Radosław Piotr Radzki
Antiosteopenic effect of alpha-ketogluterate and ipriflavone 
in ovariectomized and orchidectomized rats
Atrophic changes of bone tissue during osteopenia and osteoporosis are characterized by low bone mass, deterioration of bone tissue and disruption of bone architecture, compromised bone strength and an increase in the risk of fracture. Appear in animals and humans. One of the key factor caused development of atrophic changes is lack of the influence of the gonadal sex steroids.
Aim The purpose of the present study was to determine of the single or combined effect of the alpha-ketoglutarate (AKG) – precursor of the glutamine and ipriflavone (IP) – the synthetic analogue of phytoestrogen daidzeine, on the mineralization of bone tissue in female and male rats during development of osteopenia (Experiment I) and in condition of established osteopenia (Experiment II). In both Experiments the osteopenia has been involved by bilateral gonadectomy. The single or combined influences of AKG and IP on bone tissue in healthy animals were also verified.
Material and Methods. The experiments were conducted with the use of females and males of Wistar rats at the age of 3 months. After adaptation to the experimental conditions, the animals were randomly divided and submitted for sham-operation (SHO) and ovariectomy (OVX) or orchidectomy (ORX). In Experiment I, the experimental treatment with AKG and IP started after a 7-day period of convalescence, whereas in Experiment II the rats were submitted to 60-day period of osteopenia fixation, after which the proper part of the study was commenced. Alpha-ketoglutarate (AKG) was applied in the experimental solution for drinking, at the concentration of 1,0 mol/l, and as a control, the control solution (PLC) was used. Ipriflavone, at a daily dose of 50 mg/kg b.w. and physiological saline (PF) – as the control – were applied by the gavage. After 60 days of experimental treatment with AKG and IP, in both Experiments, the animals were killed, and the weight of uterus and senile vesicles recorded. Blood serum, as well as isolated femora and lumbar vertebrae (L2–L4) were stored for further analysis. Based on the 3-point bending test, mechanical properties such as the maximal force and the force in the resilience limit of femora were estimated. With the use of peripheral quantitative computed tomography (pQCT), analysis of trabecular bone tissue in distal methaphysis (5 mm from knee joint surface) and of cortical tissue in middiaphysis (50% of bone length) of femora were performed. Densitometric examination (DEXA) allowed for measurements of BMD, as well as, BMC of isolated femora and lumbar vertebrae (L2–L4). The activity of total alcaline phosphatase and concentration of calcium and phosphorus were determined spectrophotometrically, whereas the concentration of osteocalcin and carboxyterminal cross-linking telopeptide of bone collagen I were estimated using ELISA methods. 
Results of the present study proved the differential effect of gonadectomy on the character and intensity of metabolic bone disorders in females and males. Atrophic processes of bone tissue were more intensive in males than in females, and the differences mainly related to cortical tissue, with comparable changes in trabecular tissue. Despite the differences in gender, the decrease in mineralization of femora and lumbar vertebrae, as well as the mechanical properties of femora, were observed in both genders. Additionally, the character of the changes in architectonical properties and biochemical markers of bone metabolism indicate an intensification of resorption and structural weakness of the bone material. Alpha-ketoglutarate and ipriflavone, applied exclusively and inclusively, counteract these changes. With regard to the data obtained by tomography, densitometry, and mechanical analysis of the isolated bones, in combination with biochemical markers of bone metabolism, it can be generally assumed, that the mechanism of the AKG or IP exclusively influence the stimulation of bone tissue formation and inhibition of its resorption. The reaction on the experimental treatment depends from the degree of bone tissue degradation, which relates from the time of the lack of gonadal steroids. AKG or IP applied during osteopenia development completely inhibited the degradation of bone tissue. After the treatment with AKG or IP, in the condition of established osteopenia, the resorption was markedly limited, but not halted. The inclusive influence of AKG and IP during osteopenia development demonstrated the anabolic effect of bone metabolism, and the results obtained exceeded those in groups where the examined substances were applied alone. The synergistic effect of AKG and IP in animals with long-term lack of influence of gonadal steroids, leads to equalization between formation and resorption, and in consequence efficiently counteracts bone disorders. 
Conclusions. The obtained results prove that exclusive, and predominantly synergistic, effect of AKG and IP indicate their effectiveness in the prophylaxis and therapy of osteopenia and osteoporosis, induced by bilateral gonadectomy, both in human and animal. Additionally, the results clearly prove, that treatment with AKG or/and IP improve the quality of bone tissue in healthy animals.