Physico Chemical Characteristics Of Heparin Biology Essay
At the turn of the 20th century, attention was drawn to Heparin because of its anti-coagulant nature. Many people (Pavlov in 1887, Schmidt in 1892, Morawitz in 1905, Doyon in 1912 and Jaques in 1978) had isolated preparations with anti-coagulant properties. (D.Comper, 1981)
There is a controversy regarding the credit of discovery of Heparin. It was initially attributed to a physiologist named William H. Howell but later the authors credited the discovery to one of his former students, Jay McLean, who did research under guidance of Howell at John Hopkins Medical School. During 1916-17, Jay McLean, doing research under Howell, investigating pro-coagulant preparations, he isolated a fat-soluble phosphatide anti-coagulant in canine liver tissue with apparent in vitro anti-coagulant properties that lead to excess bleeding in the experimental animals. (Member of Heparin Science, n.d.) (McLean, 1959)
Many researchers investigated heparin during 1930s. The structure of Heparin was first published by Erik Jorpes at Karolinska Institutet in 1935, which made it possible for a Swedish company called Vitrum AB to produce first heparin product for intravenous use in 1936. (Member of news-medical.net, n.d.)
Heparin is a linear shaped, highly negatively charged bio molecule and it belongs to glycosaminoglycan (GAGs) family of polysaccharides. Heparin is a highly negatively charged molecule in tissues. It is widely distributed in mammalian tissues and fluids, the largest amounts found in lungs, spleen, liver and muscle. It exists normally in intracellular environment. (D.Comper, 1981)
The property of heparin, which has attracted most attention and resulted in universal clinical use, is its anticoagulant activity. Thus heparin is usually defined as an anticoagulant. The anticoagulant activity of Heparin is because of its binding nature with Anti Thrombin (AT), which inactivates the fibrinogen, which is responsible for coagulation or clotting. (Anon., 1995) A number of parameters can be involved in binding of Heparin, with stereo chemical properties of interacting particles and the nature of biological specificity is generally characterised by the components that participate in the reaction rather than the mode of the specific mechanism of binding. (D.Comper, 1981).
Heparin and heparan sulfate glycosaminoglycans belong to the group of acidic complex polysaccharides which are found on the cell surface and in the extracellular matrix. (Sasisekharan R, Venkataraman G., 2000). The Heparin polymer consists of repeating disaccharide units consisting of L-iduronic acid, Idu and D-glucosamine, GlcN which are joined by α(1 4) linkage. A single repeating unit in the sequence has three D-glucosamine and two uronic acid residues. The uronic acid residue can be D-glucuronic acid or L-iduronic acid. (Member of people.vcu.edu, 2008). Whereas a disaccharide unit has a total of three sulphate groups, one attached to 2-hydroxyl group of Idu and 2 links to 2-amino and 6-hydroxyl groups of GlcN. The structure of Heparin can be imagined as a ribbon with sulphate and carboxyl groups on the edges and hydroxyl and sugar ring oxygen atoms on the surface between these highly negatively charged groups. The conformation and local structure, which is responsible for the heterogeneity in the sequence and the pattern of the sulfation are all responsible for the interaction of heparin with other bio molecules. (S.Faham, R.E.Hileman, J.R.Fromm, R.J.Linhardt, D.C.Rees, 1996).
Figure 1. [A] Chemical structure of repeating unit in Heparin. [B] Structure of Heparin.
2.4 Physico-Chemical Characteristics of Heparin
The discovery of heparin happened unexpectedly while investigating the blood coagulation problem. This has resulted in an extensive research in the physico-chemical properties of heparin. Physico-Chemical properties of Heparin are discussed in this section.
The wide range of heterogeneity in the physico-chemical properties of heparin in the current market and the lack of suitable and shareable reference standards for the identification or quantification of process-related impurities caused, and are still causing, heated debates among scientific institutions, companies and authorities. (Lino Liverani, Giuseppe Mascellani, Franco Spelta, 2009)
Heparin is known to accelerate the inhibitor binding and to understand this activity its charge density is studied and how it effects the thrombin inhibition by heparin dependent protease inhibitors, antithrombin (AT) and heparin cofactor (HC II). The relation between structure and activity is not very well understood in the literature but only in AT binding that physical structure is responsible for a given function. (Robert.E, Man-Chiu Poon, Micheal J. Griffith, 1983)
2.5 Filtration: Ultrafiltration
Ultrafiltration uses a finely porous membrane to separate water or a base solution and microsolutes from macromolecules and colloids. Average pore diameter of the membrane can be in the range of 1 to 100 nanometers. (Baker, 2007) Membrane Ultrafiltration is a pressure driven process which can be used to separate macro molecules or colloidal particles in the size range of 1nm to 100nm, from a solvent or much smaller solutes. This ultrafiltration method is mostly used for concentrating dilute protein solutions. Ultrafiltration has a drawback which is due to the build-up of the solute particles at the surface of the membrane. This is known as concentration polarization effect. Physico-chemical properties are essential towards the effective application of membrane separation process of ultrafiltration. (W.Richard Bowen and Paul M. Williams, 1996).
Figure 3: Schematic diagram representing the experimental setup of filtration.
Figure 4: Schematic representation of the entire filtration setup.
2.6 Characterisation of Ultrafiltration Membranes:
Ultrafiltration membranes usually have a finely porous surface layer or skin supported on a much more open micro porous substrate. The finely porous surface layer performs the separation while the micro porous substrate provides mechanical strength. These membranes separate dissolved macro molecules of different sizes and are usually characterised by their molecular weight cut off, which is the molecular weight of the protein molecule that is 90% or more, rejected by the membrane. (Baker, 2007).
2.7 Fouling and Concentration Polarisation in Ultrafiltration:
Membrane fouling and concentration polarisation are the two aspects of a same problem which is the build-up of retained particles in the boundary layer neighbouring a membrane surface. Both phenomena are responsible for reduction of flux permeation through the membrane and change the selectivity of the process. Concentration polarisation describes the concentration profile of solutes in the liquid phase adjacent to the membrane resulting in the balance between different transport phenomena. Whereas, fouling comprises the matter which has left the liquid phase, to form a deposit on surface of the membrane, or inside the porous structure. However, during membrane processing, not only the concentration of the species from each class happens to change, but also new species can appear, either by reaction or by colloidal aggregation, protein denaturation, salt crystallisation, particles attrition or cell breakage. But this is generally ignored while analysing fouling. Experiments also proved that very small amount of fine particles or aggregates can foul the surface of a membrane. (P.Aimar, M.Meireles, P.Bacchin, V.Sanchez, 1993)
2.8 Dead-End Filtration:
In the dead-end ultrafiltration, the colloidal suspension flows perpendicularly to the membrane. Any particles in the solution that are larger than the pore size of the membrane are deposited, accumulating on the membrane surface, forming a cake of particles which is responsible for resistance of the filtration process. The liquid that passes through the membrane and collected in the container below is called as the filtrate. In a dead-end filtration process, resistance cake grows which affects the rate of filtration. The filtration rate decreases with filtration time due to the hydraulic resistance of the filter cake. Laboratory scale dead-end filtration is generally carried in a filtration cell which is simply a cylindrical vessel, fitted with a porous support onto which the membrane is placed. An exit port is provided for the filtrate which is collected and weighed on a digital balance. For accurate measurements and efficient data collection, the balance can be connected to a computer. The flux is calculated by measuring the mass of the filtrate collected which in turn gives the volume collected, if the density is known. The pressure is kept constant by connecting the cell to a compressed gas source, usually nitrogen. (BE, 2008).
Figure 2: Schematic diagram showing filtration of feed solution and separation of permeate.
2.9 Membrane Resistance:
Membrane resistance is the resistance offered by the by the membrane. This is because of the layer of particles rejected by the membrane, which is formed as a cake, which is responsible for the reduction in the flux. This is calculated using the following equation
Where µ is the viscosity of the solution, Rm is the membrane resistance, A is the cross-section area of the membrane, P is the pressure applied, t is time, V is volume, α is specific resistance of the cake, C is the concentration of the solution.
Chapter 3: Materials and Methods
The heparin used in the experiments is available as ‘Heparin Sodium’ by which is used only for experimental requirements.
3.2.1 pH Measurements:
Each experiment is carried out with a different sample with different pH and it is one of the very important factors determining the outcome of the experiment. Different pH values with which the experiments were carried out was 3, 5, 7.4 and 9. This was measured using Philips PW 9421 digital pH meter was used, with a Fisher probe (type FB68788).
3.2.2 Conductivity Measurements:
While measuring the pH, simultaneously conductivity was measured using Protec conductivity meter P.I.8140 using a Fisher Probe.
3.2.3 Particle Size Measurements:
The hydrodynamic diameter of the heparin particles is measured using the dynamic light scattering principle. Dynamic Light Scattering is used to measure size of particles and molecule size. This technique measures the diffusion of particles moving under Brownian motion, and converts this to size and a size distribution is obtained using the Stokes-Einstein relationship. Non-Invasive Back Scatter technology (NIBS) is incorporated to give the highest sensitivity simultaneously with the highest dynamic size and concentration range. (Malvern Instruments, n.d.) This is done using the High Performance Particle Sizer (HPPS) and the dispersion technology software (DTS). The Malvern High Performance Particle Sizer, HPPS can measure the particles within the size range of 0.6nm to 6µm. This can be done with a small amount of sample solution, about 5ml to 7ml. During the measurement, temperature of the sample is maintained constant at 25◦C. Measurement is done in a glass container called cell curvet. The instrument is controlled with a desktop computer running on windows XP.
3.2.4 Zeta Potential Measurements:
Zeta potential measurements are carried out on Malvern Zetasizer Nano range S. Zeta potential is measured in a zeta cell with electrodes, with a sample carrying capacity of 2ml to 3ml. When electricity is passed through the electrodes, the ions in the sample move towards oppositely charged electrodes. The electrophoretic mobility of these ions is measured using Laser Doppler Velocimetry (LDV), accumulation of the net charge at the surface of the particle affects the distribution of ions in the surrounding interfacial region, resulting in an increased concentration of counter ions close to the surface. Thus an electrical double layer exists around each particle. The liquid layer surrounding the particle exists as two parts; an inner region, called the Stern layer, where the ions are strongly bound and an outer, diffuse, region where they are less firmly attached. Within the diffuse layer there is a notional boundary inside which the ions and particles form a stable entity. When a particle moves, ions within the boundary move with it, but any ions beyond the boundary do not travel with the particle. This boundary is called the surface of hydrodynamic shear or slipping plane. The potential that exists at this boundary is known as the Zeta potential. Electrophoretic mobility is used to calculate the zeta potential of the solution using the equation
Where Ue is the electrophoretic mobility, z is zeta potential, ƞ is the viscosity, ε is the dielectric constant, f(ka) is the Henry’s function which is usually 1.5 or 1 for most of the solutions. (Member of nbtc cornell, n.d.)
Figure 4: Schematic showing zeta cell curvet.
Molecular Weight Measurements:
Molecular weight is carried out in the same instrument, Malvern Zetasizer Nano range S. Static Light Scattering (SLS) is used to determine the molecular weight of proteins and polymers. In this particular technique, the scattering intensity of multiple samples of different concentrations is measured, and used to construct a Debye plot. Using this Debye plot, average molecular weight and second virial coefficient can be calculated.
Membrane characterisation is done with AFM.