Effects of α-Ketoglutarate on Bone Homeostasis and Plasma Amino Acids in Turkeys M. R. Tatara,*,1 A. Brodzki,† W. Krupski,‡ E. S´liwa,* P. Silmanowicz,† P. Majcher,§ S. G. Pierzynowski,储 and T. Studzin´ski* *Department of Animal Physiology, Faculty of Veterinary Medicine, The Agricultural University of Lublin, ul. Akademicka 12, 20–950 Lublin, Poland; †Department and Clinic of Animal Surgery, Faculty of Veterinary Medicine, The Agricultural University of Lublin, ul. Głe˛boka 28, 20–950 Lublin, Poland; ‡II Department of Radiology, Medical University of Lublin, ul. Staszica 16, 20–081 Lublin, Poland; §Department of Rehabilitation and Physiotherapy, Medical University of Lublin, ul. Chodz´ki 7, 20–950 Lublin, Poland; and 储Department of Cell and Organism Biology, Lund University, Helgonava¨gen 3b, SE–223 62 Lund, Sweden ABSTRACT The objective of the study was to evaluate the effect of denervation and α-ketoglutarate (AKG) administration on the development of osteopenia in the turkey radius. At 22 d of age, all turkeys were subjected to neurectomy of the right radius. Control turkeys were given a saline solution into the crop each day for 97 d. Experimental turkeys were given 0.4 g of AKG/kg of BW into the crop each day. After 98 d, BW was not affected by the AKG treatment. Volumetric bone mineral density of the radius was measured by quantitative computed tomography. Mechanical properties were tested using a 3-point bending test. Cross-sectional area, second mo-
ment of inertia, and mean relative wall thickness were measured as well. Amino acid concentrations were assessed with the use of ion-exchange chromatography. Denervation had a negative effect on all bone characteristics that were measured except bone length. The AKG had a positive effect on all bone characteristics except bone length. Plasma concentrations of proline and leucine were increased by AKG, whereas concentrations of taurine and glutamine were decreased. The turkey radius appears to be a good model for studying osteopenia because its development can be affected by treatments such as denervation and AKG administration.
(Key words: α-ketoglutarate, leucine, long-term denervation of radius, proline, turkey) 2005 Poultry Science 84:1604–1609
INTRODUCTION Bone tissue in vertebrates has 3 main functions: supportive and locomotory, protective for the central nervous system and bone marrow, and metabolic by serving as a reservoir of Ca and P in the body. To provide optimal development and function of organisms, the bone tissue must fulfill all of these functions properly. Amount of bone is to a great extent (70 to 80%) genetically determined. The remaining 20 to 30% of the peak bone mass determination is ascribed to nutritional and environmental factors, which are subject to modification (Eastell and Lambert, 2002). Among many factors responsible for proper skeletal development and maintaining homeostasis, the nervous system has great importance (Edoff et al., 1997; Chenu, 2002a; Imai and Matsusue, 2002; Taylor, 2002). In recent years it has been documented that the neuronal control of bone cell metabolism is guided by
nerve-derived factors (neuropeptides) and their receptors that are present in bone cells (Chenu, 2002b; Taylor, 2002). There is evidence that next to the neuropeptides such as calcitonin gene-related peptide, vasoactive intestinal peptide, neuropeptide Y, substance P, noradrenaline, galanin, a role in bone metabolism regulation is played by glutamate (Chenu, 2002a,b; Taylor, 2002; McDonald et al., 2003). In vivo and in vitro studies provided evidence that glutamatergic signaling is necessary for normal osteoblast function (Taylor, 2002). α-Ketoglutarate (AKG) is the precursor to glutamate and glutamine and is a central molecule in the citric acid cycle. The rate of AKG formation is important in determining the overall rate of this cycle (Wire´n et al., 2002). Glutamate released from nerve fibers in bone tissue is synthesized by the reductive amination of its ketoacid (α-ketoglutaric acid) taking place in peri-vein hepatocytes (Stoll et al., 1991). The AKG functions as an energy donor and ammonium ion scavenger as well as providing a source of glutamine that stimulates protein synthesis, in-
2005 Poultry Science Association, Inc. Received for publication February 12, 2005. Accepted for publication July 8, 2005. 1 To whom correspondence should be addressed:
[email protected].
Abbreviation Key: AKG = α-ketoglutarate.
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EFFECTS OF AKG ON BONE HOMEOSTASIS AND AMINO ACIDS
hibits protein degradation in muscle, and constitutes an important metabolic fuel for cells of the gastrointestinal tract (Hixt and Mu¨ller, 1996; Jones et al., 1999). Investigations performed by Kristensen et al. (2002) in pigs revealed that AKG administered enterally increased proline synthesis, which plays a key role in collagen formation processes. The AKG, ascorbate, and Fe2+ steer the conversion of pro-collagen to collagen, which is dependent on hydroxylation of peptide-bound proline to hydroxyproline via prolyl hydrolase (Tocaj et al., 2003). Moreover, other investigations have shown that enteral feeding of AKG supplements increases circulating plasma levels of insulin, growth hormone, and insulin-like growth factor-1 (Moukarzel et al., 1994; Jeevanandam and Petersen, 1999). Imbalances between osteoblast and osteoclast activity result in disturbances of bone integrity, with severity characterized by the amount of bone mass remaining relative to peak. Osteopenia, or bone loss, is characterized by decreased mineralization and mechanical endurance of bone tissue and is generally considered to have occurred at 1 to 2.5 SD below peak bone mass and osteoporosis by 2.5 SD or more below peak bone mass (Beck and Hansen, 2004). The aim of the study has thus been to elucidate the effect of AKG administration on free plasma amino acid pool and skeletal system development under conditions of bone innervation and denervation in fast-growing turkeys. Furthermore, we aimed to propose turkeys as an experimental model for investigations of skeletal development and mineralization in poultry with the use of osteopenia induction by bone denervation.
MATERIALS AND METHODS The experimental procedures used throughout this study were in compliance with the guidelines for the care and use of animals as published by the NRC (1996) and were approved by The Local Ethics Committee on Animal Experimentation of The Agricultural University of Lublin, Poland.
Birds Thirteen HB-91 male turkeys (Holly Berry Hatcheries Ltd., Langworth, UK) were selected from a flock at the age of 3 wk, and randomly divided into 2 weight-matched groups. Throughout the course of the study they were separated from the remaining farm stock by a special wire fencing construction and kept under standard rearing conditions with free access to fresh water and appropriate feed ad libitum, supplied in accordance with the stage of production cycle, and air temperature set at the optimal level for all flock. The experiment was conducted from 22 to 119 d of age.
Surgical Procedure On d 22 of life, all experimental turkeys were subjected to surgical denervation of the right radius by neurectomy
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of the medianoulnaris and the radialis nerves. The neurectomy was performed in the region of the proximal epiphysis of the humerus under general anesthesia, with the use of ketamine (50 to 80 mg/kg of BW, Ketamina 10%, Biowet Puławy Corp., Puławy, Poland) and earlier premedication by xylasine (10 mg/kg of BW Sedazin, Biowet Puławy Corp., Puławy, Poland). To avoid nerve fiber regeneration, the procedure consisted of cutting out 2 sections of approximately 10 mm each of the radialis and the medianoulnaris nerves. All the left wings of birds were surgically untouched, and the left radius served as the control bone to the right denervated radius.
AKG Treatment and Sampling Procedure Powdered AKG (Gramineer Int. AB, Lund, Sweden) with a purity of 99% was mixed with saline and adjusted by the addition of NaOH to a final pH of 7.3. Starting from 22 d of life, the AKG-treated group of turkeys (n = 6) received 0.4 g/kg of BW of AKG per d, directly to the crop via a tube, whereas the control group (n = 7) was given the same volume of saline in the same way. Administration of AKG was continued until 118 d of life, whereas the turkeys were killed on 119 d of life. After 14 wk of the experiment, the blood samples were collected for hematological analysis and free plasma amino acids concentration assessment. Moreover, bone samples were Xrayed, isolated, and frozen at −25°C for further analyses.
Volumetric Bone Mineral Density of Radius The quantitative computed tomography method and SOMATOM AR.T – SIEMENS apparatus (Siemens Corp., Germany) supplied with VR3 software were used for determination of volumetric bone mineral density of the cortical bone of radius. The metaphyseal quantitative computed tomography scan was 2 mm thick and positioned at 5% of radius length, measuring from the distal extremity of this bone.
Analysis of Mechanical and Geometrical Properties of Radius Mechanical properties of investigated bones were estimated using a 3-point bending test in INSTRON 4302 apparatus (Instron Corp., Canton, MA) linked with a computer registering the relationship between forces perpendicular to the longitudinal axis of investigated bone and the resulting displacement. The distance between supports was set at 40% of total radius length and the measuring head loaded bone samples with a constant speed of 10 mm/min. The results were presented graphically, and the maximum elastic strength and the ultimate strength of radius were estimated (Ferretti et al., 1993a,b). On the basis of measurements of horizontal and vertical diameters of mid-diaphyseal cross-section of the radius, the cross-sectional area, the second moment of inertia and
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TATARA ET AL. Table 1. Characteristics of volumetric bone mineral density, morphological and mechanical properties in the left (control) and right (denervated) radius of turkeys1 Investigated parameter Bone weight (g) Bone length (mm) Bone mineral density (g/cm3) Cross-sectional area (mm2) Second moment of inertia (mm4) Mean relative wall thickness Maximum elastic strength (N) Ultimate strength (N)
Left radius (intact wing; n = 13) 6.5 121.2 1.556 16.6 49.0 0.513 173 224
± ± ± ± ± ± ± ±
0.2a 0.8a 0.024a 0.7a 3.3a 0.031a 15a 18a
Right radius (denervated wing; n = 13) 6.1 120.6 1.467 11.7 34.6 0.328 122 144
± ± ± ± ± ± ± ±
0.3b 1.0a 0.039b 1.2b 3.4b 0.042b 14b 18b
Means for each investigated parameter that do not share common superscripts differ (P < 0.05). Values are means ± SEM.
a,b 1
the mean relative wall thickness were derived (Ferretti et al., 1993a,b).
Morphology, Mechanics, and Volumetric Bone Mineral Density of Radius
Plasma Amino Acids Concentration and Hematological Analysis
Mean weight of the right denervated radius in turkeys reached 6.1 ± 0.3 g and was significantly lower than 6.5 ± 0.2 g obtained in the left control bone (P = 0.03). However, the length of the right and the left radius did not differ. The comparison of the right denervated radius with the left control bone showed significant decrease in the values of geometrical and mechanical parameters as well as the volumetric bone mineral density (all P < 0.01; Table 1). The comparison of the investigated properties of the radius from the AKG and the control groups of turkeys is presented in Table 2. The weight of the radius of AKGtreated turkeys reached significantly higher value than in the control group of birds (P < 0.001), whereas the length of this bone did not differ. Moreover, the volumetric bone density, and geometrical and mechanical properties of the radius were significantly increased in turkeys that were administered AKG (all P < 0.05).
Blood samples were collected from animals at the age of 17 wk to heparinized tubes, 24 h after the last dosage of AKG. Moreover, blood was collected after 3-h fasting and centrifuged rapidly, just before their storage at −25°C. Before the measurement, 1 mL of each plasma sample was treated with 1 mL of 6% sulphosalicylic acid and centrifuged at 12,000 rpm for 15 min. The samples were subjected to automated ion-exchange chromatography with 5 lithium-based buffers on an Ingos AAA 400 amino acid analyzer (Ingos Corp., Czech Republic; Iłz˙ecka et al., 2003). Moreover, hematocrit, hemoglobin concentration, red blood cells and white blood cells were assessed.
Statistical Analysis All data are presented as means ± SEM. Differences between means were tested for statistical significance with a 2-way ANOVA (bone parameters) and Student’s t-test for nondependent variables (BW, amino acid concentrations, and hematological parameters) using SAS computer software (SAS Institute, 1998). Data were found to be normally distributed and have equal variance. Differences showing P < 0.05 were considered significant.
RESULTS BW At the beginning of the experiment, turkeys assigned to the AKG-treated group had a BW of 509 g ± 16, which was not significantly different from that of the control birds for which the BW was 510 g ± 15. At the end of experiment, the AKG-treated and the control birds reached 9.8 ± 0.4 kg and 8.8 ± 0.4 kg, respectively. The obtained differences of the final BW of turkeys were not statistically different either.
Hematological Analysis and Plasma Amino Acids Concentration The red blood cell count, hematocrit value, hemoglobin content, and white blood cell count were not significantly different in the control and AKG-treated turkeys. The analysis of plasma amino acid concentration showed that AKG administration in turkeys significantly increases plasma proline and leucine concentrations by 53 and 25%, respectively, when compared with the control birds (both P = 0.01). However, AKG treatment in turkeys significantly decreased plasma glutamine and taurine concentration from 742.2 ± 52.2 and 377.0 ± 55.5 nmol/mL obtained in the control birds to the values of 456 ± 10.9 and 221.8 ± 21.4 nmol/mL, respectively (P < 0.05; Table 3).
DISCUSSION Denervation of Radius as a Model for Osteopenia Investigation performed in rats showed that peripheral denervation of the pelvic limbs may serve as the experi-
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EFFECTS OF AKG ON BONE HOMEOSTASIS AND AMINO ACIDS Table 2. Characteristics of volumetric bone mineral density, morphological and mechanical properties of the radius in turkeys from the control and α-ketoglutarate-treated groups1 Investigated parameter
Control group (n = 14)
Bone weight (g) Bone length (mm) Bone mineral density (g/cm3) Cross-sectional area (mm2) Second moment of inertia (mm4) Mean relative wall thickness Maximum elastic strength (N) Ultimate strength (N)
5.6 120.8 1.429 12.1 33.7 0.368 116 148
± ± ± ± ± ± ± ±
0.2a 1.0a 0.028a 1.1a 3.4a 0.042a 13a 19a
AKG-treated group (n = 12) 7.1 121.1 1.608 16.5 51.2 0.482 184 226
± ± ± ± ± ± ± ±
0.2b 0.9a 0.015b 0.8b 2.5b 0.043b 13b 16b
Means for each investigated parameter that do not share common superscripts differ (P < 0.05). Values are means ± SEM.
a,b 1
mental model for osteopenia induction. Furthermore, bone denervation is complementary to other models for osteopenia induction like ovariectomy, gastrectomy, or glucocorticoids treatment (Ferretti et al., 1995; Lehto-Axtelius et al., 1998; Ito et al., 2002; Tatara et al., 2005). Wings in turkeys are suitable for investigations of bones under condition of their innervation and denervation. The neurectomy of the radialis and the medianoulnaris nerves in turkeys has no influence on locomotory functions of these birds and just slightly affects animal welfare, which is unquestionably advantageous when compared with other animal models of osteopenia using bone denervation (Tatara, 2003; Tatara et al., 2005). The results obtained in turkeys showed that long-term denervation of the radius induces significantly lower values of bone weight and volumetric bone mineral density measured in the distal metaphysis. The geometrical parameters like cross-sectional area, second moment of inertia, and mean relative wall thickness were significantly decreased in the denervated radius as well. As a conseTable 3. Plasma amino acids concentration (nmol/mL) obtained in the control and α-ketoglutarate-treated turkeys1 Amino acid Cysteic acid Taurine Threonine Serine Glutamate Glutamine Proline Glycine Alanine d-Amino-n-butyrate Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine
Control group (n = 6) 116 377 510 900 384 724 610 709 1058 26 498 194 116 195 335 264 141 81 338 113 391
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
21a 56a 18a 64a 36a 52a 70a 42a 76a 1a 27a 29a 23a 8a 12a 35a 6a 4a 41a 27a 63a
AKG-treated group (n = 7) 88 222 608 855 394 456 936 705 1121 25 582 239 130 227 418 266 155 95 511 100 434
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
8a 21b 51a 42a 13a 11b 83b 77a 102a 2a 44a 7a 9a 14a 14b 20a 7a 8a 106a 10a 30a
Means that do not share common superscripts differ (P < 0.05). Values are means ± SEM.
a,b 1
quence of bone denervation, a significant decrease in the bone mineral density and the geometrical properties of the radius evoked its lower mechanical endurance. The maximum elastic strength as well as the ultimate strength of the right denervated radius of turkeys were significantly reduced when compared to the left innervated bone by 29.5 and 35.7%, respectively. Changes in mineralization and mechanical and morphological properties of the radius prove an osteopenic effect obtained in this bone after long-term denervation, and these results are in agreement with earlier studies performed on the humerus in turkeys (Tatara et al., 2005). However, as opposed to the study performed by Edoff et al. (1997) in rats, this study showed that the denervation of the radius in turkeys had no effect on the bone length.
Effect of AKG on Bone Properties Positive influence of AKG on bone tissue is highlighted when considering the significant increase in such parameters as bone weight, cross-sectional area, second moment of inertia, mean relative wall thickness, maximum elastic strength, ultimate strength, and volumetric bone mineral density in turkeys after AKG administration. Obtained results from the right denervated and the left control radius of these birds indicate that AKG induces a beneficial effect not only in physiologically innervated bones but under conditions of bone denervation that were used to cause experimental osteopenia. These findings are generally in accordance with the results obtained from femur, tibia, humerus, and denervated ulna of turkeys (Tatara, 2003; Tatara et al., 2004a,b) and similar to effects observed in lambs after AKG administration (Harrison et al., 2004). Furthermore, positive influence of AKG administration on the skeletal system was observed in rats subjected to experimental ovariectomy (Bien´ko et al., 2002; Radzki et al., 2002); however, the mechanism responsible for these effects was not explained.
Plasma Amino Acids Concentration and Hematological Examination Investigations performed in pigs showed that enteral administration of AKG increases arterial plasma proline
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level, whereas its parenteral treatment decreases plasma concentration of glutamine (Kristensen et al., 2002). A similar effect of AKG on proline and glutamine peripheral blood plasma concentration was observed in this study; however, AKG was given in turkeys directly to the crop. Moreover, as opposed to a reduction in taurine plasma level, the significant increase of leucine plasma concentration by 25% in AKG-treated turkeys was obtained, and this effect was not reported previously. The positive response to AKG may be through proline and its effect on hydroxyproline for collagen synthesis. A positive response for leucine suggests it as a candidate for regulation of bone metabolism. This statement is supported by other investigations showing leucine as a special amino acid among the branched-chain amino acids promoting muscle-protein synthesis (Shimomura et al., 2004). It can be postulated that the anabolic effect of AKG on bone tissue is evoked by increased protein synthesis and mediated by proline and leucine. The analysis of basal hematological parameters in turkeys showed no effect of AKG administration on the hemopoietic processes. The red blood cell count, hematocrit value, and hemoglobin content were not changed. Moreover, the white blood cell count was not affected. Thus, the erythropoiesis seems to be within physiological range, even after 14 wk long oral AKG administration in these birds was performed. In conclusion, this study proved that oral administration of AKG positively influences bone mineralization and mechanical properties, confirming its beneficial effect on skeletal system development and bone tissue in turkeys tested for an osteopenic factor. The AKG may have its effect on the skeletal system of turkeys through increased concentration of plasma proline and leucine. Moreover, the proposed experimental model may be used for further investigations on skeletal development and its homeostasis maintenence in poultry.
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