Radiochemical quality control of67Ga-citrate radiopharmaceuticals

A rapid, miniaturized chromatography system has been developed to determine the possible contaminants of⁶⁷Ga-citrate. This method is simple, inexpensive and suitable for laboratory routine tests. By classical paper chromatography the analysis takes several hours to complete.     

Distribution of 67Ga-citrate in 3'-methyl-4-dimethylaminoazobenzene-induced hepatoma of rats

The distribution of 67Ga-citrate in the hepatoma of rat induced by 3'-methyl-4-dimethylaminoazobenzene was studied. 67Ga uptake ratio resected specimen, autoradiography and histological specimen were compared each other. 67Ga uptake ratio of the tumor was increased 1.6 to 7.2 times (average 4.4) to control group. Regardless of the size of the tumor, macroautoradiographically observed distribution of 67Ga-citrate in the hepatoma was higher in the peripheral zone than in the central zone. Histologically the degeneration of tumor cell was low or absent in the peripheral zone of tumor, whereas it was intense in the central zone. 67Ga-citrate was highly accumulated in the zone which the degeneration was low or absent. We, however, could not demonstrate the site where 67Ga-citrate was incorporated.     

Combinatorial enzymatic synthesis of heparan sulfate

Escherichia coli K5 heparosan was enzymatically modified by Chen and colleagues to construct a library of heparan sulfate polysaccharides for evaluation, leading to the discovery that a 2-O-sulfoiduronic acid residue is not essential for antithrombin-mediated anticoagulant activity in larger oligosaccharide and polysaccharide structures.     

Subtraction scintigraphy with 67Ga-citrate and 99mTc-colloid in the diagnosis of intrahepatic masses

Subtraction scintigraphy of the liver with 67Ga-citrate and 99mTc-colloid (99mTc-phytate) was performed by subtracting the image of the latter from the image of the former in 34 patients who were suspected of having intrahepatic masses, especially hepatoma. The computer used was Scintipac 1200 (32 KW memories and 2.4MBX2 disk memories). Both images were taken in a digital form (128 X 128 elements). After normalizing both images, 4 different factors (0.6, 0.8, 1.0, and 1.2) were applied to the 99mTc image before the subtraction from the 67Ga image. We found that this technique was very useful for the detection of abnormal 67Ga accumulation in the liver.     

Heparin and heparan sulfate: structure and function

This review covers the structure and function of heparin and heparan sulfate glycosaminoglycans. Their chemical structures are discussed, including recently developed methods for sequencing picomole to nanomole quantities of heparin- and heparan sulfate-derived oligosaccharides. The biosynthesis of heparin and heparan sulfate is reviewed as it relates to their diverse and varied structures, and their biological activities and functions are discussed. The literature up to August 2001 is reviewed, and 208 references are cited.     

Utilization of 67Ga-citrate for the measurement of histamine-induced edema in anesthetized rats

Radioactivity of the histamine-induced paw edema was measured after intravenous injection of 67Ga-citrate in anesthetized rats. The radioactivity was fluctuated almost parallel with the edema rate following subcutaneous injection of histamine. Both the radioactivity and the edema rate were almost equally reduced by pretreatment with chlorpheniramine, an antihistaminic agent. There was a good correlation between the edema rate and the radioactivity. These results suggest that 67Ga-citrate is useful for the pharmacological study of antiinflammatory drugs, or is available for measuring the vascular permeability.     

High sensitivity separation and detection of heparan sulfate disaccharides

Eight Delta-disaccharide standards from heparan sulfate/heparin were derivatized with the fluorophore 4,4-difluoro-5,7- dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, hydrazide (BODIPY) via formation of a Schiff's base and separated using HPAEC on a Propac PA1 column with a linear salt gradient and isocratic 150 mM NaOH. Detection was with an in-line fluorescence detector. The standard deviation (sigma(n-1)) in retention times were 0.7-2% over nine runs. The limit of detection, was 100 fmol (100 x 10(-15)mol) of BODIPY labeled Delta-disaccharides, representing considerably improved detection compared to other fluorophore labeled derivatives and, unlike these, required no further purification steps. Separation and improved detection of BODIPY-Delta-disaccharide conjugates will assist the structural analysis of HS and the development of improved sequencing methodologies.     

Hyphenated techniques for the analysis of heparin and heparan sulfate

The elucidation of the structure of glycosaminoglycan has proven to be challenging for analytical chemists. Molecules of glycosaminoglycan have a high negative charge and are polydisperse and microheterogeneous, thus requiring the application of multiple analytical techniques and methods. Heparin and heparan sulfate are the most structurally complex of the glycosaminoglycans and are widely distributed in nature. They play critical roles in physiological and pathophysiological processes through their interaction with heparin-binding proteins. Moreover, heparin and low-molecular weight heparin are currently used as pharmaceutical drugs to control blood coagulation. In 2008, the health crisis resulting from the contamination of pharmaceutical heparin led to considerable attention regarding their analysis and structural characterization. Modern analytical techniques, including high-performance liquid chromatography, capillary electrophoresis, mass spectrometry, and nuclear magnetic resonance spectroscopy, played critical roles in this effort. A successful combination of separation and spectral techniques will clearly provide a critical advantage in the future analysis of heparin and heparan sulfate. This review focuses on recent efforts to develop hyphenated techniques for the analysis of heparin and heparan sulfate.     

Orthogonal sulfation strategy for synthetic heparan sulfate ligands

[reaction: see text] An orthogonal sulfation strategy involving six different protecting groups has been developed for generating sulfated carbohydrate libraries based on heparan. Chemoselective cleavage conditions (optimized for a heparan disaccharide) can be performed in the presence of sulfate esters as well as the remaining protecting groups.     

Novel heparan sulfate mimetic compounds as antitumor agents

Heparan sulfate glycosaminoglycans (HSGAGs) are involved in tumor cell growth, adhesion, invasion, and migration, due to their interactions with various proteins. In this study, novel HSGAG-mimetic compounds (KI compounds) were designed and synthesized. As a result of cell-based assays, KI-105 was found to exert potent inhibitory activities against migration and invasion of human fibrosarcoma HT1080 cells. The present results indicate that a novel invasion/migration inhibitor, KI-105, can increase the adherence of HT1080 cells. It was conceivable that this cellular effect was caused by an increase in the amount of cell-surface HSGAGs and focal adhesions. Although further investigations are needed to decipher the molecular mechanism of KI-105, it is suggested that heparanase and Cdc42 are involved in its biological effects.     

Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity

O-sulfotransferases (OSTs) are critical enzymes in the cellular biosynthesis of the biologically and pharmacologically important heparan sulfate and heparin. Recently, these enzymes have been cloned and expressed in bacteria for application in the chemoenzymatic synthesis of glycosaminoglycan-based drugs. OST activity assays have largely relied on the use of radioisotopic methods using [³⁵S] 3′-phosphoadenosine-5′-phosphosulfate and scintillation counting. Herein, we examine alternative assays that are more compatible with a biomanufacturing environment. A high throughput microtiter-based approach is reported that relies on a coupled bienzymic colorimetric assay for heparan sulfate and heparin OSTs acting on polysaccharide substrates using arylsulfotransferase-IV and p-nitrophenylsulfate as a sacrificial sulfogroup donor. A second liquid chromatography-mass spectrometric assay, for heparan sulfate and heparin OSTs acting on structurally defined oligosaccharide substrates, is also reported that provides additional information on the number and positions of the transferred sulfo groups within the product. Together, these assays allow quantitative and mechanistic information to be obtained on OSTs that act on heparan sulfate and heparin precursors.

Homodimerization of hyaluronan and heparan sulfate derivatives by olefin metathesis reaction

Hyaluronan and heparan sulfate disaccharides of the type β-d-glucuronic acid-(1→3)-N-acetyl-β-d-glucosamine and α-l-iduronic acid-(1→4)-N-acetyl-β-d-glucosamine, respectively, with an n-pentenyl group at the reducing end have been synthesized. Homodimerization of these derivatives using Grubbs catalyst furnished dimerized disaccharides separated by a C₈ spacer arm.     

New electrophoretic and chromatographic techniques for analysis of heparin and heparan sulfate

Heparin (HE) and heparan sulfated glycosaminoglycans are well-known mediators of tissue development, maintenance and functions; the activities of these polysaccharides are depending mainly on their sulfate substitutions. The HE structure is also a very important feature in antithrombotic drug development, since the antithrombin binding site is composed by sequences of a specific sulfation pattern. The analysis of disaccharide composition is then a fundamental point of all the studies regarding HE/heparan sulfate glycosaminoglycan (and thereby proteoglycan) functions. The present work describes two analytical methods to quantify the disaccharides constituting HE and heparan sulfate chains. The use of PAGE of fluorophore-labeled saccharides and HPLC coupled with a fluorescence detector allowed in one run the identification of 90-95% of HE disaccharides and 74-100% of rat kidney purified heparan sulfate. Moreover, the protocol here reported avoid the N-sulfation disaccharides degradation, which may affect N-sulfated/N-acetylated disaccharides ratio evaluation. These methods could be also very important in clinical treatments since they are useful for monitoring the availability kinetics of antithrombotic drugs, such as low-molecular-weight HEs.     

Clinical evaluation of 67Ga-citrate scintigraphy with reference to tumor detectability (author's transl)

The clinical usefulness of 67Ga-citrate whole body scanning is evaluated in 178 cases of the various diseases in a period of 1976-1978. The cases are 59 cases of thoracic lesions, 41 cases of head and neck lesions, 35 cases of abdominal, bone, and soft tissue lesions, and 43 cases of benign and malignant lymphoma. The clinical results and detectability of various known lesions are shown in tables 1, 2, 3 and 4. The detectability of unknown lesions of malignant lymphoma is shown in table 5. The study demonstrates the particular clinical values of 67Ga-citrate scan in detectability of unknown lesions to be in cases of malignant lymphoma.     

Chemical heterogeneity of heparan sulfate from a human neuroblastoma cell line

The chemical heterogeneity of radiolabelled neuroblastoma heparan sulfate has been studied by ion exchange chromatography and by affinity chromatography on heparan sulfate-agarose. Although the entire population of chains shows considerable homogeneity in charge density, the deaminative cleavage products ranged in size from disaccharides to eicosasaccharides. Under appropriate conditions neuroblastoma heparan sulfate could be separated into two pools of low or high affinity for lung heparan sulfate-agarose. Analyses of periodate oxidation-alkaline elimination indicated that the high affinity chains contained larger proportions of heparin-like segments, i.e. iduronate-rich and N-sulfated ones.     

Advances in the separation,sensitive detection,and characterization of heparin and heparan sulfate

Elucidation of the relationship between the structure and biological function of the glycosaminoglycans (GAGs) heparin and heparan sulfate (HS) presents an important analytical challenge mainly due to the difficulty in determining their fine structure. Heparin and HS are responsible for mediation of a wide range of biological actions through specific binding to a variety of proteins including those involved in blood coagulation, cell proliferation, differentiation and adhesion, and host–pathogen interactions. Therefore, there is a growing interest in characterizing the microstructure of heparin and HS and in elucidating the molecular level details of their interaction with peptides and proteins. This review discusses recent developments in the analytical methods used for sensitive separation, detection, and structural characterization of heparin and HS. A brief discussion of the analysis of contaminants in pharmaceutical heparin is also presented.     

Using an enzymatic combinatorial approach to identify anticoagulant heparan sulfate structures

Heparan sulfate (HS) represents a major class of glycans that perform central physiological functions. Emerging HS and glycosaminoglycan microarray techniques are used to interrogate the structure and function relationship to develop novel therapeutic agents. Availability of HS with specific sulfation patterns has been a limiting factor and impedes the accuracy of HS glycomics studies. Although organic synthesis provides oligosaccharides, these may not fully represent the biological functions of polysaccharides. Here, we present a study for developing an enzyme-based approach to synthesize a polysaccharide library with different sulfation patterns. Using different combinations of biosynthetic enzymes, we synthesized eight unique polysaccharides. We discovered that polysaccharides without the iduronic acid residue displayed strong binding affinity to antithrombin and high anti-Xa and anti-IIa activities. The enzyme-based synthetic approach could become a general method for discovering new HS structures with unique biological functions.