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Glucosamine: A Glycobiology Primer
 
Glucosamine and its epimer galactosamine are found in large structural molecules termed proteoglycans (sugar chains bound to a protein core) and glycoproteins (proteins with some sugars attached). Hyaluronan, also known as hyaluronic acid (very long copolymer of N-acetyl-D-glucosamine and glucuronic acid) and chondroitin sulphate (very long copolymer of N-acetylgalactosamine and glucuronic acid) are two such structural molecules. 
to view an enlargement, place mouse here 1. Proteoglycans and Glycoproteins
Proteoglycans and glycoproteins are found on the cell surface and in the extracellular matrix. Proteoglycans function as lubricants and support elements in connective tissue. Proteoglycans on the cell surface interact with the extracellular matrix. By absorbing large volumes of water they form solutions with high viscosity and elasticity. Glycoproteins have been shown to be determinants of the group behaviour of cells and lubricants of epithelial tissue.  Glycoproteins on the cell surface have a role in communication, cell structure, and self-recognition by the immune system. Many peptide hormones are glycoproteins. For instance follicle stimulating hormone (FSH)  is a glycoprotein secreted by the pituitary gland, which acts on the gonads. Common sugar units in the carbohydrate chains of glycoproteins include glucose, galactose, mannose, fucose, N-acetylgalactosamine and N-acetyl-D-glucosamine.  The basic structure of a proteoglycan consists of a protein core with one to hundreds of carbohydrate chains, termed glycosaminoglycans, covalently bound to a specific serine amino acid. Proteoglycans are distinguishable from glycoproteins on the basis of their bound carbohydrates. The saccharides bound to glycoproteins lack a serial repeat unit whereas glycosaminoglycans consist of repeating disaccharide subunits. An uronic acid and a hexose, most often N-acetyl-D-glucosamine or its epimer N-acetylgalactosamine, comprise the disaccharide subunit.  to view an enlargement, place mouse here Although our understanding of the biological functions of proteoglycans and glycoproteins continue to evolve, information to date establishes a role for these macromolecules as lubricants, shock absorbers, and structural components of connective tissue.2. Hyaluronan
Hyaluronan is an important class of glycosaminoglycans. Also known as hyaluronic acid, it is widely distributed in the skin, cartilage and most body fluids such as the synovial fluid of the joints, vitreous humour of the eye, and the umbilical cord.  A copolymer of N-acetyl-D-glucosamine and glucuronic acid, hyaluronan is noted for its capacity to bind huge amounts of water in excess of its own weight.  to view an enlargement, place mouse here Due to hyaluronan's large molecular weight, polyelectrolyte character and the large volume it occupies in solution, the glycosaminoglycan is thought to be a good lubricant and shock absorber. Current uses of hyaluronan include the injection of hyaluronan into osteoarthritic knees. (Brand named products include Synvisc>®<, Suplasyn>®< and Hyalgan>®<.) Though used primarily for pain relief, current research proposes a role for hyaluronan in the structural modifications of joints1. Also of growing interest is hyaluronan's role in wound healing. It has been shown to be essential in the scarless healing of fetal wounds2,  and current research aims to evaluate its potential in adult wound healing. Unlike glycosaminoglycans in general hyaluronan is synthesized as a free carbohydrate chain. Activated glucuronic acid and activated N-acetyl-D-glucosamine react in the plasma membrane to form the basic unit of hyaluronan in a reaction catalyzed by hyaluronan synthetase.  to view an enlargement, place mouse here Many growth factors and cytokines are known to activate its biosynthesis.3. Chondroitin Sulphate
Chondroitin sulphate is the main glycosaminoglycan of cartilage. It is a prominent component of tendons, ligaments, and the aorta and has been isolated from brain, kidney and lung tissue. Chondroitin is a copolymer of N-acetylgalactosamine and glucuronic acid. N-acetylgalactosamine is obtained from N-acetyl-D-glucosamine in a reversible epimerization reaction. The glycosaminoglycan is synthesized off of specific serine amino acids in a protein core. In a reaction catalyzed by glycosyltransferase, activated glucuronic acid is covalently bound to galactose. Galactose is part of a triple sugar linkage consisting of xylose-galactose-galactose, from which the chondroitin sulphate glycosaminoglycan will grow.  Another glycosyltransferase then catalyzes the addition of activated N-acetylgalactosamine to the growing carbohydrate chain. Sulphation takes place as the sugar chain is growing with the aid of sulphotransferases, which catalyze the transfer of sulphate from phosphoadenosine phosphosulphate (PAPS) to carbon 4 or 6 of N-acetylgalactosamine.  to view an enlargement, place mouse here The degree and distribution of sulphation, in addition to copolymer length, determine the chemical, physical, and biological properties of this proteoglycan. It is important to note that sulphation unlike phosphorylation is generally a permanent modification. Thus sulphation plays a determining role as opposed to a regulatory role in a molecule's biological activity. Glucose is regarded exclusively as a source of carbohydrate fuel in energy producing metabolic pathways. However, the molecule is involved in a myriad of anabolic pathways. One such pathway results in the synthesis of UDP-N-acetyl-D-glucosamine, a precursor for the diverse and widely distributed proteoglycan and glycoprotein macromolecules.  Hyaluronan and Chondroitin sulfate are just two examples of widely distributed molecules that utilize N-acetyl-D-glucosamine directly or indirectly in the form of its epimer N-acetylgalactosamine.  Mammals including humans have the molecular machinery to synthesize these nucleotide sugars endogenously from glucose. The pathway, often referred to as the hexosamine biosynthesis pathway,  involves the transamidation and N-acetylation of glucose followed by the activation of the N-acetylated amino sugar. The activated acetylated sugar e.g. N-acetyl-D-glucosamine or N-acetylgalactosamine, is then used in the synthesis of glycoproteins and proteoglycans. The synthetic steps are outlined below. Glucose undergoes phosphorylation and isomerization en route to becoming an amino sugar. Hexokinase catalyzes the irreversible phosphorylation of α-D-Glucose on carbon six. The resulting molecule, α-D-Glucose 6-phosphate, can no longer leave the cytosol due to the hydrophilicity imparted by the negatively charged phosphate ester. Phosphoglucose isomerase then catalyzes the reversible isomerization of Glucose 6-phosphate to Fructose 6-phosphate. Glucosamine 6-phosphate, an amino sugar, is then synthesized from fructose 6-phosphate via transamidation of the amide nitrogen of glutamine.  to view an enlargement, place mouse here The reaction is catalyzed by glutamine fructose-6-phosphate amidotransferase, and is considered the rate-limiting step for glucose utilization by the hexosamine pathway3. Acetyltransferase catalyzes the N-acetylation of Glucosamine 6-phosphate into N-acetyl-D-glucosamine 6-phosphate. to view an enlargement, place mouse here N-acetyl-D-glucosamine 6-phosphate isomerizes to N-acetyl-D-glucosamine 1-phophate, which upon the addition of uridine triphosphate (UTP) is converted to UDP-N-acetyl-D-glucosamine, a nucleotide diphosphate sugar. to view an enlargement, place mouse here Pyrophosphate is also formed, and its rapid hydrolysis by a pyrophosphorylase drives the reaction forward. UDP-N-acetyl-D-glucosamine can then be epimerized to UDP-N-acetylgalactosamine. to view an enlargement, place mouse here UDP coupling activates the sugar for use in the synthesis of proteoglycans and glycoproteins.For use in macromolecular assembly glucosamine and N-acetyl-D-glucosamine must be converted into UDP-N-acetyl-D-glucosamine. There are fewer steps in the conversion of N-acetyl-D-glucosamine since it enters the hexosamine biosynthetic pathway downstream of glucosamine's entrance.  Both supplements are ingested and absorbed from the gut. Feeding experiments have shown that glucosamine absorption from the intestinal tract occurs slowly whereas N-acetyl-D-glucosamine absorption occurs somewhat faster4.  Before becoming N-acetyl-D-glucosamine 6-phosphate, which can be isomerised and activated into UDP-N-acetyl-D-glucosamine for use in proteoglycan and glycoprotein synthesis, glucosamine must be phosphorylated and acetylated.  to view an enlargement, place mouse here Glucosamine's intracellular transport and phosphorylation is believed to occur by the same mechanism as glucose5. Unlike glucosamine, N-acetyl-D-glucosamine need only be phosphorylated before joining the endogenous pathway. Phosphorylation occurs in the cytosol by N-acetyl-D-glucosamine kinase6. to view an enlargement, place mouse here The rate-limiting step of N-acetyl-D-glucosamine entry in the hexosamine pathway appears to be intracellular transport7. Though, glucosamine and N-acetyl-D-glucosamine may seem equivalent in their biological utility with N-acetyl-D-glucosamine being a mere step ahead of glucosamine that one step can represent a significant hurdle. Acetylation is not as simple a task as presented in the endogenous pathway. First acetyl-Coenzyme A is synthesized from acetate, coenzyme A, and adenosine triphosphate in a reaction catalyzed by acetylcoenzyme A synthetase. The acetyl group can then be transferred to glucosamine 6-phosphate with the aid of glucosamine 6-phosphate acetyltransferase. Although acetylation of glucosamine 6-phosphate is thought to occur rapidly8, little supporting evidence has been published.  In fact common pharmaceutical agents may inhibit acetylation. The presence of salicylic acid inhibits Glucosamine-6-phosphate acetyltransferase, and at very low concentrations salicylic acid also inhibits the formation of acetyl-CoA by acetyl coenzyme A synthetase thereby preventing the conversion of Glucosamine-6-phosphate to N-acetyl-D-glucosamine-6-phosphate9.  In addition to interference of acetylation by common active agents, certain diseases and conditions have been documented in which the N-acetylation of the amino sugar is relatively deficient10 11. N-acetyl-D-glucosamine, as an acetylated version of glucosamine should be spared such regulatory ordeals. N-acetyl-D-glucosamine, as an integral precursor in the biosynthesis of proteoglycans and glycoproteins, has tremendous biological utility. Although our body engages in the endogenous production of N-acetyl-D-glucosamine, glucosamine is a popular supplement. N-acetyl-D-glucosamine is also available as a supplement. Glucosamine and N-acetyl-glucosamine supplements have shown positive clinical benefits in a variety of conditions12 13 14 15 16 17 18. Supplementation has its advantages: The exogenous administration and activation of N-acetyl-D-glucosamine from glucosamine closely resembles the endogenous pathway, only it is significantly reduced in length. Both supplements enter the endogenous pathway at a point which bypasses the rate limiting transamidation step of the hexosamine pathway19. Glucosamine enters the hexosamine pathway beyond the rate limiting step catalyzed by glutamine fructose-6-phophate amidotransferase. N-acetyl-D-glucosamine enters even further down the anabolic pathway bypassing both the rate limitng transamidation step and the acetylation step. Glucose on the other hand is usually oxidized to carbon dioxide in energy producing reactions or stored as glycogen for later use. The hexosamine biosynthetic pathway accounts for a very small amount of glucose utilization. Thus glucosamine supplements are more committed than glucose to becoming UDP-N-acetyl-D-glucosamine and ultimately glycoproteins and proteoglycans.  Glucosamine is essentially glucose with an amino group instead of a hydroxyl attached to the molecule's second carbon. N-acetyl-D-glucosamine is one step further along the metabolic pathway with the addition of an acetyl group attached to the amine group.  Glucosamine supplements are usually prepared from the shell of crustaceans. This hard chitin covering is a biopolymer of N-acetyl-D-glucosamine (chemical name: acetamido-2-deoxy-D-glucose). Chitin is the animal counterpart of cellulose, the structural polymer of the plant kingdom, which is also a glucose polymer. Chitin is insoluble in water, dilute acids, dilute and concentrated alkalies, alcohol and most other organic solvents. To prepare glucosamines from chitin the macromolecule is treated with strong acid. Chitin treatment with concentrated sulphuric acid yields glucosamine sulphate and treatment with concentrated hydrochloric acid yields glucosamine hydrochloride. Both products are glucosamine salts, which when put in solution dissociate into glucosamine free base and the sulphate or chloride anions.  Glucosamine sulphate should be clearly distinguished from chondroitin sulphate, which is not a salt. In chondroitin sulphate (and heparin sulphate), the sulphate replaces a hydroxyl group and is covalently bound to carbon, usually carbon 4 or 6 of the glucopyranose. The active part of a glucosamine salt is the free base glucosamine; the sulphate or chloride ions have no biological activity with respect to glucosamine.  to view an enlargement, place mouse here Glucosamine sulphate is a very hygroscopic substance and thus difficult to formulate. Crystallizing glucosamine sulphate with a sodium or potassium salt reduces its hygroscopicity making it possible to use in commercial preparations. The table below lists the various commercially available salts of glucosamine and the free glucosamine content.
A recent analysis of commercially available glucosamine supplements on the Canadian market was published in the Journal of Rheumatology. The authors analysed each preparation for the content of glucosamine base. Only one product out of 14 tested contained 100% of the amount of glucosamine base claimed on the label. The majority of preparations contained less than 60% of the stated amount of glucosamine20. This oversight in formulation may explain why some clinical studies have demonstrated positive outcomes while others have not: glucosamine content and not the salt content is important for biological activity. N-acetyl-D-glucosamine, because of its different physical properties, can be made available in pure powder with no additives. The acetyl group on the amine gives the molecule increased stability, and it is not hygroscopic21. Whereas the glucosamine melts at 88oC and decomposes at 110oC, N-acetyl-D-glucosamine has a melting point of 204oC and can be used in high temperature baking and hot beverages.  N-acetyl-D-glucosamine is a pleasant sweet tasting powder and can be added to hot beverages such as tea or coffee as a mild sweetener. The salts of glucosamine on the other hand are bitter and generally have an unpleasant taste, which in conjunction with their lower melting point make for poor food additives.  Chondroitin sulphate is a copolymer of N-acetylgalactosamine and glucuronic acid.  to view an enlargement, place mouse here Sulphation takes place at either carbon 4 or 6 of cyclic N-acetylgalactosamine, and the sulphate groups are covalently bound to the molecule. Covalent bonding of the sulphate group precludes sulphate dissociation, so unlike the monosaccharide salt glucosamine sulphate the sulphate groups of chondroitin contribute to the compound's biological activity. Isolated chondroitins are glycosaminoglycans chemically separated from their protein core. These polymers may contains 30 to 50 copolymer subunits and have molecular weights ranging from 15,000 to 25,000 kilo Daltons, to view an enlargement, place mouse here Commercial products containing chondroitin sulfate do not indicate the molecular size distribution of the chondroitin sulphate molecule in the preparations.Chondroitin sulphates have recently enjoyed popularity as supplements administered alone or in combination with glucosamine. Their popularity, however, is by no means an accurate reflection of biological utility; there are a number of formidable biological obstacles to the incorporation of chondroitin supplements into proteoglycans. These include, the nature of digestion and absorption, the course of endogenous sulphation, and the features of macromolecular assembly. A look at how chondroitin's physical and chemical properties hinder its involvement in each of these biological processes is warranted. As large polymers, chondroitins are not likely to be absorbed as ingested. One of the main functions of the digestive system is the break down of macromolecules into readily absorbable constituent subunits. The smallest subunit of chondroitin, which could still be considered a molecule of chondroitin would be a disaccharide of glucuronic acid and either sulphate or unsulphated N-acetylgalactosamine.  However, it is generally not possible to absorb disaccharides from the gastrointestinal tract. In fact a common disorder, lactose intolerance, results from the inability to absorb the lactose disaccharide. This molecule stays in the gut and through osmotic pressure draws water into the lumen leading to diarrhea. Supplementation with the enzyme lactase breaks down the disaccharide into its constituent parts- galactose and glucose, and these monosaccharides are readily absorbed. Thus chondroitin sulphate is most likely absorbed in the form of its constituent monosaccharides, glucuronic acid and N-acetyl-D-galactosamine. Since N-acetyl-D-glucosamine and N-acetyl-D-galactosamine are epimers, this is tantamount to N-acetyl-D-glucosamine supplementation. Even if chondroitin sulphate could be absorbed, its biological utility would still be questionable. Endogenous sulphation occurs in conjunction with polysaccharide growth, and the sulphation pattern, as dictated by the specific sulphotransferase involved, determines the overall chemical, physical, and biological properties of a proteoglycan22. The incorporation of exogenously sulphated glycosaminoglycans into proteoglycans, if it occurs at all, may not have the correct sulphation pattern to yield a molecule with the desired properties. The addition of sugars to proteins is a post-translational event. That is to say that the sugars are added after the protein chain is formed. The sugar chains are built up from pools of uridine diphosphate monosaccharides (UDP-sugars). For the purposes of efficient and thrifty macromolecular assembly, these pools of activated sugars serve as standardized sugar sources for use in the assembly of numerous classes of macromolecules. For instance, a common pool of N-acetylgalactosamine would be drawn upon in the synthesis of both proteoglycans and glycoproteins.  If this pool contained sulphated N-acetylgalactosamine the molecular properties of glycoproteins and proteoglycans would be dramatically altered. Thus any absorbed sulphated N-acetylgalactosamines from the digestion of chondroitin sulphates are likely further metabolized in the lysosomes resulting in the removal of the sulphate group. Known biochemical pathways would favour a complete breakdown of chondroitin to the level of at least the N-acetylgalactosamine. In this regard, chondroitin supplementation is an inefficient and more costly version of N-acetyl-D-glucosamine supplementation. 1 Altman, R. D. (2003). "Status of Hyaluronan Supplementation Therapy in Osteoarthritis." Curr Rheumatol Rep 5(1): 7-14. 2 Anderson, I. (2001). "The Properties of Hyaluronan and its Role in Wound Healing." Prof Nurse 17(4): 234-5. 3 Ashcroft, S. J. (1980). "Glucoreceptor mechanisms and the control of insulin release and biosynthesis." Diabetologia 18(1): 5-15. 4 Gaulden, E. C. and W. C. Keating (1964). "The effect of intravenous N-acetyl-D-glucosamine on the blood and urine sugar concentrations of normal subjects." Metabolism 13(5): 466-72. 5 Virkamaki, A. and H. Yki-Jarvinen (1999). "Allosteric regulation of glycogen synthase and hexokinase by glucosamine-6-phosphate during glucosamine-induced insulin resistance in skeletal muscle and heart." Diabetes 48(5): 1101-7. 6 Hinderlich, S., M. Berger, et al. (2000). "Molecular cloning and characterization of murine and human N-acetylglucosamine kinase." Eur J Biochem 267(11): 3301-8. 7 Ashcroft, S. J. (1980). "Glucoreceptor mechanisms and the control of insulin release and biosynthesis." Diabetologia 18(1): 5-15. 8 Virkamaki, A. and H. Yki-Jarvinen (1999). "Allosteric regulation of glycogen synthase and hexokinase by glucosamine-6-phosphate during glucosamine-induced insulin resistance in skeletal muscle and heart." Diabetes 48(5): 1101-7. 9 Kent, P. W. and A. Allen (1968). "The biosynthesis of intestinal mucins. The effect of salicylate on glycoprotein biosynthesis by sheep colonic and human gastric mucosal tissues in vitro." Biochem J 106(3): 645-58. 10 Burton, A. F. and F. H. Anderson (1983). "Decreased incorporation of 14C-glucosamine relative to 3H-N-acetyl glucosamine in the intestinal mucosa of patients with inflammatory bowel disease." Am J Gastroenterol 78(1): 19-22. 11 Pouyssegur, J. and I. Pastan (1977). "Mutants of mouse fibroblasts altered in the synthesis of cell surface glycoproteins. Preliminary evidence for a defect in the acetylation of glucosamine 6-phosphate." J Biol Chem 252(5): 1639-46. 12 Reginster, J. Y., R. Deroisy, et al. (2001). "Long-term effects of glucosamine sulphate on osteoarthritis progression: a randomised, placebo-controlled clinical trial." Lancet 357(9252): 251-6. 13 Pavelka, K., J. Gatterova, et al. (2002). "Glucosamine sulfate use and delay of progression of knee osteoarthritis: a 3-year, randomized, placebo-controlled, double-blind study." Arch Intern Med 162(18): 2113-23. 14 Das, A., Jr. and T. A. Hammad (2000). "Efficacy of a combination of FCHG49 glucosamine hydrochloride, TRH122 low molecular weight sodium chondroitin sulfate and manganese ascorbate in the management of knee osteoarthritis." Osteoarthritis Cartilage 8(5): 343-50. 15 Noack, W., M. Fischer, et al. (1994). "Glucosamine sulfate in osteoarthritis of the knee." Osteoarthritis Cartilage 2(1): 51-9. 16 Thie, N. M., N. G. Prasad, et al. (2001). "Evaluation of glucosamine sulfate compared to ibuprofen for the treatment of temporomandibular joint osteoarthritis: a randomized double blind controlled 3 month clinical trial." J Rheumatol 28(6): 1347-55. 17 Breborowicz, A., M. Kuzlan-Pawlaczyk, et al. (1998). "The effect of N-acetylglucosamine as a substrate for in vitro synthesis of glycosaminoglycans by human peritoneal mesothelial cells and fibroblasts." Adv Perit Dial 14: 31-5. 18 Salvatore, S., R. Heuschkel, et al. (2000). "A pilot study of N-acetyl glucosamine, a nutritional substrate for glycosaminoglycan synthesis, in paediatric chronic inflammatory bowel disease." Aliment Pharmacol Ther 14(12): 1567-79. 19 Virkamaki, A. and H. Yki-Jarvinen (1999). "Allosteric regulation of glycogen synthase and hexokinase by glucosamine-6-phosphate during glucosamine-induced insulin resistance in skeletal muscle and heart." Diabetes 48(5): 1101-7. 20 Russell, A. S., A. Aghazadeh-Habashi, et al. (2002). "Active ingredient consistency of commercially available glucosamine sulfate products." J Rheumatol 29(11): 2407-9. 21 Log Book File # PAT 1.2.01 22 Brockhausen, I. (2003). "Sulphotransferases acting on mucin-type oligosaccharides." Biochem Soc Trans 31(2): 318-25. | |
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Notice: The information is intended to provide the reader with a comprehensive understanding of the science underlying the role of glucosamine and N-acetylglucosamine in the mammalian body. The scientific information on this site has been compiled from many different scientific sources. No specific therapeutic claims are made for any product. The information provided on this site is for general information purposes only and is not intended to qualify, supplement or replace that of your medical professional. These statements have not been evaluated by the Food and Drug Administration. This product is not intended to diagnose, treat, cure, or prevent any disease. Your healthcare giver should undertake diagnosing and treating your medical condition.
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