Nitrous Acid Digestion Research Articles

Comparative Studies on Branded Dalteparin with Two Generic Versions: Hepagumin and Fluzepamin

Dalteparin is a low molecular weight heparin (LMWH) prepared by nitrous acid digestion. Recently generic versions of the product have become available. The purpose of this study was to compare the molecular and functional profile of Dalteparin and two generic versions from Japan (Hepagumin and Fluzepamin). The molecular profile was determined using the HPLC before and after heparinase‐1 digestion. The anticoagulant activity was measured using the whole blood ACT‐Hemochron and plasma‐based APTT and Heptest, and amidolytic anti‐Xa and anti‐IIa assays. Thrombin generation studies were performed using a kinetic method. By HPLC, the molecular weight of the three products was comparable and ranged from 6.3 to 6.8 kDa. After heparinase digestion all three products give comparable result (molecular weight ranging from 2.1 to 2.3 kDa). The oligosaccharide components after digestion were also comparable. In the whole blood anticoagulant studies no differences were noted between products at the 1.25 U/ml. In the APPT and anti‐Xa assay, there were no differences between three products. However, minor differences were noted in the Heptest and factor IIa inhibition. These results show that Hepagumin and Fluzepamin do not significantly differ from the branded dalteparin. The minor differences noted in clot based assays were not statistically different.

Characterization of Sulfated Oligosaccharides in Mucopolysaccharidosis Type IIIA by Electrospray Ionization Mass Spectrometry

Heparan sulfate is a linear glycosaminoglycan with considerable structural diversity that binds a myriad of growth factors and proteins that play pivotal roles in a variety of biological processes. We have investigated the structural complexity of partially degraded fragments of heparan sulfate in mucopolysaccharidosis type IIIA in which there is a defect in heparan sulfate catabolism. Mono- to hexadecasaccharides were isolated from the urine of a mucopolysaccharidosis IIIA patient and shown to have non-reducing end glucosamine N-sulfate residues, reflecting the catabolic deficiency in heparan N-sulfatase (sulfamidase) activity. The use of nitrous acid digestion (pH 1.5) combined with separation by reverse-phase high-performance liquid chromatography and analysis by electrospray ionization-mass spectrometry identified multiple forms of these oligosaccharides with some N-acetylated glucosamine residues and one to three sulfates per disaccharide. Furthermore, we demonstrated that each oligosaccharide existed in multiple sulfated forms. Many structural isomers were present, suggesting a complex mixture of oligosaccharides present in the urine as a consequence of a defect in heparan sulfate degradation.

Discordance between the 1st and 2nd Low Molecular Weight Heparin Standards. Implications for Dosing.

Soon after the introduction of low molecular weight heparins (LMWHs) as antithrombotic agents, a LMWH standard to cross-reference the potency of commercially available LMWHs was developed. For the past 15 years, this standard has been used by manufacturers to cross-reference their branded LMWHs. The 1st LMWH standard (85/600) was produced by nitrous acid digestion of porcine mucosal heparin. This standard had characteristics that were comparable to dalteparin. Because of the widespread use of the first standard, the supplies are now depleted necessitating the introduction of new standard material. The WHO has introduced a 2nd LMWH standard which is also produced by nitrous acid digestion and which mimics the commercially available LMWH reviparin. The molecular weight profile and the biologic activities, including SERPIN affinity, of these two standards are very distinct. When used to cross-reference commercially available LMWHs such as enoxaparin, the new standard consistently overestimates the anti-Xa potency by 5–10% in comparison to the first standard. Such an overestimation may lead to a reduction in dosage and have a significant impact on the clinical dosing of LMWH in various indications. As many generic versions of LMWHs are currently being introduced, a systematic study was carried out to cross-reference three commercially available generic products from India and South America against the first and second LMWH standards using amidolytic anti-Xa and anti-IIa assays. Differences were observed in the slopes of the concentration-response curves for these standards in both the anti-Xa and anti-IIa assays. The potencies of the generic LMWHs ranged from 141 to 152 U/mg in relation to the first standard and from 132 to 143 U/mg in relation to the second standard. On average, the anti-Xa potency was 6.5% lower when calculated with the 2nd standard. The differences in anti-IIa potency were further amplified and exhibited wider variation. On average, the anti-IIa potency was 18.9% lower when calculated with the 2nd standard. These studies clearly demonstrate that the limitations in the use of the first and second LMWH standards for cross-referencing commercially available LMWHs and their potential generic versions. If the 2nd standard is used, it should first be cross-referenced against the first standard in order to properly assign the anti-Xa and anti-IIa activities. This study also underscores the need for developing individual standards for each of the branded products. Such standards should be defined in terms of not only anti-Xa and anti-IIa activity, but also in terms of anticoagulant activity in global clotting assays such as the USP assay, activated clotting time (ACT), aPTT and Heptest. Such designations will be particularly useful in evaluating the potential equivalence of generic versions of branded LMWHs.

Biochemical and pharmacologic characteristics of Reviparin, a low-molecular-mass heparin.

The introduction of low-molecular-mass heparins (LMMHs) has added a new dimension to the prophylactic and therapeutic management of thromboembolic disorders. These agents are now globally accepted as drugs of choice for postsurgical prophylaxis of deep vein thrombosis (DVT). Currently, the LMMHs are being developed for various therapeutic and cardiovascular indications. Reviparin is an optimized LMMH prepared by controlled nitrous acid digestion of porcine mucosal heparin. This drug has been developed utilizing validated procedures and exhibits a relatively narrow molecular mass distribution in contrast to most other commercially available LMMHs. The specific activity in the anticoagulant assays is approximately 40 U/mg whereas the specific activity in amidolytic anti-Xa assays is approximately 100 anti-Xa U/mg. Reviparin is capable of producing dose- and time-dependent antithrombotic effects in animal models of thrombosis. Although ex vivo anticoagulant effects are initially observed at dosages that are antithrombotic, this agent has been found to produce sustained antithrombotic effects when ex vivo anticoagulant actions are not measurable. Repeated administration of this LMMH induces progressively stronger antithrombotic effects. This drug has also been found to release tissue factor pathway inhibitor (TFPI) following both intravenous (IV) and subcutaneous (SC) administration. The studies included in this article are designed to provide additional data on the molecular profile using new calibration methods and additional results on pharmacologic studies. In particular, the release of TFPI following IV and SC administration to nonhuman primates is described. The effect of repeated administration of Reviparin mimicking the postsurgical prophylaxis of DVT is also reported in terms of any augmentation of the antithrombotic or hemorrhagic effects of this agent.

Preclinical studies on a low molecular weight heparin

Some major developments in the area of antithrombotic therapy have occurred during the past decade. Of these, the concept of fractionation of heparin has resulted in the development of several products from this agent. The introduction of low molecular weight heparins (LMWHs) has added a new chapter to the prophylactic and therapeutic management of thromboembolic disorders. These agents are now globally accepted as drugs of choice for post-surgical prophylaxis of deep vein thrombosis (DVT). Currently, the LMWHs are being developed for various therapeutic and cardiovascular indications. Reviparin is an optimized LMWH prepared by controlled nitrous acid digestion of porcine mucosal heparin. This drug has been developed using validated procedures and exhibits a relatively narrow molecular weight distribution in contrast to most other commercially available LMWHs. The specific activity in anticoagulant assays is approximately 32 U/mg whereas the specific activity in terms of anti-Xa units is 120 anti-Xa U/mg. Reviparin is capable of producing a dose- and time-dependent antithrombotic effect in animal models of thrombosis. While the ex vivo effects initially occur at dosages that are antithrombotic, this agent has been found to produce sustained antithrombotic effects without any detectable ex vivo anticoagulant actions. This agent has also been found to release tissue factor pathway inhibitor (TFPI) after both intravenous and subcutaneous administration. Repeated administration of reviparin produces progressively stronger antithrombotic effects. The current studies are designed to provide additional data on its molecular profile using new calibration methods and additional results on the pharmacological studies in a dose-dependent manner. In particular, the release of TFPI following i.v. and s.c. administration in a primate model is described. The effect of repeated administration mimicking the post-surgical prophylaxis of DVT is also reported in terms of any increase in the antithrombotic or haemorrhagic effects of this agent. Comparative antithrombotic and pharmacological studies are also reported to compare the pharmacological profiles of reviparin, nadroparin and enoxaparin.

Sulphated macromolecules produced by in vivo labelling in the rat gastric mucosa.

The aim of this study was to investigate the nature and distribution of sulphated macromolecules of the extracellular matrix in rat gastric mucosa. This was achieved by developing an in vivo labelling system. An intraperitoneal injection of 1 mCi [35S]-sulphate was given for either 4 h (0.01% incorporation into macromolecular fraction) or 8 h (0.13% incorporation). At the end of the labelling period the stomach was removed and the mucosa and submucosa was either taken as a single combined sample or separated into four layers by blunt dissection. Each sample was papain digested and analysed by ion-exchange chromatography. This analysis revealed sulphated species of differing charge existing in differing proportions throughout the mucosa. These sulphated species eluted at NaCl concentrations of approximately 0 (A), 0.19 (B), 0.34 (C) and 0.78 mol/L (D) from a Q-Sepharose ion exchange column. Further analysis by size exclusion chromatography and chemical and enzymatic digestion showed that peaks B and C had molecular weights of 2.4 x 10(5) and 2.8 x 10(5), respectively and were resistant to chondroitinase ABC, heparitinase and nitrous acid digestion. Peak D was found to contain a polydisperse population of molecules with a molecular weight range of approximately 1 x 10(4) to 6 x 10(4). This sample was susceptible to nitrous acid and chondroitinase ABC digestion and was found predominantly in the sample isolated from deeper in the tissue. We have thus developed an in vivo labelling technique for sulphated macromolecules that can be used in the further study of injury to the gastric mucosa.

Spectroscopic Characterisation of Disaccharides Derived from Keratan Sulfates

Skeletal keratan sulfates have been degraded by three independent techniques and the resultant, borohydride-reduced, disaccharides have been characterised by NMR spectroscopy. The 1H and 13C (where available) chemical shifts are reported for the following substances, where GalNAc-ol represents N-acetyl-galactosaminitol, GlcNAc-ol represents N-acetyl-glucosaminitol, GlcNAc(6S)-ol represents N-acetyl-glucosaminitol 6-O-sulfate and 2,5AnMan(6S)-ol represents 2,5-anhydro-D-mannitol 6-O-sulfate. (a) GlcNAc(6S)beta(1-3)Gal-ol, isolated after keratanase (from Pseudomonas sp.) digestion. (b) Gal beta(1-4)GlcNAc(6S)-ol and Gal(6S)beta(1-4) GlcNAc(6S)-ol, the 1H chemical shifts have been reported previously [Brown, G. M., Huckerby, T. N., Morris, H. G., Abram, B. L. & Nieduszynski, I. A. (1994) Biochemistry 33, 4836-4846; Brown, G. M., Huckerby, T. N. & Nieduszynski, I. A. (1994) Eur. J. Biochem. 224, 281-308], GlcNAc(6S)beta(1-6)GalNAc-ol, [formula: see text], [formula: see text], all isolated after keratanase II digestion. (c) Gal beta(1-4)2,5AnMan(6S)-ol and Gal(6S)beta(1-4)2,5AnMan(6S)-ol, isolated after hydrazinolysis and nitrous acid digestion. In addition, the model compounds Gal beta(1-4)GlcNAc-ol and Gal beta(1-6)GlcNAc-ol have also been examined by 1H and 13C NMR spectroscopy. The value of these data for microstructural analysis of keratan sulfate samples is discussed.

Spectroscopic characterisation of disaccharides derived from keratan sulfates.

Skeletal keratan sulfates have been degraded by three independent techniques and the resultant, borohydride-reduced, disaccharides have been characterised by NMR spectroscopy. The 1H and 13C (where available) chemical shifts are reported for the following substances, where GalNAc-ol represents N-acetyl-galactosaminitol, GlcNAc-ol represents N-acetyl-glucosaminitol, GlcNAc(6S)-ol represents N-acetyl-glucosaminitol 6-O-sulfate and 2,5AnMan(6S)-ol represents 2,5-anhydro-D-mannitol 6-O-sulfate. (a) GlcNAc(6S)beta(1-3)Gal-ol, isolated after keratanase (from Pseudomonas sp.) digestion. (b) Gal beta(1-4)GlcNAc(6S)-ol and Gal(6S)beta(1-4) GlcNAc(6S)-ol, the 1H chemical shifts have been reported previously [Brown, G. M., Huckerby, T. N., Morris, H. G., Abram, B. L. & Nieduszynski, I. A. (1994) Biochemistry 33, 4836-4846; Brown, G. M., Huckerby, T. N. & Nieduszynski, I. A. (1994) Eur. J. Biochem. 224, 281-308], GlcNAc(6S)beta(1-6)GalNAc-ol, [formula: see text], [formula: see text], all isolated after keratanase II digestion. (c) Gal beta(1-4)2,5AnMan(6S)-ol and Gal(6S)beta(1-4)2,5AnMan(6S)-ol, isolated after hydrazinolysis and nitrous acid digestion. In addition, the model compounds Gal beta(1-4)GlcNAc-ol and Gal beta(1-6)GlcNAc-ol have also been examined by 1H and 13C NMR spectroscopy. The value of these data for microstructural analysis of keratan sulfate samples is discussed.

Pharmacologic validation of the clinical effects of an optimized low-molecular-weight heparin-reviparin.

Reviparin is a low-molecular-weight heparin (LMWH) prepared by controlled nitrous acid digestion of porcine mucosal heparin. The trade name designation for this agent is Clivarin. This agent has been released in Germany and France for the prophylaxis of deep venous thrombosis (DVT) in surgical patients. This agent is developed utilizing optimized procedures and exhibits a uniform, narrow-molecular-weight distribution in comparison to the other commercially available LMWHs. The specific activity in the anticoagulant assays is approximated to be 32 U/mg whereas the specific activity in terms of anti-Xa units is designated 120 aXa U/mg. Reviparin is capable of producing a dose- and time-dependent antithrombotic effect in animal models of thrombosis. While the ex vivo effects are initially presented at antithrombotically active dosages, this agent has been found to produce antithrombotic effects without any detectable ex vivo actions. This agent is also known to release tissue factor pathway inhibitor (TFPI) after both intravenous (IV) and subcutaneous (SC) administration. Repeated administration of Reviparin produces progressively stronger antithrombotic effects. Similarly, the bleeding as measured by rabbit ear blood loss is also progressively increased. However, the ratio between the dosage producing these effects is quite large. The current studies are designed to provide additional data on the molecular profile using new calibration methods and additional results on the pharmacologic studies in a dose-dependent manner. In particular, the release of TFPI following IV and SC administration in a primate model is described. The effect of repeated administration mimicking the post-surgical prophylaxis of DVT is also reported in terms of any augmentation of the antithrombotic or hemorrhagic effects of these agents.

Specific heparan sulfate saccharides mediate the activity of basic fibroblast growth factor.

In a previous study, we showed that heparitinase releases a 14-saccharide sequence (Oligo-H) from heparan sulfate (HS) with the structure delta GlcUA beta 1,4GlcNSO3-alpha 1,4[IdceA(2S)alpha 1,4GlcNSO3]5 alpha 1,4IdceA alpha 1,4GlcNAc (where IdceA(2S) represents iduronic acid 2-sulfate), which binds to basic fibroblast growth factor (bFGF) with high affinity (Turnbull, J. E., Fernig, D., Ke, Y., Wilkinson, M. C. & Gallagher, J. T. (1992) J. Biol. Chem. 267, 10337-10341). This paper describes further work on the binding properties of HS saccharides and their capacity to mediate bFGF activity in a mitogenesis assay in which responsiveness is dependent on the addition of HS or heparin. Saccharides prepared by heparinase or nitrous acid digestion and heparitinase-resistant fragments five disaccharide units (degree of polymerization (dp) = 10) or less in size were unable to activate bFGF. However, heparitinase-resistant saccharides of dp12-16 were active in the assay; the dp14 and dp16 fractions were equivalent in activity to heparin and more active than the parent HS. Saccharides of the same size and basic structure as the active fractions (> or = dp12) bound to bFGF with high relative affinity. Active saccharides were composed mainly of N-sulfated disaccharides, the predominant unit being IdceA(2S)-GlcNSO3. This was enriched at least 5-fold in the active saccharides by comparison with the original HS. In addition, the dp12 and dp14 active fractions had a notably low content of trisulfated disaccharides (IdceA(2S)-GlcNSO3(6S)) (where GlcNSO3(6S) represents N-sulfated glucosamine 6-sulfate), which are the major repeat units of heparin. The data show that sequences similar in size and basic structure to Oligo-H can mediate the mitogenic activity of bFGF. Overall, the results provide further evidence that specific HS sequences are generated biosynthetically in order to fulfill particular biological functions such as activation of bFGF.

Effects of 4-deoxy- l- threo-pentose, a novel carbohydrate, on neural cell proteoglycan synthesis and function

A novel carbohydrate, 4-deoxy- l- threo-pentose (4-deoxyxylose), was synthesized by way of reductive dechlorination of a chlorodeoxy sugar. This carbohydrate, an analogue of xylose which is required for the initiation of glycosaminoglycan (GAG) synthesis, was used to explore the function of GAG side chains in neurite outgrowth on a laminin substrate. 4-Deoxyxylose inhibited the incorporation of 35SO 4 into the GAGs of neuronal and astrocytic proteoglycans, with no effect being seen on the incorporation of [ 3H]glucosamine into proteoglycan. Direct analysis of the heparan sulphate fraction from such cells using nitrous acid digestion confirmed that the GAGs were undersulphated. No inhibition of either 35SO 4 or [ 3H]glucosamine incorporation was observed in primary mouse hepatocytes exposed to 4-deoxyxylose. 4-Deoxyxylose produced a direct dose-dependent inhibition of neurite outgrowth by sensory neurons, and medium conditioned by neurons or astrocytes in the presence of 4-deoxyxylose displayed less laminin-complexed neurite-promoting activity than medium conditioned in its absence. These data suggest that 4-deoxyxylose inhibits neurite outgrowth by altering the sulphation of the GAGs of heparan sulphate proteoglycans.

Effects of sized heparin oligosaccharide on the interactions of Chinese hamster ovary cell with thrombospondin.

Binding and degradation of TSP by CHO cells and adhesion of CHO cells to substrate-adsorbed TSP are mediated by cell surface PGs and inhibitable by heparin. In order to learn how these three processes are related, we studied the effects of defined heparin oligosaccharides up to 18-mer produced by nitrous acid digestion. There was a complex correlation among oligosaccharide chain length, affinity of oligosaccharide for TSP in a solid phase binding assay, and potencies of oligosaccharide in inhibition of the three cellular processes. Inhibition of degradation was more sensitive to shorter oligosaccharides than inhibition of binding. For instance, the 10-mer inhibited binding of TSP to cells by 10% and degradation by 70%. Punctate immunofluorescence of cell surface bound TSP was replaced by a diffuse pattern after incubation in the presence of the 10-mer. These results suggest that the clustering of TSP on the cell surface may trigger endocytosis and degradation. Inhibition of binding of TSP to cells, in turn, was more sensitive to midsized oligosaccharides than inhibition of cell adhesion to adsorbed TSP. Inhibition of adhesion correlated with the ability of oligosaccharides to block binding of 125I-heparin to adsorbed TSP.

Comparison of proteoglycans synthesized by porcine normal and polycystic renal tubular epithelial cells in vitro

Newly synthesized porcine tubular epithelial cell proteoglycans were labeled in vitro with Na 2[ 35S]SO 4. At the beginning of the labeling period (24 h) [ 35S]sulfate incorporated into macromolecules was measured following PD-10 chromatography. There was a significant reduction in the amount of 35S-labeled macromolecules isolated from polycystic cells compared to that from normal cells. The distribution of recovered radiolabeled material among the medium, cell surface, and intracellular fractions was similar for both normal and polycystic cells. Analysis of the proteoglycans in polycystic cells demonstrated that 86 and 73% of 35S-labeled macromolecules were of the heparan sulfate type in polycystic and normal cells, respectively. The remainder was chondroitin sulfate. Proteoglycans were characterized using DEAE-Sephacel ion-exchange chromatography, chondroitinase ABC, heparitinase, and nitrous acid digestion followed by Sepharose CL-4B gel permeation chromatography. The majority of radiolabeled material in the medium, cell surface, and intracellular fractions eluted between 0.35 and 0.39 m NaCl. However, a second peak (peak II) that eluted at 0.25 m NaCl was found in the medium from polycystic cells. This peak accounted for 27% of the total macromolecules secreted into the medium. Proteoglycans in the major peak were susceptible to nitrous acid and chondroitinase ABC digestion. A similar proportion of peak II was degraded by chondroitinase ABC. However, the remainder was only slightly susceptible to treatment with nitrous acid or heparitase. In normal cells a small amount of material eluted at a similar low charge; the proteoglycans were the same as those found in the major peak and appeared as a shoulder on this peak.

Altered patterns of proteoglycan deposition during maturation of the fetal mouse lung

Previous studies have shown that β-xyloside inhibits maturation of the fetal mouse lung (Smith et al., Dev. Biol. 138, 42–52, 1990). Insofar as this drug inhibits proteoglycan deposition, the present studies were undertaken to examine the chemical composition and tissue distribution of proteoglycans in order to determine, more precisely, their role during lung morphogenesis. Autoradiography of labeled 16- and 19-day embryonic lungs demonstrated greater incorporation over the mesenchyme. Treatment with β-xyloside did not alter the autoradiographic appearance; however, β-xyloside treatment followed by nitrous acid digestion, eliminated most silver grains. Isolation of proteoglycans from extracellular, membrane and intracellular pools over the 16- to 19-day interval demonstrated redistribution of heparan sulfate proteoglycan from an intracellular to a membrane location, while chondroitin sulfate proteoglycan redistributed from intracellular to extracellular. Only the synthesis of chondroitin sulfate proteoglycan was inhibited by β-xyloside. On the basis of these results we suggest that a chondroitin sulfate proteoglycan is required for lung maturation and that inhibition of its synthesis results in inhibition of septa formation and subsequent failure of morphogenesis and differentiation.

Subendothelial extracellular-matrix heparan sulfate proteoglycan-degrading activity of human monocyte macrophages.

At the early stage of atherogenesis, circulating monocyte macrophages appear to adhere to the endothelial cell surface and migrate subendothelially to become foam cells. The mechanism of these macrophage-endothelial cell interactions was investigated. Adherent macrophages isolated from human blood were plated on [35S]O4-prelabeled extracellular matrix-coated dishes prepared from cultured porcine aortic endothelial cells. During incubation for 2-3 days at pH 7.4 either in the presence or absence of serum, macrophages solubilized the labeled extracellular matrix to a lower molecular weight component (Kav approximately equal to 0.5) than the materials (Kav = 0) released into the medium containing no cells. The degrading activity was not stored intracellularly but instead was found pericellularly, requiring continuous cell-matrix contact. Heparin (10 micrograms/ml) inhibited this degrading activity of macrophages. Degradation products were precipitated with cetylpyridinium chloride and were resistant to further digestion with alkali, pronase, or chondroitinase ABC, but were converted to further lower molecular weight fragments (Kav = 0.84) after nitrous acid digestion or heparitinase treatment. The intact glycosaminoglycan side chains determined by subjecting the extracellular matrix to cleavage with alkali or pronase were larger (Kav congruent to 0.20) than those of degradation products released by macrophages. These results suggest that the attachment and subsequent invasion of endothelial cells by monocyte macrophages may involve the production of extracellular-matrix heparan sulfate proteoglycan-degrading activity by these cells.

Accumulation and distribution of sulfated materials in the maturing mouse lens capsule.

Lenses of late gestational and postnatal normal-eyed mice were tested for accumulated sulfated materials by using Spicer's high-iron-diamine staining method and also for newly incorporated sulfate autoradiographically following administration of 35SO4 either in vivo or in isolated and organ-cultured lenses. Accumulated and newly incorporated sulfate was observed in all lenses for each age group tested. Discrete regional differences were seen in histochemical staining patterns for sulfate on the lens capsule in specimens of all ages, and distinct laminar zonations were seen in the various regions of the capsule in older specimens. Typically, the anterior and equatorial regions of the capsule demonstrated three histochemically distinct laminar zones while the posterior capsule usually demonstrated two laminar zones. Autoradiographic results indicated that sulfate was indeed being incorporated into these regions, and in the same general pattern as seen with histochemistry. The materials were largely insensitive to testicular hyaluronidase but were preferentially sensitive to nitrous acid digestion, indicating the presence of capsular heparan sulfates. Autoradiographic results from organ-cultured lenses indicated that this tissue itself is a primary source of these materials.

Abnormal accumulation of sulphated materials in lens tissue of mice with the aphakia mutation

Sulphated materials were tested for in the eyes of late gestational and postnatal normal mice and mice with the aphakia mutation using Spicer's high iron diamine staining method. Qualitative identification of these materials was attempted with bovine testicular hyaluronidase and nitrous acid digestion methods. The grossly abnormal morphology of the aphakia lens made it necessary to confirm identification of lens-derived tissue by testing for lens crystallins using standard immunohistological methods. As seen in normal mouse lens maturation, accumulated inter- and intracellular sulphated materials were observed in aphakia lens tissue from just before birth through juvenile maturation. Large cyst-like structures consisting of lens-derived tissue were commonly seen in the eyes of young postnatal mutant mice. Sulphated materials formed basal lamina-like structures on many of these lens-derived units, but a well-defined lens capsule never formed. Abnormal fibrillar structures rich in sulphated materials were seen in the intraocular cavity in many older mutant specimens, most of which were largely resistant to both digestion methods. These results indicate that the potential to elaborate sulphated materials qualitatively similar to those seen in normal mouse lens maturation is present in the aphakia mutant, although the mode of accumulation is grossly disturbed.

Ultrastructural localization of glycosaminoglycans in human term placenta.

Sulfated glycoconjugates were stained in normal human term placentas using Spicer's high-iron diamine (HID) method with thiocarbohydrazide and silver proteinate (TCH-SP) enhancement. Specific identification of glycosaminoglycans (GAG) was accomplished by digestion of the stained material with chondroitinase ABC or AC for removal of chondroitin sulfates and nitrous acid for removal of N-sulfated GAGs. The syncytiotrophoblast apical surface demonstrated moderate to intense staining with HID-TCH-SP, which was removed by prior digestion with the chondroitinases, but not by nitrous acid. The syncytiotrophoblast basal surface and endothelial cell surfaces lacked sulfate staining. A few cytoplasmic granules in syncytiotrophoblast cells demonstrated staining similar to the apical surface. Three layers of the basal lamina were identified in these preparations. The lamina lucida immediately beneath the syncytiotrophoblast and the majority of the lamina densa stained weakly or not at all, whereas the underlying lamina diffusa and stroma demonstrated moderate to intense staining. The majority of lamina diffusa staining was removed by chondroitinase ABC or AC; the remaining material was removed by nitrous acid digestion. Thus the syncytiotrophoblast surface contains a chondroitin sulfate and the basal lamina contains a mixture of intensely stained chondroitin sulfate and a weakly stained N-sulfated GAG.