Effects of oral glucosamine and chondroitin sulfate alone and in combination on the metabolism of SHR and SD rats

Glucosamine (G), often combined with chondroitin sulfate (CS), is a popular natural supplement used widely to treat osteoarthritis. However, use of glucosamine has been linked to development of insulin resistance. To assess the association between glucosamine and insulin resistance more closely, we challenged two rat strains highly sensitive to sugarinduced insulin resistance – SpragueDawley (SD) and Spontaneously Hypertensive (SHR) rats. Since elevations of systolic blood pressure (SBP) have been found to be an early and highly sensitive sign of insulin resistance in these two rat strains, we used this parameter as our primary endpoint. Four groups of both rat strains received either no agent (control), G, CS, or a combination of both for 9 weeks. The intake of each agent was calculated to be approximately 3–7 times comparable to human dose. Throughout the study, SBP of both strains consuming the two ingredients alone and in combination were not elevated. Rather, they were significantly lower than control, contrary to what is found in glucoseinduced insulin resistance in rats. Over the study period, body weights of the four groups of SD and SHR did not vary significantly. Furthermore, no consistent trends in circulating glucose concentrations were found among the four different groups in the two strains after oral challenge with glucose. Finally, no significant histological differences were found in hearts, kidneys, and livers among the various groups of SHR and SD. From the above result, we conclude that glucosamine and chondroitin sulfate given alone or together do not produce insulin resistance or other related perturbations in two rat strains highly sensitive to sugarinduced insulin resistance.     

Antihypertensive and metabolic effects of whole Maitake mushroom powder and its fractions in two rat strains

Maitake mushroom has been reported to favorably influence hypertension and diabetes mellitus. The purpose of this study was to compare the effects of whole Maitake mushroom powder and two extracts designated as ether soluble (ES) and water soluble (WS) on Zucker fatty rats (ZFR), a model of insulin resistance, and on spontaneously hypertensive rats (SHR), a model of genetic hypertension. In the initial study, we followed four groups of eight ZFR and SHR receiving special diets: a baseline diet (BD), BD + whole Maitake mushroom powder (20% w/w), BD + fraction ES (0.10% w/w), and BD + WS (0.22% w/w). Different effects of these dietary regimens on the 2 rat strains were found. At 35 days, only consumption of the ES diet significantly decreased systolic BP (SBP) in SHR (average 197 vs. 176 mm Hg, p < 0.001), while in ZFR only the groups consuming the whole Maitake and WS diets showed significantly decreased SBP (138 vs. 120–125 mm Hg, p < 0.001). A challenge test with losartan (an angiotensin II receptor blocker) indicates that angiotensin II does not play a major role in SBP regulation of ZFR, but does in SHR where consumption of ES relative to other groups significantly lowered activity of this system. In SHR, glucose, cholesterol, circulating insulin and HbA1C were virtually similar among all dietary groups; but whole Maitake (–22%), ES (–120%) and WS (–80%) diets were associated with decreased triglycerides, and the ES diet with lowered serum creatinine (–29%). In ZFR, circulating insulin and HbA1C were significantly decreased in the whole Maitake powder and ES groups, and tended to be lower in the WS group compared to control. In the ensuing studies, we gavaged ZFR once daily with water (control), 44 mg fraction WS, or 44 mg fraction WS plus 100 g niacin-bound chromium (NBC). Oral gavage of WS clearly lowered SBP and circulating glucose concentrations, more so with the addition of chromium. We conclude that the examined forms of Maitake mushroom have antihypertensive and antidiabetic potential which differ among rat strains. The ES fraction may decrease SBP in SHR via alteration in the renin-angiotensin system.     

Blood pressure lowering effects of niacin-bound chromium(III) (NBC) in sucrose-fed rats: renin-angiotensin system

Excessive intake of sugars significantly elevates systolic blood pressure (SBP) in susceptible rats. Although the exact pathological mechanisms behind sugar-induced hypertension are uncertain and may be multiple, disturbances in the renin-angiotensin system (RAS) manifested by elevated circulating levels of angiotensin-2 may be involved. We attempted to confirm that the RAS was significantly involved in sugar-induced BP elevations and examined whether the ability of niacin-bound chromium (NBC) to ameliorate sugar-induced SBP elevations was due, at least in part, through effects on the RAS. Initially, 40 mature Sprague-Dawley rats (SD), male and female, were involved in the study comparing two methods to estimate rat blood pressure indirectly. Then 13 were selected to examine the effects of NBC on the RAS. All rats eventually ingested a diet heavy in sucrose (30% w/w). In addition to blood pressure readings, the following procedures were implemented: insulin and losartan challenges, evaluation of serum ACE activity, measurement of serum angiotensin-2 levels, blood chemistries, and LNAME challenge. While dietary sucrose raised SBP significantly in control, adding NBC to the treatment group lowered it back toward baseline. The treatment group was more sensitive to exogenous insulin challenge and showed decreased activity of the RAS estimated by less lowering of SBP after losartan challenge, decreased serum ACE (angiotensin-converting enzyme) activity, and lower levels of circulating angiotensin-2. The former two parameters showed statistical significance; and the latter, a trend toward statistical significance. A separate group receiving captopril served as a positive control and showed decreased ACE activity and circulating levels of angiotensin-2 compared to the control group. Our data suggest that the RAS plays a significant role in sugar-induced hypertension and that NBC lowers SBP, at least in part, via actions on the RAS. Other findings suggest that the NO system is important in sucrose-induced BP elevations as well.     

Glycosaminoglycans of Rat Cerebellum: II. A Developmental Study

Total and individual glycosaminoglycans (GAGs) were determined in rat cerebellum in tissue explants at various postnatal ages. The major constituents of GAGs were chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). Dermatan sulfate (DS) and keratan sulfate (KS) could not be detected and therefore each amounts to less than 5% of all GAGs at all ages studied. HA was the prominent GAG during postnatal development and only a minor constituent at adult ages, whereas CS was the predominant GAG in adulthood. HS remained relatively constant throughout development. The incorporation of [3H]glucosamine into individual GAGs was highest for HS at postnatal day 6, whereas HA showed intermediate and CS the lowest levels of incorporation during the first postnatal week. All major GAGs showed the lowest incorporation values at adult ages.     

Identification and functions of chondroitin sulfate in the milieu of neural stem cells

The behavior of cells is generally considered to be regulated by environmental factors, but the molecules in the milieu of neural stem cells have been little studied. We found by immunohistochemistry that chondroitin sulfate (CS) existed in the surroundings of nestin-positive cells or neural stem/progenitor cells in the rat ventricular zone of the telencephalon at embryonic day 14. Brain-specific chondroitin sulfate proteoglycans (CSPGs), including neurocan, phosphacan/receptor-type protein-tyrosine phosphatase beta, and neuroglycan C, were detected in the ventricular zone. Neurospheres formed by cells from the fetal telencephalon also expressed these CSPGs and NG2 proteoglycan. To examine the structural features and functions of CS polysaccharides in the milieu of neural stem cells, we isolated and purified CS from embryonic day 14 telencephalons. The CS preparation consisted of two fractions differing in size and extent of sulfation: small CS polysaccharides with low sulfation and large CS polysaccharides with high sulfation. Interestingly, both CS polysaccharides and commercial preparations of dermatan sulfate CS-B and an E-type of highly sulfated CS promoted the fibroblast growth factor-2-mediated proliferation of neural stem/progenitor cells. None of these CS preparations promoted the epidermal growth factor-mediated neural stem cell proliferation. These results suggest that these CSPGs are involved in the proliferation of neural stem cells as a group of cell microenvironmental factors.     

Characterization of proteoglycans synthesized by rat thyroid cells in culture and their response to thyroid-stimulating hormone

A Fisher rat thyroid cell line was maintained in culture and the cells were labeled with [3H]glucosamine, [35S]sulfate, and [35S]cysteine to examine the synthesis of proteoglycans. 3H and 35S radioactivity from these precursors were incorporated into both chondroitin sulfate (CS) and heparan sulfate (HS) proteoglycans. CS proteoglycans were almost exclusively secreted into the medium while HS proteoglycans remained mainly associated with the cell layer. Single chain glycosaminoglycans released by papain digestion or alkaline borohydride treatment of either the CS or HS proteoglycans had average molecular weights of approximately 30,000 on Sepharose CL-6B chromatography. Both CS and HS proteoglycans were relatively small and contained only one or two glycosaminoglycans chains. 3H and 35S incorporation into both CS and HS proteoglycans were increased by thyroid-stimulating hormone (TSH) in a dose-dependent manner, which is in part explained by an adenylate cyclase-dependent mechanism as indicated by a similar effect in response to dibutyryl cAMP. TSH enhanced the incorporation of 35S into CS from [35S]cysteine about 1.5-fold and that from [35S]sulfate about 2-fold. This result demonstrated that the increased 35S incorporation from the [35S]sulfate precursor reflects an actual increase in sulfate incorporation and is not simply a result from an apparent increase in specific activity of the phosphoadenosine phosphosulfate donor. Analysis of disaccharides from chondroitinase digests revealed that the proportion of non-sulfated, 4-sulfated, and 6-sulfated disaccharides was not altered appreciably by TSH. These results, together with the disproportionate increase in 3H incorporation into CS from [3H]glucosamine, indicated that TSH increased the specific activity of the 3H label as well. Chase experiments revealed that CS proteoglycans were rapidly (t1/2 = 15 min) secreted into the medium and that the degradation of cell-associated proteoglycans was enhanced by TSH.     

Studies on the chemistry of arterial wall, XVII. Metabolic characteristics of different types of chondroitin sulfate-dermatan sulfate hybrids in arterial tissue.

1) Chondroitin sulfate and dermatan sulfate of bovine arterial tissue exist as copolymers with a varying degree of hybridization between chondroitin and dermatan sulfates. A fraction rich in dermatan sulfate hybridized with 20% chondroitin sulfate (termed DS-rich hybrid) and a fraction rich in chondroitin sulfate containing 17% DS as copolymer constituent (CS-rich hybrid) can be isolated by the subfractionation of the arterial tissue CS-DS preparation. 2) When arterial tissue segments were preincubated with [14C]glucosamine, 95% of the radioactivity incorporated into the glycosaminoglycans was found to be present in the galactosamine moiety of all of the CS-DS subfractions, whereas the relative proportion of 14C radioactivity incorporated into the galactosamine and uronic acid components was 51:49 following preincubation with [14C]glucose. In both experiments the specific radioactivity of the DS-rich hybrids was twice as high as that of the CS-rich hybrids. 3) Enzymatic degradation of the hybrid CS-DS subfractions by chondroitin AC and ABC lyases revealed that the specific radioactivity of the CS and DS disaccharide units released from the DS-rich hybrids was twice as high as those isolated from the CS-rich hybrids, but within each hybrid fraction the galactosamine moieties of the CS and DS units and their glucuronic and iduronic acid components exhibited equal specific radioactivities. 4) The results strongly support the assumption that distinct compartments exist for the formation of hybrid CS-DS proteoglycans with different proportions of CS and DS.     

Simultaneous detection of submicrogram quantities of hyaluronic acid and dermatan sulfate on agarose-gel by sequential staining with toluidine blue and Stains-All

A new discontinuous agarose-gel electrophoresis in 0.05 M HCl/0.04 M barium acetate combined with the highly sensitive visualization technique using toluidine blue/Stains-All has been developed for the simultaneous assaying of hyaluronic acid (HA) and dermatan sulfate (DS) with a detection limit at submicrogram level greater than other conventional procedures. Furthermore, this procedure also separates and reveals chondroitin sulfate (CS). The densitometric analysis of bands resulted in a linear response between 0.01 and 0.5 microg of glycosaminoglycans (GAGs) with correlation coefficients greater than approximately 0.94. Hyaluronic acid and dermatan sulfate extracted and purified from the abdominal skin of six rats were separated and quantified in comparison with the evaluation made by treatment of chondroitin ABC lyase and separation of Delta-disaccharides from hyaluronic acid (DeltadiHA) and dermatan sulfate/chondroitin sulfate (Deltadi4s and Deltadi6s) by HPLC. The total amount of rat skin polysaccharides (hyaluronic acid and dermatan sulfate) was 1.24+/-0.26 microg/mg of tissue by discontinuous agarose-gel electrophoresis and 1.20+/-0.33 microg/mg by HPLC with hyaluronic acid and dermatan sulfate percentages of 50.32+/-2.38 and 49.66+/-2.53, respectively. The analyses also confirmed that hyaluronic acid and dermatan sulfate are the main rat abdominal skin polysaccharides with chondroitin sulfate present in trace amounts. This new agarose-gel electrophoresis could be particularly useful in the study of the distribution of glycosaminoglycans in the skin from different body sites of animals and normal human subjects and may be of importance in understanding the changes that occur in the skin, especially the metabolism of extracellular matrix constituents, in connective tissue disorders.     

Glycosaminoglycans in Hydra magnipapillata (Hydrozoa, Cnidaria): demonstration of chondroitin in the developing nematocyst, the sting organelle, and structural characterization of glycosaminoglycans

The hydrozoan is the simplest organism whose movements are governed by the neuromuscular system, and its de novo morphogenesis can be easily induced by the removal of body parts. These features make the hydrozoan an excellent model for studying the regeneration of tissues in vivo, especially in the nervous system. Although glycosaminoglycans (GAGs) and proteoglycans (PGs) have been implicated in the signaling functions of various growth factors and play critical roles in the development of the central nervous system, the isolation and characterization of GAGs from hydrozoans have never been reported. Here, we characterized GAGs of Hydra magnipapillata. Immunostaining using anti-GAG antibodies showed chondroitin or chondroitin sulfate (CS) in the developing nematocyst, which is a sting organelle specific to cnidarians. The CS-PGs might furnish an environment for assembling nematocyst components, and might themselves be components of nematocysts. Therefore, GAGs were isolated from Hydra and their structural features were examined. A considerable amount of CS, three orders of magnitude less heparan sulfate (HS), but no hyaluronan were found, as in Caenorhabditis elegans. Analysis of the disaccharide composition of HS revealed glucosamine 2-N-sulfation, glucosamine 6-O-sulfation, and uronate 2-O-sulfation. CS contains not only nonsulfated and 4-O-sulfated N-acetylgalactosamine (GalNAc) but also 6-O-sulfated GalNAc. The average molecular size of CS and HS was 110 and 10 kDa, respectively. It has also been established here that CS chains are synthesized on the core protein through the ubiquitous linkage region tetrasaccharide, suggesting that indispensable functions of the linkage region in the synthesis of GAGs have been conserved during evolution.     

Mutational study of heparan sulfate 2-O-sulfotransferase and chondroitin sulfate 2-O-sulfotransferase

Heparan sulfate (HS) and chondroitin sulfate (CS) are highly sulfated polysaccharides with a wide range of biological functions. Heparan sulfate 2-O-sulfotransferase (HS-2OST) transfers the sulfo group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the 2-OH position of the hexauronic acid that is adjacent to N-sulfated glucosamine, whereas chondroitin sulfate 2-O-sulfotransferase (CS-2OST) transfers the sulfo group to the hexauronic acid that is adjacent to N-acetylated galactosamine. Here we report a systematic mutagenesis study of HS-2OST and CS-2OST based on their structural homology to estrogen sulfotransferase and HS 3-O-sulfotransferase isoform 3 (3-OST3), for which crystal structures exist. We have identified six residues possibly involved in binding to PAPS. HS-2OST carrying mutations of these residues lacks sulfotransferase activity and the ability to bind 3'-phosphoadenosine 5'-phosphate, a PAPS analogue, as determined by isothermal titration calorimetry. Similar residues involved in binding to PAPS were also identified in CS-2OST. Additional residues that participate in carbohydrate substrate binding were also identified in both enzymes. Mutations at these residues led to the loss of sulfotransferase activity but maintained the ability to bind to phosphoadenosine 5'-phosphate. The catalytic function of HS-2OST appears to involve two histidine residues (His140 and His142), whereas only one histidine (His168) of CS 2-OST is likely to be critical. This unique feature of HS 2-OST catalytic residues directed us to characterize the Drosophila heparan sulfate 2-O-sulfotransferase. The results from this study provide insight into the differences and similarities various residues play in the biological roles of the HS-2OST and CS-2OST enzymes.     

Proteoglycan and glycosaminoglycan synthesis by cultured rat mesangial cells

The synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfate/dermatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer. In summary, approximately 60% of total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.     

Effects of wheat germ agglutinin and concanavalin A on the accumulation of glycosaminoglycans in pericellular matrix of human dermal fibroblasts. A comparison with insulin.

The effect of insulin, wheat germ agglutinin (WGA), peanut agglutinin (PNA) and concanavalin A (ConA) on [3H]glucosamine incorporation into pericellular glycosaminoglycans (GAGs) was investigated in two lines of cultured human dermal fibroblasts. Insulin and WGA stimulated [3H]glucosamine incorporation into hyaluronic acid (HA) and heparan sulphate (HS) without any alteration of chondroitin sulphate (CS) and dermatan sulphate (DS) contents. ConA increased [3H]glucosamine incorporation into HS, CS and DS, but had no effect on [3Hglucosamine incorporation into HA. PNA affected neither the content, nor the composition of GAGs. In contrast to PNA, ConA and WGA stimulated glycolysis and demonstrated an evident antiproliferative effect on dermal fibroblasts. Thus, both the insulin-like action of WGA and ConA on cultured dermal fibroblasts and the differences between the effects of lectins on modulation of GAGs synthesis appear to be determined by their chemical structure.     

Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library

Placental malaria, a serious infection caused by the parasite Plasmodium falciparum, is characterized by the selective accumulation of infected erythrocytes (IEs) in the placentas of the pregnant women. Placental adherence is mediated by the malarial VAR2CSA protein, which interacts with chondroitin sulfate (CS) proteoglycans present in the placental tissue. CS is a linear acidic polysaccharide composed of repeating disaccharide units of d-glucuronic acid and N-acetyl-d-galactosamine that are modified by sulfate groups at different positions. Previous reports have shown that placental-adhering IEs were associated with an unusually low sulfated form of chondroitin sulfate A (CSA) and that a partially sulfated dodecasaccharide is the minimal motif for the interaction. However, the fine molecular structure of this CS chain remains unclear. In this study, we have characterized the CS chain that interacts with a recombinant minimal CS-binding region of VAR2CSA (rVAR2) using a CS library of various defined lengths and sulfate compositions. The CS library was chemo-enzymatically synthesized with bacterial chondroitin polymerase and recombinant CS sulfotransferases. We found that C-4 sulfation of the N-acetyl-d-galactosamine residue is critical for supporting rVAR2 binding, whereas no other sulfate modifications showed effects. Interaction of rVAR2 with CS is highly correlated with the degree of C-4 sulfation and CS chain length. We confirmed that the minimum structure binding to rVAR2 is a tri-sulfated CSA dodecasaccharide, and found that a highly sulfated CSA eicosasaccharide is a more potent inhibitor of rVAR2 binding than the dodecasaccharides. These results suggest that CSA derivatives may potentially serve as targets in therapeutic strategies against placental malaria.     

Separation and properties of five glycosaminoglycan sulfatases from rat skin.

Chondroitin sulfates, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, and oligosaccharides derived from these sulfated glycosaminoglycans have been used for the measurement of sulfatase activity of rat skin extracts. Chromatographic fractionation of the extracts followed by specificity studies demonstrated the existence of five different sulfatases, specific for 1) the nonreducing N-acetylglucosamine 6-sulfate end groups of heparin sulfate and keratan sulfate, 2) the nonreducing N-acetylgalactosamine (or galactose) 6-sulfate end groups of chondroitin sulfate (or keratan sulfate), 3) the nonreducing N-acetylgalactosamine 4-sulfate end groups of chondroitin sulfate and dermatan sulfate, 4) certain suitably located glucosamine N-sulfate groups of heparin and heparan sulfate, or 5) certain suitably located iduronate sulfate groups of heparan sulfate and dermatan sulfate. Two arylsulfatases, one of which was identical in its chromatographic behaviors with the third enzyme described above, were also demonstrated in the extracts. These results taken together with those previously obtained from studies on human fibroblast cultures suggest that normal skin fibroblasts contain at least five specific sulfatases and diminished activity of any one may result in a specific storage disease.     

Glucosamine and glucose induce insulin resistance by different mechanisms in rat skeletal muscle

It has been hypothesized that glucose-induced insulin resistance is mediated by accumulation of UDP-N-acetylhexosamines (UDP-HexNAcs). In a previous study on rat epitrochlearis muscles incubated with high concentrations of glucose and insulin (Kawanaka K, D-H Han, J Gao, LA Nolte, and JO Holloszy. J Biol Chem 276: 20101-20107, 2001), we found that insulin resistance developed even when the increase in UDP-Hex-NAcs was prevented. Furthermore, actinomycin D completely prevented glucose-induced insulin resistance despite a greater accumulation of UDP-HexNAcs. In the present study, we used the same epitrochlearis muscle preparation, as well as the rat hemidiaphragm, to determine whether, like glucose, glucosamine causes insulin resistance by an actinomycin D-inhibitable process. Incubation of diaphragm muscles with 10 mM glucosamine for 3 h resulted in an approximately fivefold increase in UDP-HexNAcs, an approximately 50% reduction in insulin responsiveness of glucose transport, and a 58% reduction in ATP concentration. These effects of glucosamine were not prevented by actinomycin D. Incubation of epitrochlearis muscles with 20 mM glucosamine for 3 h or with 10 mM glucosamine for 5 h also caused large decreases in insulin responsiveness of glucose transport but with no reduction in ATP concentration. Actinomycin D did not prevent the glucosamine-induced insulin resistance. The insulin-induced increases in tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the binding of PI 3-kinase to IRS-1 were decreased approximately 60% in epitrochlearis muscles exposed to glucosamine. This is in contrast to glucose-induced insulin resistance, which was not associated with impaired insulin signaling. These results provide evidence that glucosamine and glucose induce insulin resistance by different mechanisms.     

SHR的胰岛素抗性及骨胳肌的形态学特征

本文研究了自发性高血压大鼠及其对照品系－正常血压大鼠的胰岛素抗性和肌肉组织的形态学差异。结果表明：ＳＨＲ的基础胰岛素水平,糖耐量实验时胰岛素曲线下的面积均高于ＷＫＹ。而基础血糖水平,ＧＴＴ时血糖曲线下的面积两者相似。组织形态学研究表明：ＳＨＲ内胳肌中对胰岛素长一智Ⅰ型纤维比例低于ＷＫＹ。本研究发现：基础胰岛素水平或ＧＴＴ时胰岛素曲线下的面积与血压之间有正相关关系,而基础胰岛素水平与骨胳肌中Ｉ型纤维     

Glycosaminoglycans of Rat Cerebellum: I. Quantitative Analysis of the Main Constituents at Postnatal Day 6

Abstract: Isolated glycosaminoglycans (GAGs) were quantified biochemically in the cerebella of 6-day-old rats. ¹⁴C-Labeled hyaluronic acid (HA) and chondroitin-4-sulfate (C-4-S), added prior to isolation of GAGs from tissue, served as internal standards to allow correction for unknown losses during the purification procedure and exact quantification of GAGs in the intact tissue. Three main constituents—HA, chondroitin sulfate (CS), and heparan sulfate (HS)—were found at concentrations of 1.82, 1.52, and 0.76 μg/mg protein amounting to 44%, 37%, and 19% of the total GAG fraction, respectively. Incorporation of [³H]glucosamine precursor into GAGs was higher for HS (56% of incorporated precursor) and lower for HA (29%) and CS (15%). The specific activities of individual GAGs were 64.7 nCi/μg for HS, 14.2 for HA, and 8.3 for CS.     

The effect of aging on the synthesis of hexosamine-containing substances from rat costal cartilage. A decrease in sulfation of chondroitin sulfate with aging.

The amount of glycosaminoglycan (GAG) in dry costal cartilage tissue of rats decreased with aging, while the GAG content in mg DNA (unit cartilage cell) remained the same with aging. These results can be explained by the finding that the total number of cartilage cells decreased with aging. Electrophoretic analysis showed that chondroitin 4-sulfate was the major GAG in rat costal cartilage of various ages. Rat costal cartilage of different ages was incubated with radioactive precursors, and newly synthesized GAG was prepared and the radioactivity analyzed to determine the biosynthetic activity. As to changes in the radioactivity uptake with aging per mg dry cartilage tissue, aging influenced [35S]sulfate incorporation into GAG more significantly than [3H]glucosamine incorporation into GAG. There was a significant decrease in the specific radioactivity of [35S]sulfate per mg DNA (unit cartilage cell), whereas the specific radioactivity of [3H]glucosamine per mg DNA did not change significantly with aging. Both the total sulfotransferase activity and the specific activity per mg DNA decreased significantly with aging. Analysis of disaccharide units formed after chondroitinase ABC digestion of labeled GAG isolated from young and old cartilage showed that the percentage of incorporation of [3H]glucosamine into deltaDi-OS increased significantly with aging. These results suggested that the appearance of nonsulfated positions in the structure of the chondroitin sulfate chain increased with aging. On the basis of gel chromatography on Bio-Gel A-1.5 m no significant difference in the approximate molecular size of chondroitin sulfate was observed between the young and old GAG samples. The present study indicated that the sulfation of chondroitin sulfate chains from rat costal cartilage decreased with the process of aging.