INFLUENCE OF ALPHA-KETOGLUTARATE ON BIOSYNTHESIS OF
COLLAGEN, BONE MINERAL DENSITY AND OSTEOPOROSIS.
Hans Jungvid M.D., Protista International AB, Sweden
Under physiological conditions, maintenance of skeletal mass is the result of a tightly coupled process of complex multi-interactions between genetic, hormonal, physiological and mechanical factors.
Diseases of the skeletal system, including osteoporosis, arise when the delicate balance between these factors is disrupted, as for example, in postmenopausal women when oes-trogen level decreases, often very dramatically with the cessation of ovarian function. In quite many studies oestrogen deficiency is considered one of the most important factors leading to loss of bone mass. A better understanding of these relationships is very impor-tant and also necessary for the identification of the deterioration of the skeleton and, in future, risk of fracture Ref 1-16.
Alpha-ketoglutarate (AKG) is the nitrogen-free portion of the amino acids known as glutamine and glutamic acid. Both are natural compounds formed in Krebs cycle, the en-ergy-producing process that occurs in most cells. Glutamine requirements are increased during injury, in particular to sustain the needs of rapidly growing cells in healing of e.g. muscle tissue. AKG has been proved to be a potent precursor of glutamine Ref 17-21.
When it comes to cells that produce and maintain e.g. osteoblasts (bone forming cells that secrete unmineralised collagen), nothing is yet reported about the AKG mode of action. However, the aim of this review is to present data that proves the efficacy of AKG in im-proving bone mineral density (BMD), thereby significantly enhances the possibility of a reduced number of bone fractures Ref 31.
The skeleton contains 99% of the body’s calcium, which is stored in the bone as hy-droxyapatite [Ca10(PO4)6 (OH)2]. In addition to this mineral component, the bone tissue matrix also contains type I collagen, a type of protein. There are two different types of bone, cortical bone and trabecular bone. Cortical bone is denser and more calcified than trabecular bone and is found on the outside of bones and along the length of the long bones (i.e. the arms and legs).
A healthy adult bone is composed by e.g.:
• ~65% of hydroxyapatite.
• ~10% of collagen.
• A small amounts of magnesium, sodium, and bicarbonate.
• Water approximately 25%.
Biomechanics of Trabecular Bone
Trabecular bone, the porous type of bone, found in the spine and at all articulating joints, can be classified as a porous cellular solid, consisting of an irregular three-dimensional array of bony rods and plates, called trabeculae, which themselves are composed of a composite material. Bone marrow fills the spaces of the pores. In addition, because all free bone surfaces are covered with bone cells, bone is a living tissue that is self-healing and has the ability to adjust its morphology in response to changes in its mechanical envi-ronment, the so-called but poorly understood phenomenon of bone remodelling. As such, the mechanical complexity of this two-phase biological tissue surpasses any engineering material, making it a fascinating subject of study regardless of clinical applications. Trabe-cular bone is more susceptible to fracture later in life.
Bone Mineral Turnover
The skeleton is continually renewed through a process known as remodelling; a sequence of events whereby old bone is replaced by new bone (bone turnover). Modelling is the process by which bone growth occurs and where there is a higher rate of bone formation relative to bone loss.
Three types of cells produce and maintain bone:
• Osteoblasts (bone-forming cells) work at bone surfaces where they secrete osteoid (unmineralised collagen), modulate the crystallisation of hydroxyapatite and influ-ence the activity of osteoclasts.
• Osteoclasts (bone-resorbing cells) are responsible for the resorption (destruction) of old worn out bone, which is necessary for the repair of bone surfaces and the remodelling of bone.
• Osteocytes are osteoblasts, which have become embedded within the mineralised regions of bone. They are involved in the sensing and translation of information about the internal bone environment.
During bone growth, formation is higher than breakdown. After peak bone mass is achieved, the rates of breakdown and formation are equal and bone mass is thought to remain constant. As we get older, the rate of breakdown increases and exceeds the rate at which bone is formed. Overall, bone is lost and the skeleton becomes more fragile and prone to fractures Ref 22.
Picture 1 Ref. MRC-HNR:Research:Bone Health:Bone&Calcium
Classification of Osteoporosis
Osteoporosis can be classified in primary and secondary. Primary osteoporosis is a con-dition of reduced bone mass and fractures found in menopausal women (type I) or in older men and women (type II). Other causes of primary osteoporosis include idiopathic osteoporosis of childhood and hereditary conditions such as osteogenesis imperfecta and Marfan’s syndrome.
Type I osteoporosis is six times more frequent in women than in man, in women often beyond the age of 50. It represents a loss of trabecular bone after menopause, related to loss of estrogens. On the other hand, type II osteoporosis is only two times more frequent in women than in men, and occurs in more advanced ages (75 years or more). It is an age-related process.The loss of bone mass is slower and affects both cortical and trabe-cular bones. Low levels of active vitamin D will decrease intestinal absorption of calcium, causing secondary hyperparathyroidism and consequently bone atrophy Ref 23.
Low risk related factors in osteoporosis: strength and muscular resistance, multiparity, high calcium intake, moderate physical activity, obesity, fluor ingestion (added to water), drugs (estrogen, thiazides, diuretics and calcium supplementation).
High risk related factors in osteoporosis: smoking, alcoholism, long period of immobiliza-tion and inactivity, physical inactivity, primary amenorrhea, secondary amenorrhea, pre-cocious menopause (idiopathic, ophorectomy, hysterectomy), nuliparity, nutritional factors (low calcium intake, high caffeine consumption, low protein, fiber and sodium intake), drugs ( glucocorticoids, anticonvulsant, heparin, thyroxine), family history, low stature and small bones Ref 23.
How to Measure Bone Mineral Density?
In people the strength of bone is mostly determined using a technique called dual energy xray absorptiomety, or DEXA. Studies have shown that bone mineral density of the hip as measured by DEXA helps to predict whether a person will have a hip fracture. The risk of a fracture will double if the DEXA decreases by approximately 12%. The strength of the bone is largely determined by the bone mineral density (BMD) Ref 23.
Bone Turnover Markers
Aside from other osteometabolic diseases, such as renal osteodystrophy, osteoporosis is characterized by only slight increases in bone turnover; so, the evaluation of osteoporosis requires highly sensitive markers. In general, these substances represent either a me-tabolite of bone matrix breakdown, such as pyrridinoline or have an enzymatic activity related to bone formation, such as alkaline phosphatase. It is thought that these markers, along with densitometric studies, would help the identification of women with rapid loss of bone mass, allowing an earlier diagnosis.
Markers of bone formation include osteocalcin, alkaline phosphatase and type I procolla-gen extension peptide. All of them are secretory products of osteoblasts during bone ma-trix synthesis. Of these, the first two are available for clinical use and show correlation with bone formation rate.
Alkaline phosphatase is the most used marker to estimate bone formation, but it is not specific for the bone as it includes other sites of production, such as the liver and small intestine. In the absence of other conditions that interfere with alkaline phosphatase activ-ity, this marker will indirectly represent bone formation. Nowadays the measurement of a specific alkaline phosphatase derived from osteoblasts it is available. Several studies showed that osteocalcin is a more sensitive marker than total alkaline phosphatase in de-termining bone formation.
Markers of bone reabsorption include urinary hydroxyproline and piridinoline, both of which reflect collagen breakdown. Hydroxyproline is an aminoacid essentially unique to collagen and is not catabolized in the body. It is derived from various types of collagen and thus it is not specific of bone tissue. It is neither a sensitive method as it is metabo-lized in the liver. Pyrridinoline and desoxipyrridinoline are specific for bone turnover and are not metabo-lized in vivo, thus having more specificity and sensitivity than hydroxyproline. The simultaneous study of bone reabsorption and formation by these multiple markers has more applicability than the study of one unique marker Ref 23.
Table 1 Common osteoporosis therapy strategies
Strategy Decreased resorbtion Increased formation
Effect on bone mass Stabilize Increase
Effect on bone cells Decrease osteoclast activity Increase osteoblast activity
Examples of strategy Estrogen, calcitonin, Fluoride, Vitamin D3
Bisphosphonates parathyroid hormone (PTH)
Strontium ranelate Mixed effects
Picture 2. This image shows trabecular bone structure in the lower spine of a young adult compare to an osteoporotic elderly adult
Role of Diet in the Prevention of Osteoporosis
In recent years, much attention has been directed toward the prevention of osteoporosis, since this disease has become a leading cause of morbidity and mortality in elderly woman. Research has demonstrated that the prevention of osteoporosis and osteoporo-sis-related fractures may best be achieved by initiating sound health behaviours early in life and continuing them throughout life. Several reports have shown that the adequate consumption of nutrients, calcium in particular, during the pre-pubertal and early post-pubertal years of females contribute to increased peak bone mass. In fact, healthy early life practices, including adequate consumption of most nutrients, regular physical activity, and other health behaviours, contribute to greater bone mineral measurements and opti-cal peak bone mass by the fourth decade of life of females, and, perhaps, also of males .
The role of dietary protein in bone health is controversial whereas a high intake of certain proteins may be beneficial for bone health.
Several reports discuss the positive effect of vegetable protein, or high level of protein intake, whereas low levels of protein intake or protein of animal origin may be unfavourable Ref 24-27.
Almost no reports exist, on the effect on osteoporosis of increased dietary levels of certain amino-acids in comparison with low dietary protein levels.
The Role of Collagen in Bone Strength
Bone is a complex tissue of which the principal function is to resist mechanical forces and fractures. Bone strength depends not only on the quantity of bone tissue but also on the quality, which is characterized by the geometry and the shape of bones, the micro archi-tecture of the trabecular bones, the turnover, the mineral, and the collagen. Different de-terminants of bone quality are interrelated, especially the mineral and collagen, and analysis of their specific roles in bone strength is difficult. The interactions of type I colla-gen with the mineral and the contribution of the orientations of the collagen fibers when the bone is submitted to mechanical forces is of course very important. Different proc-esses of maturation of collagen occur in bone, they result either from enzymatic or non enzymatic processes. The enzymatic process involves activation of lysyl oxidase, which leads to the formation of immature and mature crosslinks that stabilize the collagen fibrils. Two types of non enzymatic processes are described in type I collagen: the formation of advanced glycation end products due to the accumulation of reducible sugars in bone tissue, and the process of racemization and isomerization in the telopeptide of the colla-gen. These modifications of collagen are age-related and may impair the mechanical properties of bone. The role of the crosslinking process of collagen in bone strength, clini-cal disorders associated with bone collagen abnormalities and bone fragility, such as os-teogenesis imperfecta and osteoporosis is evident Ref 28-30.
Alpha-ketoglutarate and Bone Strength
Studies in man and animals (pigs) have shown that Alpha-ketoglutarate (AKG) increases serum proline, the precursor of hydroxyprolin and the main component of bone collagen type I Ref 31-33. In a randomized double-blind, six month clinical, phase II, study conducted at the Osteoporosis Outpatient Department of Institute of Agriculture Medicine (IAM) in Lub-lin, Poland, the effect of Ca-AKG in comparison to CaCO3 on bone mass, the C-terminal cross-linking telopeptide of type I collagen (CTX) and Osteocalcin (OC) serum levels in post menopousal women with Ostopenia was investigated. In total 76 post menopousal women between the age of 45-65 were randomly selected to have either Ca-AKG (1,68g CaCO3 and 6,00g AKG) or 1,68g Ca (CaCO3) daily. The total daily intake of Ca was estimated and standardized. The primary end-points were CTX and OC serum levels. The secondary end-points were spine body-mass density (BMD) meas-ured by DEXA.
The maximum decrease of the mean CTX level in the Ca-AKG group was observed after 24 weeks (37,0%, p=0,006). The differences in CTX between the groups were statistically significant after 3 and 6 month (p=0,03). The OC serum level was not affected by Ca-AKG. The BMD in the Ca-AKG group increased by 1,6% (p=0,04) after six month.
Additional studies in pigs, lambs and turkeys, all confirm a significant improvement on bone mineral density (BMD) and bone mineral content (DEXA method) when AKG or Or-nithine-AKG were given orally at a level of 0,4 g/kg BW and day Ref 35-40.
Alpha-ketoglutarate (AKG) is the nitrogen-free portion of the amino acids known as glutamine and glutamic acid. It is formed in the Krebs cycle, the energy-producing proc-ess that occurs in most body cells. AKG is used by the cells during growth and in healing of injuries and other wounds, and is especially important in the healing of muscle tissue. A controlled study found that intravenous AKG prevented a decline in protein synthesis in the muscles of patients recovering from surgery. For these reasons, it has been specu-lated that oral AKG supplements might help improving strength or muscle-mass gains by weightlifters, but no research has been done to test this theory Ref 17-20.
Intravenous AKG has also been used during heart surgery to protect the heart muscle from damage but no research has revealed whether oral AKG might be an effective treatment for heart disease or an effective drug in improving heart function . Chemical structure and other characteristics (encl. A).
Ornithine alpha-ketoglutarate (OKG) is a salt formed of two molecules ornithine and one molecule of alpha-ketoglutarate.
The Glutamate Family
The amino acids glutamate, glutamine, proline and arginine are members of this family. Proline is generated by a ring formation under consumption of one molecule of NADH + H+, NADPH + H+ and ATP each. This points out how energy-consuming biosyntheses are. Since it will not be mentioned explicitly in the following discussion, it should be kept in mind that every comparable biosynthetic pathway is fuelled by energy amounts of the same scale. Proline contains no primary but a secondary amino group and is therefore actually an alpha-imino acid (the name-giving feature is the >NH group), but it is nevertheless referred to as an amino acid. It is the one exception to the general structure of amino acids.
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Picture 3 Glutamate dehydrogenase reaction: The starting compounds are alpha-ketoglutaric acid and NH4+
Formation of Proline from Alpha-Ketoglutarate via Glutamate and Glutamine. The conver-sion of proline to hydroxyproline occurs only at proline residues that are part of a polypeptide chain .
Picture 5, Biosynthesis of proline from glutamate
Chemical and biological safety of AKG
Food and Drug Administration FDA
The EAFUS list (Food Additive Database) contains information on Alpha-ketoglutarate (EAF 3316 under the synonym 2-oxopentanedioic acid). Product classified under EAF means that use of the product is reported but it has not yet been assigned for a toxicology literature search (encl B).
FAO/WHO Expert Committee on Food Additives
Summary of evaluation performed (FEMA No 3891, JECFA No 634, Functional class; fla-vouring agent) comments that there is “no safety concern at current levels of intake when used as a flavouring agent”. Report TRS 896-ECFA 53/67 (encl C).
Efficacy of Alpha-ketoglutarate on bone mineral density (BMD) is covered by a world wide patent application (encl D).
Increased bone loss is associated with a continuous hyper-metabolic state in bone tissue, with a collagen-related protein catabolism , . Increased bone collagen type I breakdown is one of the key factors responsible for the dramatic decrease of bone mass observed in women after menopause. In elderly the common reduced energy and protein intake have a crucial impact on the prevalence of the disease. Nutritional treatment plays an important role in preventing and treating osteoporosis, though most of the dietary recommendations focus on calcium and vitamin D intake 26, , .
It is possible that conventional diets or metabolic processes in the gut of especially elderly (postmenopausal) people are unable to provide the required amount of specific amino acids, which are of an extraordinary importance in patients with rapid loss of bone. While the availability of proline is essential for bone pro-collagen synthesis, this amino acid might easily be underestimated in the modern diet, based on vegetables or low amounts of certain amino-acids, especially proline. Hydroxyproline, one of the most desired amino-acids in synthesis of pro-collagen - common in high protein diets -, is easily absorbable, however metabolically inert and can not participate in the anabolic process aimed to in-crease the pro-collagen availability. Therefore the need for proline, possibly biosynthe-sised from AKG, as an energy donor or catalyst in conversion of pro-collagen to collagen during hydroxylation of proline to hydroxyproline is an obvious choice.
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Rafal S. Filip1, Birger Lindegard 2, Jan Wernerman3, Agnieszka Haratym-Maj4, Tadeusz Studzinski5, Malgorzata Podgurniak6, Eva Linné Larsson7, Karl-Johan Öhman7, Stefan G. Pierzynowski8. To be published in Bone.
1 Department of Bone Metabolich Diseases, Inst of Agr Med, Lublin Poland
2 Department of Nephrology, University Hospital, Lund Sweden
3 Department of Anaesthesia and Intensive Care, Karolinska Inst, Huddinge Univ. Hospital, Sweden
4 Departments of Pathophysiology, Dep. of Gynaecology, Inst. of Agr. Med. Lublin Poland
5 Departments of Animal Physiology, Agr. University, Lublin Poland
6 Departments of Statistics, Warsaw Agr University, Lublin Poland
7 Gramineer International AB, Lund Sweden
8 Departments of Cell and Organism Biology, Lund University, Sweden.
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