Thirteen-Week Oral Toxicity Study of L-Lysine Hydrochloride in Rats

Abstract

l-Lysine hydrochloride (Lys) is an essential amino acid in humans and animals, and it is used in animal feeds, in prevention of herpes simplex recurrence, and cereal fortification in some developing countries. This study evaluated toxicological and behavioral effects of Lys during a dosing study with male and female Sprague-Dawley rats. The amino acid was incorporated into a standard diet at doses equal to 1.25%, 2.5%, and 5.0% (w/w). A control group of rats received a standard diet. All diets were administered ad libitum for 13 consecutive weeks. To examine stability of any potential effects, the administration period was followed by a 5-week recovery period, during which only the standard diet was provided to all animals. In male and female rats in each concentration group, treatment-related changes were not observed in the clinical signs, body weights, diet consumption, water intake, ophthalmology, gross pathology, organ weights, or histology. A Lys-related drop in serum concentration and an increase in urine excretion of chlorides was a compensatory reaction to the ingested hydrochloride. No functional, biochemical, or histological changes in renal function were found. The no-observed-adverse-effect level (NOAEL) for Lys was estimated at 5.0% for both genders (male, 3.36 ± 0.12 g/kg/day; female, 3.99 ± 0.28 g/kg/day).

Currently, approximately 45 tons of l-lysine hydrochloride (Lys) is employed in human dietary supplements each year (Anderson and Raiten 1992), while an additional 225 thousand tons is employed yearly in animal feed ( Wall Street Journal 1995). Besides prophylaxis of herpes simplex recurrence (McCune et al. 1984; Thein and Hurt 1984), other potential therapeutic uses of Lys supplements are suggested by research reports and case histories dealing with cardiovascular diseases (Pauling 1993), stroke (Yamori 1993), osteoporosis (Mack et al. 1962), and stress-induced pathologies (Smriga and Torii 2003). Because cereal proteins are low in Lys, Lys dietary inadequacy is common in developing countries where low socioeconomic groups depend on wheat for their protein supply (Young and Pellet 2000). Although controversy exists on the enhancement of cereal protein quality with Lys (Ghai and Chaudhuri 1971; Scrimshaw, Taylor, and Young 1973; Kurpad et al. 2001, 2003), the use of this amino acid to fortify low-quality proteins is a possibility (Hussain et al. 2004).

In view of the already existing and potential roles of Lys supplements and their use without medical supervision, as well as the possibility of new dietary uses, we evaluated the subchronic toxicity of diet-incorporated Lys in rats. The tested amino acid was mixed into a standard diet at concentrations of 0%, 1.25%, 2.5%, and 5.0% (w/w), and the diets were orally administered ad libitum to male and female rats for 13 continuous weeks. To find out whether Lys-induced changes (if any) were recoverable, this administration period was followed by a 5-week-long recovery, during which only a standard diet was provided to all tested rats.

MATERIALS AND METHODS

Animals and Feeding Protocols

Seventy-five 4-week-old male and an equal number of 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 (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 am to 7:00 pm). Following the acclimatization period (2 weeks), healthy animals of both genders showing a normal increase in body weight were selected at 6 weeks of age and used throughout the study. The selected rats were divided by a computer block-placement method into four separate groups (12 rats per group) based on their weight, to eliminate the homogeneity of the group mean body weight. The control group was fed a standard diet, whereas the remaining three groups received a diet supplemented with Lys (Lot.n. 303VKBC, Ajinomoto, Tokyo, Japan). Lys was supplied at three various doses (1.25%, 2.5%, and 5.00%, w/w). Control and Lys-supplemented diets did not differ in color and odor. All diets were administered ad libitum for 13 weeks. Following the treatment period, six rats from the control and high-dose groups were randomly selected for continuation in the recovery period. During the recovery, only a standard unsupplemented diet was provided. The study was conducted in compliance with the Good Laboratory Practice Standards for Safety Studies on Drugs (Notification No. 313, March 31, 1982) and Guidelines for Toxicity Studies Required for Applications for 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 test article administration period, and once daily (morning) during the 5-week recovery period. The rats were weighted twice a week at a specified time (9:00 am to 12:00 noon). Twenty-four-hour diet intake was measured twice per week, and water intake once per week. Estimated intake of each amino acid (mg/kg/day) for each week was calculated based on the mean consumption for 1 day for that week.

Ophthalmologic Examination

Ophthalmologic examination was performed prior to the start of administration. Animals in which abnormalities of the external appearance of the eyes, the anterior part, the vitreous body, and the fundus oculi of the eyes were observed using an ophthalmoscope were not included in the study. The ophthalmologic examination was repeated in week 13 of administration (six randomly selected rats from each group), and week 5 of recovery (all rats).

Urinalysis

Urinalysis was conducted in all rats in weeks 5 and 13 of the administration period, and in week 5 of the recovery, as follows. Animals were placed in metabolic cages for 4 h, provided with water, and deprived of diet. Urine samples were collected. Immediately thereafter, rats were given a diet, and further 20-h urine samples were collected while providing both diet and water. The following parameters were evaluated only from the 4-h samples: pH, protein, ketone body, glucose, occult blood, bilirubin, urobilinogen (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). The following parameters were measured: red blood cell count (RBC) (Coulter 8 Item Automatic Blood Cell Analyzer T890; Japan Scientific Instrument, Tokyo, Japan), mean corpuscular volume (MCV) (ACL100), hemoglobin (Hb) (cyanmethemoglobin method; ACL100) to reticulocyte ratio (Brecher method), platelet and white blood cell count (ACL100), differential leucocyet count (May-Giemsa staining), prothrombine and activated partial thromboplastin time (PT and APTT) (automatic analyzer, Monarch), and fibrinogen (thromboplastin method; 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 heparinized tubes. 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. 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 using 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 Lys were observed during the administration period. Swelling of the hind limbs (tarsal joints) was observed in one female rat in the control group, gryposis of the upper jaw in one female rat in 1.25% concentration group, fracture of the incisors in one male rat of both the control and the 5.0% concentration group, and malocclusion in one male in the control group. All above changes were incidental. No clinical signs were observed during the recovery period.

Body weights in male and female rats in each concentration group were comparable to those recorded in controls, and no statistically significant difference was observed (Table 1). Administration-related changes in diet consumption were not observed (data not shown). Sporadic changes were seen. In the high dose, this included increased intake on day 42 in males and day 3 in females. However, those were temporary, dose-independent phenomena and the total consumption did not differ among the groups. The average intake of Lys over the course of the study is shown in Table 2. No significant administration-related changes in water intake were recorded (data not shown).

Ophthalmology

There were no abnormalities observed in any of the animals.

Urinalysis

Chloride excretion is illustrated in Table 3. A dose-dependent tendency for decreased pH was seen in both genders that received Lys diets. Males ingesting 5.0% concentration diet were characterized by significantly higher urine volume (controls, 15.3 ± 5.8 ml/day; 5.0% group, 21.3 ± 8.1 ml/day). This change was not observed at the end of the recovery period.

Hematology and Blood Chemistry

At the end of the administration period, a significant increase in the hemoglobin level was seen in females in the 5.0% concentration group (controls, 14.7± 0.6 g/dl; 5.0% group, 15.5 ± 0.6 g/dl). A significant decrease in serum chlorides was observed in males of all concentrations groups (controls, 111.9 ± 0.7 mEq/L; 1.25% group, 109.9 ± 1.1 mEq/L; 2.5% group, 110.3 ± 1.5 mEq/L; 5.0% group, 109.8 ± 1.3 mEq/L), and females of the 5.0% concentration group (controls, 112.7 ± 1.2 mEq/L; 5.0% group, 110.1 ± 1.2 mEq/L). Additionally, a significant increase in total bilirubin was observed in females in the 2.5% concentration group (controls, 0.09 ± 0.01 mg/dl; 2.5% group, 0.11 ± 0.02 mg/dl), but the change was not observed in the 5.0% concentration group.

Pathology and Histopathology

There were no significant treatment-related pathologies; minor changes were few and dose independent. At the end of the administration period, there were no treatment-related changes in absolute (Tables 4A and 4B) or relative organ weights. At the end of the recovery period, a significant increase in the absolute weight of the heart in males of the 5.0% concentration group, a significant increase in unilateral weight of the kidneys in males and females in the 5.0% concentration group, and a decrease in the absolute bilateral weight of the adrenals in males of the same group were seen. As the changes were not observed at the end of the administration period, they were considered to be accidental ones.

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 recorded at the end of the administration period are listed below. Slight focal myocarditis was observed in one male in each of the 1.25% and 2.5% concentration groups. Focal pneumonia was observed in one female in the 5.0% concentration group. Mild erosion in the glandular stomach wall (macroscopic red spots in the glandular area) was seen in one or two females in each group, including the control group. Slight cellular infiltration of the lamina propria mucosae was observed in one female in the control group, and one male and one female in the 2.5% concentration group. Minor focal necrosis was detected in two females in the 1.25% concentration group, and one male in the 5.0% concentration group. A small cyst was found in the kidney in one female in the control group. Additionally, one female in the 5.0% concentration group had dilated renal pelvis, and one female in the 2.5% concentration group had slight hyaline casts. Weak retinal atrophy was observed in one male in the 5.0% concentration group, and minor retinal degeneration in one female in both the control and the 2.5% concentration group. There were no abnormalities in other organs and tissues.

DISCUSSION

This trial evaluated the subchronic toxicity of diet-incorporated Lys. Lys was mixed into a standard diet at three concentrations, and the diets were orally administered ad libitum to male and female rats for 13 continuous weeks. The administration period was followed by a 5-week recovery, during which only a standard diet was offered. No deaths were observed throughout the administration and recovery periods. In male and female rats in each concentration group, treatment-related changes were not observed in the clinical signs, body weights, diet consumption, water intake, ophthalmology, gross pathology, organ weights, or histology. These results agree with the previous chronic observation, in which Lys (3.0%, w/w) effects were investigated in a 2-year ad libitum feeding paradigm (Anderson and Raiten 1992). Moreover, Yamori (1993) did not report any adverse effects of increasing Lys intakes by about 150% from the requirement in stroke-prone, spontaneously hypertensive rats over a 25-week period.

An increase in the level of hemoglobin in females of the 5.0% concentration group was the main hematological change. However, this increase was within both the range of the concurrent and historical laboratory values observed in the experimental facility. A drop in serum chlorides is thought to be a compensatory response to an increase in ingested hydrochloride. This adaptive reaction was linked to a significant increase of chloride excretion for 1 day in both weeks 5 and 13 of the administration period seen in both genders. We hypothesize that the level of excreted urine chlorides increased, and the urine pH dropped, as a consequence of hydrochloride presence. The values of urea nitrogen and creatine, and the lack of histological abnormalities, indicate absence of renal dysfunction. A slight rise in urine volume was found in females in the 5.0% concentration group, but it was considered as an adaptive reaction to large increases in ingested chlorides.

Based on the above data, we estimate the no-observed-adverse-effect level (NOAEL) for Lys at 5.0% for both genders (males 3.357 ± 0.115 g/kg/day; females, 3.986 ± 0.283 g/kg/day). These relatively high NOAELs are consistent with human observations (Flodin 1997) that Lys is a safe and well-tolerated dietary substance for long-term use.