Anders Hjerpe

Ion-pair high-performance liquid chromatography for determining disaccharide composition in heparin and heparan sulphate.

In this report we describe a convenient and sensitive HPLC method for separating and determining the non- and variously sulphated delta-disaccharides derived from heparan sulphate, heparin and Fragmin, using heparin- and heparan sulphate lyases. This method is superior to others since it can separate and determine twelve different non-, mono-, di- and trisulphated delta-disaccharides containing either N-sulphated, N-acetylated or unsubstituted glucosamine in a single HPLC run. The various types of delta-disaccharides are separated by an ion-pair reversed-phase chromatographic procedure on a Supelcosil LC-18 column, using a binary acetonitrile gradient system with tetrabutylammonium as the ion-pairing reagent. The eluted peaks were recorded by dual wavelength at 232 and 226 nm and a linear detector response was obtained over the entire interval tested, i.e., to 50 micrograms of delta-disaccharides. As little as 0.8-5 ng of delta-disaccharides can be reliably detected and accurately determined. Following separate digestion with the heparin- and heparan sulphate lyases (heparin lyases I, II and III), the characteristic heparin delta-disaccharides in the heparan sulphate chain, as well as the heparan sulphate delta-disaccharides in the heparin polymer, can be identified. Using combined digestions with these three lyases, the glycosaminoglycan chains are degraded almost completely (> 90%) to delta-disaccharides, which are then determined by direct injections into the HPLC system and thus an almost complete spectrum of disaccharide composition can be obtained. By this method, it is possible to analyse and confirm that the heparan sulphate chain is defined as a glycosaminoglycan dominated by GlcNAc(+/- 6S)-GlcA disaccharides and by some copolymeric disaccharides, such as GlcNS-IdoA2S and GlcNS6S-IdoA2S, otherwise most common in heparin. Fragmin, which is a controlled cepolymerized heparin fragment of M(r) 5000, is made up mainly of trisulphated disaccharides of the GlcNS6S-IdoA2S type (88.8%). Using separate digestions with the specific heparin lyases, one can also distinguish between heparin and heparan sulphate.

Proteoglycans of mineralizing rib and epiphyseal cartilage.

Rib cartilage from growing guinea pigs and epiphyseal cartilage from Beagle puppies were separated into three fractions, representing non-mineralized, low mineralized, and high mineralized, tissue, by centrifuging finely ground material in acetone/bromoform density gradients. Following extraction under dissociative conditions, the proteoglycans were fractionated by density gradient ultracentrifugation under associative and dissociative conditions. With the onset of mineralization, the cartilage lost approximately half its content of proteoglycans. The proteoglycans remaining in the calcified cartilage differed in composition and in size from those of nonmineralized tissue. With the increased mineral content of the tissues the ratios of protein to polysaccharide, of chondroitin sulfate to keratan sulfate, and of 4-sulfate to 6-sulfated chondroitin sulfate increased in the proteoglycan fraction. Furthermore, gel chromatograms indicated decreased proportions of very high molecular weight proteoglycans, in mineralized tissue.

Determination of sulphated disaccharides from chondroitin suphates by high-performance liquid chromatography

A sensitive method for the determination of chondroitin 4- and 6-sulphate is presented. After chondroitinase digestion of the chondroitin sulphate preparations, the obtained disaccharides are separated on a weak anion-exchange resin in a high-performance liquid chromatography system. The method was used to study 4-sulphate to 6-sulphate ratios in chondroitin sulphates prepared from bovine nasal cartilage and human nucleus pulposus. The results show clearly that these two preparations contain considerable amounts of both isomers.

Determination of hyaluronan and galactosaminoglycan disaccharides by high-performance capillary electrophoresis at the attomole level. Applications to analyses of tissue and cell culture proteoglycans

A rapid, highly sensitive and reproducible HPCE method is described for the determination of all non- and variously sulphated disaccharides present in hyaluronan and vertebrate chondroitin sulphates and dermatan sulphates. Following chondroitinase digestion of glycosaminoglycans or proteoglycans, the non-, di- and tri-sulphated delta-disaccharides are completely separated and readily determined within 14 min on a fused-silica capillary in 15 mM sodium dihydrogen orthophosphate, pH 3.00, using reversed polarity at 20 kV and detection at 232 nm. The determination of the various delta-disaccharides derived from either glucuronic or iduronic acid and the presence of glucuronic and iduronic clustered structures in dermatan sulphate can also easily be made, using digests with chondroitinase AC or B. A linear detector response was obtained for the entire interval tested (up to 10 mg/l of delta-disaccharides). Concentrations as small as 32, 65, 100 and 250 pmol/l (22, 38, 50 and 98 ng/l) of tri-, di- and nonsulphated delta-disaccharides, respectively, can be reliably detected.

Persistence of human papillomavirus (HPV) infections preceding cervical carcinoma

A persistent genital infection with an oncogene‐type of human papillomavirus (HPV) is considered to be essential for the development of most cervical carcinomas. Therefore, HPV analysis has been proposed as a possible complementary cytological screening program. The authors have developed a technique to analyze archival Pap smears, which has enabled them to study the relation between persistent HPV infection and the development of cervical cancer.

Determination of twelve heparin- and heparan sulfate-derived disaccharides as 2-aminoacridone derivatives by capillary zone electrophoresis using ultraviolet and laser-induced fluorescence detection

In quest for high sensitivities, we developed an ultrahigh capillary electrophoresis (CE) method for the structural analysis of heparin and heparan sulfate (HS) in biologic samples. Heparin and HS were digested with an equi‐unit mixture of heparin lyases I, II and III and the obtained Δ‐disaccharides were derivatized with the fluorophore 2‐aminoacridone. All known twelve non‐, mono‐, di‐ and trisulfated Δ‐disaccharides were completely resolved in a single run, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV. Relative standard deviation in migration times and peak areas as well as day‐to‐day variance ranged from 0.9 to 2.4%, suggesting a reproducible and precise method. Detection of 2‐aminoacridone (AMAC)‐derivatives of Δ‐disaccharides by UV at 255 nm showed 2.8 and 10 times higher sensitivity than that of derivatized and nonderivatized ones at 232 nm. Laser‐induced fluorescence detection with an Ar‐ion laser source showed an approximately 100 times higher sensitivity than that obtained at 232 nm of the nonderivatized species. Application of this method to quantitative analysis of Δ‐disaccharides derived from porcine intestinal mucosa heparin and bovine kidney HS showed excellent agreement with previously published methods, suggesting an accurate method. The developed method can be easily applied for the disaccharide analysis of heparin/HS at the attomole level with high accuracy, for distinguishing between heparin and HS and may be of value for studying their interactions with matrix effective molecules.

Serglycin Constitutively Secreted by Myeloma Plasma Cells Is a Potent Inhibitor of Bone Mineralization in Vitro

Although the biological significance of proteoglycans (PGs) has previously been highlighted in multiple myeloma (MM), little is known about serglycin, which is a hematopoietic cell granule PG. In this study, we describe the expression and highly constitutive secretion of serglycin in several MM cell lines. Serglycin messenger RNA was detected in six MM cell lines. PGs were purified from conditioned medium of four MM cell lines, and serglycin substituted with 4-sulfated chondroitin sulfate was identified as the predominant PG. Flow cytometry and confocal microscopy showed that serglycin was also present intracellularly and on the cell surface, and attachment to the cell surface was at least in part dependent on intact glycosaminoglycan side chains. Immunohistochemical staining of bone marrow biopsies showed the presence of serglycin both in benign and malignant plasma cells. Immunoblotting in bone marrow aspirates from a limited number of patients with newly diagnosed MM revealed highly increased levels of serglycin in 30% of the cases. Serglycin isolated from myeloma plasma cells was found to influence the bone mineralization process through inhibition of the crystal growth rate of hydroxyapatite. This rate reduction was attributed to adsorption and further blocking of the active growth sites on the crystal surface. The apparent order of the crystallization reaction was found to be n = 2, suggesting a surface diffusion-controlled spiral growth mechanism. Our findings suggest that serglycin release is a constitutive process, which may be of fundamental biological importance in the study of MM.

Determination of iduronic acid and glucuronic acid in glycosaminoglycans after stoichiometric reduction and depolymerization using high-performance liquid chromatography and ultraviolet detection.

The reduction of uronic acids in glycosaminoglycans (GAGs) prior to depolymerization reactions is one way in which the uronic acid content of polysaccharides can be studied without major losses. The obtained monosaccharides can be recovered from the subsequent depolymerization with a yield better than 95%. Following reduction, depolymerization, and lyophilization, D-glucuronic acid is converted to D-Glc and L-iduronic acid to 1,6-anhydro-idose. Per-O-benzoyl derivatives of these monosaccharides can be separated and detected in nanogram amounts using reversed phase HPLC. A linear detector response was obtained for injections up to 22 nmol (4 micrograms) of Glc and 1,6-anhydro-idose and the detection limit was 5 and 7 pmol, respectively. Reduction, depolymerization, and derivatization with subsequent chromatography of various GAGs can be readily performed in the 1- to 30-micrograms range.

A prospective study on the risk of cervical intra-epithelial neoplasia among healthy subjects with serum antibodies to HPV compared with HPV DNA in cervical smears

To estimate the risk of developing cervical intra‐epithelial neoplasia (CIN) among women exposed to human papillomavirus (HPV) type 16, we performed a prospective study in a population‐based cohort of more than 15,000 women followed for 34.9 months. Seventy‐four women developed CIN during follow‐up and were matched for age, time of sampling and area of residence with 148 women who remained CIN‐free during follow‐up. The blood samples taken at enrollment were tested for serum antibodies to HPV types 16, 18 and 33 capsids. Cervical smears or biopsies were analyzed for the presence of HPV DNA by nested PCR using HPV general primers and by HPV 16‐ and 18‐type‐specific PCR. HPV serology and HPV‐PCR were in good agreement, particularly when the blood sample and the Pap smear were taken less than 6 months apart. HPV DNA was found in 88% of cases and 4% of controls, whereas HPV 16 DNA was present in 44% of cases and in 1 of 142 controls. HPV‐16‐seropositive women had a 3‐fold increased risk of developing CIN. The risk was highest among women younger than 35 years of age, of whom an estimated 3.4% of HPV‐16‐seropositive and 0.5% of seronegative women developed CIN. Since the risk associated with HPV‐16 seropositivity (a measure of past or present infection) was 35‐fold lower than that of HPV DNA (present infection), most infections appear to be eliminated before CIN develops. In conclusion, HPV 16 infection does confer an excess risk of CIN development, and HPV DNA detection has a high predictive value for the presence of high‐grade CIN.

Ultrasensitive capillary electrophoresis of sulfated disaccharides in chondroitin/dermatan sulfates by laser-induced fluorescence after derivatization with 2-aminoacridone.

An ultrasensitive capillary electrophoretic method for separating the variously sulfated chondroitin/dermatan sulfate-derived delta-disaccharides after digestion with chondro/dermatolyases and derivatization with the fluorophore 2-aminoacridone is described. All known mono-, di- and tri-sulfated delta-disaccharides were completely separated using 15 mM orthophosphate buffer (pH 3.0) at 20 kV without any interference of the excess derivatizing reagent. They were detected at the anode (reversed polarity) using either an Ar-ion laser-induced fluorescence (LIF) detector (excitation wavelength 488 nm) or a UV detector. The sensitivity obtained by LIF (0.51 pmol/l) was at least 100 and 10 times higher as compared to those obtained by UV detection at 232 nm of underivatized delta-disaccharides and at 254 nm of those derivatized with aminoacridone, respectively. The method has been easily applied to the analysis of chondroitin/dermatan sulfates from various tissues at the attogram level, including chondrotin/dermatan sulfates from normal and aneurysmal human abdominal aortas.