfunction observed in hyperprolinemia. In this study, even in the 5% L-Pro group, no clinical signs or symptoms suggesting neurotoxicity of L-Pro (such as excitement, inanimation, tremor, convulsion) were observed, and the pathological studies showed no histopathological changes in central nervous system of proline-treated rats. In conclusion, the no-observed-adverse-effect-level (NOAEL) for L-Pro was determined to be at least a dietary dose of 5.0% (2772.9 mg/kg body weight/day for males and 3009.3 mg/kg body weight/day for females) under the present experimental conditions.
Acknowledgments The authors thank Dr. Hideki Wanibuchi (Department of Pathology, Osaka City University Medical School, Osaka, Japan) for helpful suggestions. This work was supported in part by a Health and Labour Sciences Research Grant (awarded to Dai Nakae) from the MHLW.
References Abernathy, R.P., Miller, J., 1965. Effects of imbalances or antagonisms among nonessential amino acids on growth and nitrogen utilization by rats. J. Nutr. 86, 231–235. Baxter, C.F., Baldwin, R.A., Davis, J.L., Flood, J.F., 1985. High proline levels in the brains of mice as related to specific learning deficits. Pharmacol. Biochem. Behav. 22, 1053–1059.
Delwing, D., Bavaresco, C.S., Wannmacher, C.M., Wajner, M., Dutra-Filho, C.S., Wyse, A.T., 2003. Proline induces oxidative stress in cerebral cortex of rats. Int. J. Dev. Neurosci. 21, 105–110. Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids (Macronutrients), 2005. Proline. Available from: