Heparin procedures

Better prospects for the synthesis of HCP for long-term use might have been anticipated with the introduction of the heparinization procedure involving covalent... 

Polyethylene catheters were surface primed either with chromic acid solution or with oxygen plasma treatment, then dipped into a 0.6 % chitosan solution in 1 % acetic acid. After drying, the catheters were exposed to ammonia and then soaked in a pH 7.0 phosphate buffer containing 1 % heparin. About 40 yg of chitosan were deposited per square cm. The heparinization procedure added 2-3 units of heparin per square cm. This heparin can be removed by soaking in 25 % sodium chloride, however, if the heparinization is performed in the presence of sodium cyanoborohydride, the brine removes only a minor part of heparin. Negligible amounts of the coating were removed by blood at 37°C after 24 hr contact. 

The translational diffusion coefficient in Eq. 11 can in principle be measured from boimdary spreading as manifested for example in the width of the g (s) profiles although for monodisperse proteins this works well, for polysaccharides interpretation is seriously complicated by broadening through polydispersity. Instead special cells can be used which allow for the formation of an artificial boundary whose diffusion can be recorded with time at low speed ( 3000 rev/min). This procedure has been successfully employed for example in a recent study on heparin fractions [5]. Dynamic fight scattering has been used as a popular alternative, and a good demonstra-... 

The theory and application of this fluorescence method have been discussed in detail by LePecq and others (3,8). The assay requires that there is sufficient ionic strength to minimize ionic binding (e.g., O.IM sodium chloride), that the pH is 4-10, that no heavy metals are present, that the fluorescence is not enhanced on binding to other excipients (e.g., proteins) and that at least portions of the nucleic acids are not complexed. These requirements can usually he met when dealing with recombinant products in some cases the samples must he manipulated to create the appropriate conditions. In the intercalative method of dye binding, proteins rarely interfere with the assay, and procedures have been developed to remove the few interferences they may cause (e.g., the use of heparin or enzymatic digestion of the protein 9). 

A typical procedure is shown in Figure 2. Other dyes besides ethidium can be used, although ethidium has an advantage in that its excitation emission bands are well removed from any protein absorbances. A standard curve can be constructed for the nucleic acid of concern and the limits of detection established. In Step 3, proteolytic enzymes may be substituted for heparin, or the step may be bypassed in the case of proteins which do not interfere. After measurement of the unknown sample the nucleic acid concentration may be simply calculated or read from the standard curve. 

Our method for NMOR determination. The following procedure (carried out in a single day) was developed for blood, stomach contents, the homogenized whole mouse, and diet [(a standard semisynthetic diet prepared as in (9)]. After the rats were killed with C0 , we collected the blood (with a heparinized syringe from the heart) and the entire stomach contents. The whole mouse was frozen in liquid N2 and homogenized as in (2) ... 

Most of the common methods of isolation of heparin (described in sufficient detail in monographs128-30) are based on a procedure, developed by Charles and Scott,31 involving autolysis of the tissue (originally beef liver and beef lung), extraction with alkali, coagulation of proteins by heating, and precipitation of a heparin - protein complex by acidification. Heparin is recovered from the complex by reprecipitation with ethanol, or acetone, or both. Fats are removed by extraction with ethanol, and proteins by treatment with trypsin. Modifications of this proce-... 

The most common source of commercial heparin has become pig intestinal-mucosa, from which somewhat more than 100 mg of heparin per kg of tissue is usually obtained.32 Data on the content of heparin in organs of different animal species (summarized in Refs. 7 and 14) are probably not strictly comparable with each other, because of different extraction and purification procedures used in different laboratories, and supposedly different recoveries from different tissues. 

From the foregoing discussion, it is apparent that heparin may be subdivided into an unlimited number of fractions when different separation approaches are applied to each fraction obtained by another procedure. More than a hundred fractions have been obtained by sequential use of affinity chromatography on antithrombin, precipitation with barium, and isoelectrofocusing.214 Although these fractions can scarcely be referred to as species, such an extensive fractionation stresses the concept of the heterogeneity of heparin, and the influence of minor differences in chemical constituents, or chain length, or both, on the physicochemical (and, conceivably, biological) properties of this polysaccharide. 

Clinical chemistry, particularly the determination of the biologically relevant electrolytes in physiological fluids, remains the key area of ISEs application [15], as billions of routine measurements with ISEs are performed each year all over the world [16], The concentration ranges for the most important physiological ions detectable in blood fluids with polymeric ISEs are shown in Table 4.1. Sensors for pH and for ionized calcium, potassium and sodium are approved by the International Federation of Clinical Chemistry (IFCC) and implemented into commercially available clinical analyzers [17], Moreover, magnesium, lithium, and chloride ions are also widely detected by corresponding ISEs in blood liquids, urine, hemodialysis solutions, and elsewhere. Sensors for the determination of physiologically relevant polyions (heparin and protamine), dissolved carbon dioxide, phosphates, and other blood analytes, intensively studied over the years, are on their way to replace less reliable and/or awkward analytical procedures for blood analysis (see below). 

The analysis of human plasma for acetaminophen, the active ingredient in some pain relievers, involves a unique extraction procedure. Small-volume samples (approximately 200 fiL) of heparinized plasma, which is plasma that is treated with heparin, a natural anticoagulant found in biological tissue, are first placed in centrifuge tubes and treated with 1 N HC1 to adjust the pH. Ethyl acetate is then added to extract the acetaminophen from the samples. The tubes are vortexed, and after allowed to separate, the ethyl acetate layer containing the analyte is decanted. The resulting solutions are evaporated to dryness and then reconstituted with an 18% methanol solution, which is the final sample preparation step before HPLC analysis. The procedure is a challenge because the initial sample size is so small. 

Randy Karl of MDS Pharma Services in Lincoln, Nebraska, examines a centrifuge tube containing a heparinized plasma sample prior to performing an extraction procedure using ethyl acetate. 

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