Research Article |
Corresponding author: Ivanka Kostadinova ( i.kostadinova@pharmfac.mu-sofia.bg ) Academic editor: Plamen Peikov
© 2023 Ivanka Kostadinova, Nikolai Danchev, Boycho Landzhov, Lyubomir Marinov, Ivalina Ivanova, Dobrina Tsvetkova.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Kostadinova I, Danchev N, Landzhov B, Marinov L, Ivanova I, Tsvetkova D (2023) Creatine lysinate – part I: investigation of the toxicity and the influence on some biochemical parameters in mice. Pharmacia 70(4): 895-899. https://doi.org/10.3897/pharmacia.70.e109446
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In our study we investigated the acute toxicity of а newly synthesized creatine lysinate as well as its effect on the biochemical parameters in mice. Creatine lysinate exerts better solubility in water (3.3%) in comparison to creatine monohydrate (1.4%) at 20 °C and it is determined as a non-toxic after intraperitoneal (LD50 – 4543 mg/kg) and oral administration (LD50 > 8000 mg/kg). Oral administration of creatine lysinate at doses of 3 g/kg/day and 6 g/kg/day for 2 weeks reduced the creatine kinase levels, which indicates muscle protection. An increased levels of liver enzymes like alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) was observed after the supplementation with creatine lysinate at both administered doses and the level of lactate was comparable both in the studied and the control group.
Biochemistry, creatine derivatives, mice, LD50
Creatine deserves a special place among ergogenic supplements, as it is one of the most studied and scientifically supported supplements on the market. Creatine is a naturally occurring non-protein nitrogen compound synthesized in the liver and kidney from amino acids arginine, glycine and methionine (
The mechanism of action of creatine is based on effects that trigger improvement in muscle energy metabolism, in which phosphorylated creatine has the ability to re-synthesize ATP from adenosine diphosphate, thus increasing their deposits (
In the study where rats that have been supplemented with supraphysiological doses of creatine (5 g/kg bw/day for 1 week, thereafter 1 g/kg bw/day for 3 to 7 weeks; equivalent to 350 g/day and 70 g/day for a 70-kg adult) had higher plasma levels of alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), g-glutamyltransferase (GGT) and alkaline phosphatase (AP) and demonstrated some structural changes indicating hepatic damage. Interestingly, creatine supplementation in combination with physical activity decreased the levels of liver enzymes (
In our study we revealed a data about the toxicity of a newly synthesized creatine lysinate (CrLys) and its influence on some biochemical parameters such ASAT, ALAT, creatine kinase (CK) and lactate after 2 weeks of administration.
CrLys (Mw – 277.32 g/mol) was synthesized and provided by Prof. Lyubomir Vezenkov from the Department of Organic Chemistry at the University of Chemical Technology and Metallurgy - Sofia (Patent for invention N 66 511, Creatine salts, LT Vezenkov, PT Angelov, LL Vezenkov, published in Bulletin 11 on 30.11.2015). The solubility of creatine lysinate in water is 3.3%. Creatine monohydrate (Biogame Co.) was used as standard for comparison. CrM (Mw –149.15 g/mol) is well-known food supplement with solubility in water about 1.4% at 20 °C (
Lethal dose 50 (LD50) is one of the ways to measure the short-term toxic potential (acute toxicity) of the substances. LD50 values can be compared using toxicity scales. The most commonly used in practice are the Hodge & Sterner Scale and the Gosselin, Smith & Hodge Scale (
Male albino mice, line H with body weight 28–32 g were divided into five groups of six animals per group (n=6). Food and water were available ad libitum. During the whole experiment the animals were maintained at room temperature 22 ± 3 °C, humidity 30%, lighting schedule 12 h light/dark cycle. Experiments were performed during the light part of the cycle. The animals are divided into five groups of six animals (n=6) depending on the administered substances and their doses:
1st group – control animals that received only drinking water; 2nd group – animals that received CrM at a dose of 1.5 g/kg/day (CrM 1.5 g/kg/day); 3rd group – animals that received CrM at a dose of 3 g/kg/day (CrM 3 g/kg/day); 4th group – that received CrLys at a dose of 3 g/kg/day (CrLys 3 g/kg/day); 5th group – animals that received CrLys at a dose of 6 g/kg/day (CrLys 6 g/kg/day). All substances were dissolved and administered to the experimental animals for 2 weeks with the drinking water. On the 1st, 7th and 14th days were performed tail suspension and Rotarod tests and at the end of the experiment histological evaluation of the soleus muscle was performed. The latest experiments are subject of our next publication. The experiments were conducted in accordance with Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes and approved by the Local Animal Care Ethics Committee (№ 329/01.06.2022).
After 2 weeks of administration of creatine derivatives, the blood samples were collected via decapitation in tubes containing dipotassium-ethylenediaminetetraacetic acid (K2-EDTA). For the biochemical analysis the blood was centrifuged for 5 minutes at 7000 rpms. Afterwards the serum was analyzed on Mindray BS-120 biochemistry analyzer, counting the following parameters: ALAT, ASAT and CK. Lactate was measured with StatStrip Lactate Xpress Meter.
LD50 values were determined with the Origin computer program by the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949). Statistical processing of the obtained results was done with the program GraphPad Prism 6.0. The arithmetic mean and the standard errors of the arithmetic mean (SEM) were determined for the biochemistry data. A statistically significant difference between the compared means was checked using the One-way ANOVA and the Tukey test. A p-value of 0.05 or lower was considered statistically significant. For graphical presentation of the data, Microsoft Office Excel 2019 was used.
CrM and CrLys were administered i.p. and p.o. in order to establish LD50. The results for the p.o. toxicity are presented in Table
Data on the toxicity of creatine monohydrate (CrM) and creatine lysinate (CrLys) after p.o. administration.
Compound | Administered dose p.o. (mg/kg b.w.) and the survival percentage | |
---|---|---|
8000 mg/kg | 10 000 mg/kg | |
CrM | 0/6 (100%) | 0/6 (100%) |
CrLys | 0/6 (100%) | – |
For CrLys, the signs of toxicity appeared after the i.p. administration of 4000 mg/kg (percentage of survival 75%) and the percentage of survival decreased to 26.7% at 5000 mg/kg (Fig.
On Table
Biochemical analysis after 2 weeks of administration of creatine derivatives. Results are presented as mean ± SEM. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001 – statistical difference between the creatine derivatives at different doses and the control group.
Group | ASAT U/L | ALAT U/L | CK U/L | Lactate, mmol/L |
---|---|---|---|---|
Control group | 195.70 ± 14.72 | 57.65 ± 5.61 | 3724 ± 400.5 | 6.96 ± 0.69 |
CrM 1.5 g/kg/day | 332.30 ± 29.18 ** | 74.10 ± 4.20 | 828.8 ± 157.9 **** | 7.44 ± 1.02 |
CrM 3 g/kg/day | 307.60 ± 22.91* | 73.58 ± 5.59 | 1311 ± 138.5 *** | 6.98 ± 0.60 |
CrLys 3 g/kg/day | 360.80 ± 26.36 ** | 87.40 ± 5.95 ** | 1833 ± 419.8 ** | 7.24 ± 0.94 |
CrLys 6 g/kg/day | 286 ± 19.18 | 60.15 ± 3.24 | 1086 ± 112 **** | 6.15 ± 1.21 |
In our study, we examined the toxicity and the influence of newly synthesized CrLys on the biochemical parameters in mice after 2 weeks of administration. Our results revealed that CrLys has lower LD50 value in mice (4543 mg/kg after i.p. administration) than the standard CrM, but nevertheless the value is above 4000 mg/kg and CrLys cannot be classified as a toxic substance according to the Hodge and Sterner Toxicity Scale (
In the acute toxicity studies performed by
In our study after 2 weeks of administration, some of the creatine derivatives (CrM 1.5 g/kg/day, CrM 3 g/kg/day and CrLys 3 g/kg/day) showed increased levels of ASAT when compared with the control group. There is also an elevation in another transaminase – ALAT, especially in the group treated with CrLys 3 g/kg/day in comparison to the control group. Furthermore, there were significant lower levels of CK in the groups treated with creatine derivatives at both doses in comparison to the control group.
In our study, the blood lactate concentration of the groups treated with creatine derivatives didn’t change significantly in comparison to the control group. The lowest lactate level was registered in the group, supplemented with CrLys at a dose of 6 g/kg/day – 6.15 vs. 6.96 mmol/L in the control group.
The major finding that can be reported from this research is related to the low toxicity of a newly synthesized CrLys. After 2 weeks of administration of creatine derivatives, an increase in the levels of liver transaminases and decreased level of creatine kinase were detected. Our next article will be focused on the effect of newly synthesized creatine derivatives on the tail suspension test and rotarod test and influence on the histology of the skeletal muscles.
Conceptualization, I.K. and N.D.; methodology, I.K., N.D.; investigation, I.K., B.L., L.M., I.I. and D. T.; writing—original draft preparation, I.K.; writing—review and editing, I.K., N.D.; visualization, I.K.; supervision, N.D.; project administration, I.K.; funding acquisition, I.K. All authors have read and agreed to the published version of the manuscript.
This article was prepared with financial support from Grant 2021, Project № D-106/04.06.2021 to the Council of Medical Science of Medical University of Sofia, Bulgaria.
Acknowledgements to Prof. Lyubomir Vezenkov from the University of Chemical Technology and Metallurgy, Sofia, Bulgaria for the synthesis and provision of the creatine derivatives and to the Medical University of Sofia for the financial support.