Weak and Strong type anion and cation exchangers
\r\n\tThe aim of this book will be to describe the most common forms of dermatitis putting emphasis on the pathophysiology, clinical appearance and diagnostic of each disease. We also will aim to describe the therapeutic management and new therapeutic approaches of each condition that are currently being studied and are supposed to be used in the near future.
",isbn:null,printIsbn:"979-953-307-X-X",pdfIsbn:null,doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"278931ae110500350d8b64805c70f193",bookSignature:"Dr. Eleni Papakonstantinou",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/7934.jpg",keywords:"Atopic eczema, Interleukin, Topical corticosteroids, Hand eczema, Blisters, Pruritus, Irritant contact dermatitis, Allergic contact dermatitis, Discoid eczema, Sebaceous glands, Inflammatory dermatitis, Facial rash",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"February 5th 2019",dateEndSecondStepPublish:"March 19th 2019",dateEndThirdStepPublish:"May 18th 2019",dateEndFourthStepPublish:"August 6th 2019",dateEndFifthStepPublish:"October 5th 2019",remainingDaysToSecondStep:"2 years",secondStepPassed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:null,coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"203520",title:"Dr.",name:"Eleni",middleName:null,surname:"Papakonstantinou",slug:"eleni-papakonstantinou",fullName:"Eleni Papakonstantinou",profilePictureURL:"https://mts.intechopen.com/storage/users/203520/images/system/203520.jpg",biography:"Dr. med. Eleni Papakonstantinou is a Doctor of Medicine graduate and board certified Dermatologist-Venereologist. She studied medicine at the Aristotle University of Thessaloniki, in Greece and she continued with her dermatology specialty in Germany (2012-2017) at the University of Magdeburg and Hannover Medical School, where she completed her dissertation in 2016 with research work on atopic dermatitis in children. During this time she gained wide experience in the whole dermatological field with special focus on the diagnosis and treatment of chronic inflammatory skin diseases and also the prevention and treatment of melanocytic and non-melanocytic skin tumors. Her research interests were beside atopic dermatitis and pruritus also the pathophysiology of blistering dermatoses. In addition to lectures at german and international congresses, she has published several articles in german and international journals and her work has been awarded with various prizes (poster prize of the German Dermatological Society for the project: 'Bullous pemphigoid and comorbidities' (DDG Leipzig 2016), 'Michael Hornstein Memorial Scholarship' (EADV Athens 2016), travel grant (EAACI Vienna 2016). Since 2017, she works as a specialist dermatologist in private practice in Dortmund, in Germany. Parallel she co-administrates an international dermatologic network, Wikiderm International and she writes a dermatology public guide for patients, as she is convinced that evidence-based knowledge has to be shared not only with colleagues but also with patients.",institutionString:"Private Practice, Dermatology and Venereology",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"1",totalChapterViews:"0",totalEditedBooks:"0",institution:null}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"16",title:"Medicine",slug:"medicine"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"270941",firstName:"Sandra",lastName:"Maljavac",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/270941/images/7824_n.jpg",email:"sandra.m@intechopen.com",biography:"As an Author Service Manager my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review, to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. 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Ion chromatography separation is based on ionic (or electrostatic) interactions between ionic and polar analytes, ions present in the eluent and ionic functional groups fixed to the chromatographic support. Two distinct mechanisms as follows; ion exchange due to competitive ionic binding (attraction) and ion exclusion due to repulsion between similarly charged analyte ions and the ions fixed on the chromatographic support, play a role in the separation in ion chromatography. Ion exchange has been the predominant form of ion chromatography to date [2]. This chromatography is one of the most important adsorption techniques used in the separation of peptides, proteins, nucleic acids and related biopolymers which are charged molecules in different molecular sizes and molecular nature [3-6]. The separation is based on the formation of ionic bonds between the charged groups of biomolecules and an ion-exchange gel/support carrying the opposite charge [7]. Biomolecules display different degrees of interaction with charged chromatography media due to their varying charge properties [8].
The earliest report of ion-exchange chromatography date back to 1850, Thompson studied the adsorption of ammonium ions to soils [9-11]. Spedding and Powell published a series of papers describing practical methods for preparative separation of the rare earths by displacement ion-exchange chromatography in 1947. Beginning in the 1950s, Kraus and Nelson reported numerous analytical methods which are used for metal ions based on separation of their chloride, fluoride, nitrate or sulfate complexes by anion chromatography [12]. In order to separate proteins an ion exchange chromatographic method was reported by Peterson and Sober in 1956. In modern form ion exchange chromatography was introduced by Small, Stevens and Bauman in 1975 [3]. Gjerde et al. published a method for anion chromatography in 1979 and this was followed by a similar method for cation chromatography in 1980 [12]. Ion-exchange chromatography has been used for many years to separate various ionic compounds; cations and anions and still continues to be used. The popularity of ion exchange chromatography has been increased in recent years because this technique allows analysis of wide range of molecules in pharmaceutical, biotechnology, environmental, agricultural and other industries [2].
Ion-exchange chromatography which is designed specifically for the separation of differently charged or ionizable compounds comprises from mobile and stationary phases similar to other forms of column based liquid chromatography techniques [9-11]. Mobil phases consist an aqueous buffer system into which the mixture to be resolved. The stationary phase usually made from inert organic matrix chemically derivative with ionizable functional groups (fixed ions) which carry displaceable oppositely charged ion [11]. Ions which exist in a state of equilibrium between the mobile phase and stationary phases giving rise to two possible formats, anion and cation exchange are referred to as counter ion (Figure 1) [1,13]. Exchangeable matrix counter ions may include protons (H+), hydroxide groups (OH-), single charged mono atomic ions (Na+, K+, Cl-), double charged mono atomic ions (Ca2+, Mg2+), and polyatomic inorganic ions (SO42-, PO43-) as well as organic bases (NR2H+) and acids (COO-) [11]. Cations are separated on cation-exchange resin column and anions on an anion exchange resin column [10]. Separation based on the binding of analytes to positively or negatively charged groups which are fixed on a stationary phase and which are in equilibrium with free counter ions in the mobile phase according to differences in their net surface charge (Figure 1) [13-14].
Types of ion exchangers
Ion exchange chromatography involves separation of ionic and polar analytes using chromatographic supports derivatized with ionic functional groups that have charges opposite that of the analyte ions. The analyte ions and similarly charged ions of the eluent compete to bind to the oppositely charged ionic functional group on the surface of the stationary phase. Assuming that the exchanging ions (analytes and ions in the mobile phase) are cations, the competition can be explained using the following equation;
In this process the cation M+ of the eluent replaced with the analyte cation C+ bound to the anion X- which is fixed on the surface of the chromatographic support (S).
In anion exchange chromatography, the exchanging ions are anions and the equation is represented as follow;
The anion B- of the eluent replaced with the analyte cation A- bound to the positively charged ion X+ on the surface of the stationary phase. The adsorption of the analyte to the stationary phase and desorption by the eluent ions is repeated during their journey in the column, resulting in the separation due to ion-exchange [2].
Molecules vary considerably in their charge properties and will exhibit different degrees of interaction with charged chromatography support according to differences in their overall charge, charge density and surface charge distribution. Net surface charge of all molecules with ionizable groups is highly pH dependent [13]. Therefore pH of the mobile phase should be selected according to the net charge on a protein of interest within a mixture is opposite to that of matrix functional group, that it will displace the functional group counter ion and bind the matrix. On the other hand oppositely charged proteins will not be retained. Adsorbed protein analytes can be eluted by changing the mobile phase pH which effect the net charge of adsorbed protein, so its matrix binding capacity. Moreover increasing the concentration of a similarly charged species within the mobile phase can be resulted in elution of bound proteins. During ion exchange chromatography for example in anion exchange as illustrated in Figure 2, negatively charged protein analytes can be competitively displaced by the addition of negatively charged ions. The affinity of interaction between the salt ions and the functional groups will eventually exceed that the interaction exists between the protein charges and the functional groups, resulting in protein displacement and elution by increasing gradually the salt concentration in the mobile phase [11].
Separation steps in anion exchange chromatography (GE Healthcare)
Complex mixtures of anions or cations can usually be separated and quantitative amounts of each ion measured in a relatively short time by ion exchange chromatography [10]. In classical ion-exchange chromatography separations have been performed in the open-column mode. Column which is loosely packed with stationary phase as small particles made of 1-2 cm diameter glass. The mobile phase or eluent contains the competing ion and is passed continuously into the column and percolates through it under gravity. Sample mixture is applied to the top of the column and allowed to pass into the bed of ion- exchange material. Eluent flow is then resumed and fractions of eluent are collected at regular intervals from the column outlet. Open column ion-exchange chromatography is very slow due to low eluent flow-rates. Increasing flow rate may result in deteriorated separation efficiency (Figure 3). In modern ion-exchange chromatography the usage of high efficiency ion exchange materials combined with flow-through detection have overcome of these challenges. Separations are performed on the column which is filled with ion-exchanger as particles in uniform size. The particles of ion-exchange material are generally very much smaller than those used for classical open column ion-exchange chromatography [1]. However ion-exchange resins used in modern chromatography have lower capacity than older resins [10]. The eluent must be pumped through the column due to the small particle size of stationary phase. The sample mixture is applied into eluent by the injection port. Finally the separated ions are detected with a flow-through detection instrument [1].
Ion exchange chromatography technique
This technique has been used for the analyses of anions and cations, including metal ions, mono- and oligosaccharides, alditols and other polyhydroxy compounds, aminoglycosides (antibiotics), amino acids and peptides, organic acids, amines, alcohols, phenols, thiols, nucleotides and nucleosides and other polar molecules. It has been successfully applied to the analysis of raw materials, bulk active ingredients, counter ions, impurities, and degradation products, excipients, diluents and at different stages of the production process as well as for the analysis of production equipment cleaning solutions, waste streams, container compatibility and other applications [2]. Wide applicability including high performance and high-throughput application formats, average cost, powerful resolving ability, large sample handling capacity and ease of scale-up as well as automation allow the ion exchange chromatography has become one of the most important and extensively used of all liquid chromatographic technique [11].
Although the extensive use of ion exchange chromatography the mechanism of the separation has not completely been elucidated. A considerable effort has been made to describe the ion exchange process theoretically [3,9]. One of the important disadvantages of this technique is that this method provides no direct information on events occurring at the surface of the stationary phase, because the ion-exchange equilibrium is always determined by the balance between the solute interaction and the eluent interaction with the active sites of resin [3]. Ion exchange is similar to sorption, since in both cases a solid takes up dissolved sample. The most important difference between them is in stoichiometric nature of ion exchange. Each ion removed from the solution is replaced by an equivalent amount of another ion of the same charge, while a solute is usually taken-up non-stoichiometrically without being replaced in sorption [15]. Stoichiometric displacement based on the mass action law and describes the retention of a solute ion as an exchange process with the counter ion bound to the surface [9]. According to this model, the retention of a protein under isocratic, linear conditions is related to counter ion concentration and can be represented by equilibrium as follow;
k is the retention factor and Cm is the concentration of the counter ion in the mobile phase. Zp/Zs (= Z) is the ratio of the characteristic charge of the protein to the value of the counter ion and presents a statistical average of the electrostatic interactions of the protein with the stationary phase as it migrates through the column. The behavior of ion exchange chromatographic system can be explained by stoichiometric models. However, the mechanism of the ion exchange separation is more complex and stiochiometric consideration is inapplicable to long-range mechanisms, such as electrostatic interactions due to the distribution of ions in solution is also influenced by the electrostatic potential [3,6]. Other interactions between solute-solute, solute-solvent and solvent-solvent also contribute to retention and selectivity in ion exchange. For example ion-dipole and dispersion interaction, should be included as important mechanisms. Additionally entropic contribution originating from solvent, such as water, structures around ion exchange sites should also be regarded as important [3]. In addition to these the primary separation mechanism is the electrostatic interaction between ion-exchange sites and counter ions in ion exchange chromatography [6].
An important feature differentiating the ion exchange resins from other types of gels is the presence of functional groups. The groups are attached to the matrix. The ion exchange process between the ions in the solution takes place on these functional groups. The exchange of ions between the ion exchange resin and the solution is governed by two principles:
The process is reversible, only rare exceptions are known
The exchange reactions take place on the basis of equivalency in accordance with the principle of electro neutrality. The number of milimoles of an ion sorbed by an exchange should correspond to the number of milimoles of an equally charged ion that has been released from the ion exchange [16].
Equilibrium is established for each sample component between the eluent and stationary phases when a sample is introduced into the ion-exchange chromatography. The distribution of component (A) between the two phases is expressed by the distribution coefficient, “DA”.
The value of DA is dependent on the size of the population of molecules of component A in the stationary and eluent phases [1]. As the equilibrium is dynamic, there is a continual, rapid interchange of molecules of component A between the two phases. The fraction of time, fm, that an average molecule of A spends in the mobile phase is given by:
fm = Amount of A in the mobile phase / Total amount of A
fm = [A]m Vm / [A]m Vm + [A]r w
= 1/ 1+ DA (w / Vm)
k\' = DA (W / Vm)
fm = 1 / 1 + k’
w: Weight of the stationary phase
Vm: Volume of the mobile phase [1]
The mechanism of the anion and cation exchange are very similar. When analytes enter to the ion exchange column, firstly they bind to the oppositely charged ionic sites on the stationary phase through the Coulombic attraction [2]. In accordance with Coulomb’s law, the interactions between ions in the solute and oppositely charged ligands on the matrix in ion-exchange chromatography are due to the electrostatic forces. Coulomb’s law is given by the equation as follow;
f: Interaction electrostatic force
q1q2: The charge on ions
: Dielectric constant of the medium
r: The distance between charges.
If the charges on both ions are same (both are positive or negative) the force is repulsive, if they are different (one positive and the other negative) the force is attractive. When the ion charge of the species increase (Divalent ion should interact more strongly than a monovalent ion) and when the dielectric constant decrease (Two oppositely charged molecules increased more strongly in an organic solvent than in water), the interactions increase. On the other hand the distance between the charges increases the interactions decrease. Additionally, other interactions, especially, van der Waals forces participate to the Coulombic forces [2,17].
Ion exchange chromatography, which is also known as adsorption chromatography, is a useful and popular method due to its;
high capacity,
high resolving power,
mild separation conditions,
versatility and widespeared applicability,
tendency to concentrate the sample
relatively low cost [17].
General components of an ion-exchange chromatography are presented as below (Figure 4).
A high pressure pump with pressure and flow indicator, to deliver the eluent
An injector for introducing the sample into the eluent stream and onto the column
A column, to separate the sample mixture into the individual components
An oven, optional
A detector, to measure the analyte peaks as eluent from the column
A data system for collecting and organizing the chromatograms and data
Ion-exchange Chromatography System
In ion-exchange chromatography, adsorption and desorption processes are determined by the properties of the three interacting entities;
The stationary phase,
The constituents of the mobile phase
The solute [18].
Selection of a suitable ion-exchange matrix probably is the most important in ion exchange protocol and is based on various factors such as; ion exchanger charge/strength, linear flow rate/sample volume and sample properties [11]. In ion-exchange chromatography, numerous stationary phases are available from different manufacturers, which vary significantly in a number of chemical and physical properties [6,18]. Stationary phases comprised of two structural elements; the charged groups which are involved in the exchange process and the matrix on which the charged groups are fixed [18]. Ion exchangers are characterized both by the nature of the ionic species comprising the fixed ion and by the nature of the insoluble ion-exchange matrix itself [1].
Ion exchangers are called cation exchangers if they have negatively charged functional groups and possess exchangeable cations. Anion exchangers carry anions because of the positive charge of their fixed groups [15]. The charged groups determine the specifity and strength of protein binding by their polarity and density while the matrix determines the physical and chemical stability and the flow characteristics of the stationary phase and may be responsible for unspecific binding effects [18].
General structure (fibrous or beaded form), particle size and variation, pore structures and dimensions, surface chemistry (hydrophilic or hydrophobic), swelling characteristics of matrix are important factors which effect chromatographic resolution [11,18]. Porosity of ion exchange beads can be categorized as non-porous, microporous and macroporous. (Figure 5 and Figure 6) [14]. High porosity offers a large surface area covered by charged groups and so provides a high binding capacity [13]. However when compared with beaded matrix fibrous ion exchangers based on cellulose exhibit lower chromatographic resolution [14]. On the other hand high porosity is an advantage when separating large molecules [13] and prefractionation [14]. Non-porous matrices are preferable for high resolution separations when diffusion effects must be avoided [13]. Micropores increase the binding capacity but cause to a band broadening. Another disadvantage of microporous beads is that protein can bind to the surface of the beads near to the pores, so penetration of proteins into the pores can prevent or slow down. These problems are overcome by using macroporous particles with pore diameters of about 600-800 nm which are introduced recently. These kinds of particles behave differently compared to microporous materials with respect to microflow characteristics the new term perfusion chromatography has been created [14].
Schematic presentation of different matrix types (a) non-porous beads (b) microporous beads (c) macroporous beads
(a) Non-porous beads (b) Porous beads
Furthermore a new matrix type which has been recently introduced is based on a completely new principle and exhibits improved chromatographic features when compared with conventional ion exchangers. This matrix which is known as continuous bed does not consist of ion exchange beads or fibers. The matrix is synthesized in the column by polymerization and established from continuous porous support consisting of a nodule chains (Figure 7). The advantages of that matrix are mainly due to the more homogeneous mobile phase flow and short diffusion distances for the proteins. This is explained by the non-beaded form and the unique pore structure of the support [14].
Size, size distribution and porosity of the matrix particles are the main factors which affect the flow characteristics and chromatographic resolution. Small particles improved chromatographic resolution. Stationary phases with particle of uniform size are superior to heterogenous materials with respect to resolution and attainable flow rates. The pore size of ion exchange bead directly effect the binding capacity for a particular protein dependent on the molecular weight of the protein because it determines the access of proteins to the interior of the beads. Binding of large proteins can be restricted to the bead surface only so that the total binding capacity of the ion exchanger is not exploited Pore diameter of 30 nm is optimal for proteins up to a molecular weight of about 200.000 Da [14].
Continuous bed matrix
In order to minimize non-specific interactions with sample components inert matrix should be used. High physical stability provides that the volume of the packed medium remains constant despite extreme changes in salt concentration or pH for improving reproducibility and avoiding the need to repack columns. High physical stability and uniformity of particle size facilitate high flow rates, particularly during cleaning or re-equilibration steps, to improve throughput and productivity [13]. There are pH and pressure limits for each stationary phases. For example pH values higher than 8 should not used in silica based materials which are not coated with organic materials. Matrix stability also should be considered when the chemicals such as organic solvents or oxidizing agents should be required to use or when they are chosen for column cleaning [14].
Matrices which are obtained by polymerization of polystyrene with varying amounts of divinylbenzene are known as the original matrices for ion exchange chromatography. However these matrices have very hydrophobic surface and proteins are irreversibly damaged due to strong binding. Ion exchangers which are based on cellulose with hydrophilic backbones are more suitable matrices for protein separations. Other ion exchange matrices with hydrophilic properties are based on agarose or dextran [14].
Several matrix types and their important properties can be listed as follow;
Matrix materials;
Cellulose; Hydrophilic surface, enhanced stability by cross-linking, inexpensive
Dextran; Considerable swelling as a function of ionic milieu, improved materials by cross-linking)
Agarose; Swelling is almost independent of ionic strength and pH, high binding capacity obtained by production of highly porous particles
Polyacrylamide; Swelling behavior similar to dextran
Acrylate-copolymer; High pH stability
Polystyrene-divinilybenzene; Hydrophobic surface, low binding capacity for proteins
Coated polystyrene-divinilybenzene; Hydrophilic surface
Silica; Unstable at pH > 8, rigid particles
Coated Silica; Hydrophilic surface [14]
In addition to electrostatic interactions between stationary phase and proteins, some further mechanisms such as hydrophobic interactions, hydrogen bonding may contribute to protein binding. Hydrophobic interactions especially occur with synthetic resin ion exchangers such as which are produced by copolymerization of styrene and divinylbenzene. These materials are not usually used for separation of proteins. However new ion exchange materials that consist of styrene-divinylbenzene copolymer beads coated with hydrophilic ion exchanger film were introduced. According to the retention behavior of some proteins, it is considered that coating of the beads so efficient that unspecific binding due to hydrophobic interactions cannot be observed. Silica particles have also been coated with hydrophilic matrix. Acrylic acid polymers are also used for the protein separation in ion exchange chromatography. These polymers are especially suitable for purification of basic proteins [14].
The functional groups substituted onto a chromatographic matrix determine the charge of an ion exchange medium; positively-charged anion exchanger or a negatively-charged cation exchanger [13]. Both exchangers can be further classified as strong and weak type as shown in Table 1. The terms weak and strong are not related to the binding strength of a protein to the ion exchanger but describe the degree of its ionization as a function of pH [14]. Strong ion exchangers are completely ionized over a wide pH range, while weak ion exchangers are only partially ionized a narrow pH range [1,11]. Therefore with strong ion exchangers proteins can adsorb to several exchanger sites. For this reason strong ion exchangers are generally used for initial development and optimization of purification protocols. On the other hand weak ion exchangers are more flexible in terms of selectivity and are a more general option for the separation of proteins that retain their functionality over the pH 6-9 range as well as for unstable proteins that may require mild elution conditions [11]. Alkylated amino groups for anion exchangers and carboxy, sulfo as well as phosphato groups for cation exchangers are the most common functional groups used on ion exchange chromatography supports [14]. Sulfonic acid exchangers are known as strong acid type cation exchangers. Quaternary amine functional groups are the strong base exchangers whereas less substituted amines known as weak base exchangers [1]. Number and kind of the substituents are determined the basicity of amino-groups. Immobilized tertiary and quaternary amines proved to be useful for ion exchange chromatography. Immobilized diethylaminoethyl and carboxymethyl groups are the most widely used ion exchangers [11].
The ion exchange capacity of an ion-exchanger is determined by the number of functional groups per unit weight of the resin [13]. The total ionic capacity is the number of charged functional groups per ml medium, a fixed parameter of each medium and can be given as mval/ml for small ions. Density and accessibility of these charged groups both on the surface and within the pores define the total binding capacity. Ionic medium and the presence of other proteins if a particular protein is considered also affect the binding capacity. However, under defined conditions, the amount of the certain protein which is bound to ion exchanger is more suitable parameter for determining and comparing the capacity of ion exchange chromatography. Albumin for anion exchangers and hemoglobin for cation exchangers is usually used for this purpose. Determination of the binding capacity before the experiment is generally recommended because the capacity for a particular protein depends on its size and also on the sample composition. The binding capacity of a column can be increased for proteins which are retained on the column at high salt concentrations. The salt concentration is adjusted to a suitable concentration in which the protein of interest tightly bound to the ion exchanger while others which have lower affinity pass through the column without occupying binding sites [14].
\n\t\t\t\tExchange Type\n\t\t\t | \n\t\t\t\n\t\t\t\tIon exchange group\n\t\t\t | \n\t\t\t\n\t\t\t\tBuffer counter ions\n\t\t\t | \n\t\t\t\n\t\t\t\tpH range\n\t\t\t | \n\t\t\t\n\t\t\t\tCommercial samples\n\t\t\t | \n\t\t
Strong cation | \n\t\t\tSulfonic acid (SP) | \n\t\t\tNa+, H+, Li+\n\t\t\t | \n\t\t\t4-13 | \n\t\t\tCapto®S | \n\t\t
SP Sepharose®\n\t\t\t | \n\t\t||||
SP Sephadex®\n\t\t\t | \n\t\t||||
TSKgel SP_5PW | \n\t\t||||
Weak cation | \n\t\t\tCarboxylic acid | \n\t\t\tNa+, H+, Li+\n\t\t\t | \n\t\t\t6-10 | \n\t\t\tCM Cellulose | \n\t\t
CM Sepharose®\n\t\t\t | \n\t\t||||
CM Sephadex®\n\t\t\t | \n\t\t||||
CM Sepharose® CL6B | \n\t\t||||
TSKgel CM-5PW | \n\t\t||||
Strong anion | \n\t\t\tQuaternary amine (Q) | \n\t\t\tCl-, HCOO3\n\t\t\t\t-, CH3COO-, SO4\n\t\t\t\t2-\n\t\t\t | \n\t\t\t2-12 | \n\t\t\tQ Sepharose®\n\t\t\t | \n\t\t
Capto®Q | \n\t\t||||
Dowex®1X2 | \n\t\t||||
Amberlite® / Amberjet®\n\t\t\t | \n\t\t||||
QAE Sephadex®\n\t\t\t | \n\t\t||||
Weak anion | \n\t\t\tPrimary amine Secondary amine Tertiary amine (DEAE) | \n\t\t\tCl-, HCOO3\n\t\t\t\t-, CH3COO-, SO4\n\t\t\t\t2-\n\t\t\t | \n\t\t\t2-9 | \n\t\t\tDEAE-Sepharose®\n\t\t\t | \n\t\t
Capto® DEAE | \n\t\t||||
DEAE Cellulose | \n\t\t
Weak and Strong type anion and cation exchangers
In ion exchange chromatography generally eluents which consist of an aqueous solution of a suitable salt or mixtures of salts with a small percentage of an organic solvent are used in which most of the ionic compounds are dissolved better than in others in. Therefore the application of various samples is much easier [1,3]. Sodium chloride is probably the most widely used and mild eluent for protein separation due to has no important effect on protein structure. However NaCl is not always the best eluent for protein separation. Retention times, peak widths of eluted protein, so chromatographic resolution are affected by the nature of anions and cations used. These effects can be observed more clearly with anion exchangers as compared to cation exchangers [14]. The salt mixture can itself be a buffer or a separate buffer can be added to the eluent if required. The competing ion which has the function of eluting sample components through the column within reasonable time is the essential component of eluting sample. Nature and concentration of the competing ions and pH of the eluent are the most important properties affecting the elution characteristics of solute ions [1].
The eluent pH has considerable effects on the functional group which exist on the ion exchange matrix and also on the forms of both eluent and solute ions. The selectivity coefficient existing between the competing ion and a particular solute ion will determine the degree of that which competing ion can displace the solute ion from the stationary phase. As different competing ions will have different selectivity coefficients, it follows that the nature of competing ion will be an important factor in determining whether solute ions will be eluted readily. The concentration of competing ion exerts a significant effect by influencing the position of the equilibrium point for ion-exchange equilibrium. The higher concentration of the competing ion in the eluent is more effectively displace solute ions from the stationary phase, therefore solute is eluted more rapidly from the column. Additionally elution of the solute is influenced by the eluent flow-rate and the temperature. Faster flow rates cause to lower elution volumes because the solute ions have less opportunity to interact with the fixed ions. Temperature has relatively less impact, which can be change according to ion exchange material type. Enhancement of the temperature increases the rate of diffusion within the ion-exchange matrix, generally leading to increased interaction with the fixed ions and therefore larger elution volumes. At higher temperatures chromatographic efficiency is usually improved [1].
Eluent degassing is important due to trap in the check valve causing the prime loose of pump. Loss of prime results in erratic eluent flow or no flow at all. Sometimes only one pump head will lose its prime and the pressure will fluctuate in rhythm with the pump stroke. Another reason for removing dissolved air from the eluent is because air can get result in changes in the effective concentration of the eluent. Carbon dioxide from air dissolved in water forms of carbonic acid. Carbonic acid can change the effective concentration of a basic eluent including solutions of sodium hydroxide, bicarbonate and carbonate. Usually degassed water is used to prepare eluents and efforts should be made to keep exposure of eluent to air to a minimum after preparation. Modern inline degassers are becoming quite popular [10].
For separation the eluent is pumped through the system until equilibrium is reached, as evidenced by a stable baseline. The time required for equilibrium may vary from a couple of minutes to an hour or longer, depending on the type of resin and eluent used [10]. Before the sample injection to the column should be equilibrated with eluent to cover all the exchange sites on the stationary phase with the same counter ion. When the column is equilibrated with a solution of competing ion, counter ions associated with the fixed ions being completely replaced with competing ions. In this condition the competing ions become the new counter ions at the ion exchange sites and the column is in the form of that particular ion [1].
Isocratic elution or gradient elution can be applied for elution procedure. A single buffer is used throughout the entire separation in isocratic elution. Sample components are loosely adsorbed to the column matrix. As each protein will have different distribution coefficient separation will achieved by its relative speeds of migration over the column. Therefore in order to obtain optimum resolution of sample components, a small sample volume and long exchanger column are necessary. This technique is time consuming and the desired protein invariably elutes in a large volume. However no gradient-forming apparatus is required and the column regeneration is needless. Alteration in the eluent composition is needed to achieve desorption of desired protein completely. To promote desired protein desorption continuous or stepwise variations in the ionic strength and/or pH of the eluent are provided with gradient elution. Continuous gradients generally give better resolution than stepwise gradients [11].
Additives which are protective agents found in the mobile phase are generally used for maintain structure and function of the proteins to be purified. This is achieved by stimulating an adequate microenvironment protection against oxidation or against enzymatic attacks [14]. Any additives used in ion exchange chromatography, should be checked for their charge properties at the working pH in order to avoid undesired effects due to adsorption and desorption processes during chromatography [13-14]. It is recommended to include in the elution buffer those additives in a suitable concentration which have been used for stabilization and solubilization of the sample. Otherwise precipitation may occur on the column during elution [14]. For example; zwitterionic additives such as betaine can prevent precipitation and can be used at high concentrations without interfering with the gradient elution. Detergents are generally useful for solubilization of proteins with low aqueous solubility. Anionic, cationic, zwitterionic and non-ionic (neutral) detergents can be used during ion exchange chromatography. Guanidine hydrochloride or urea, known as denaturing agents can be used for initial solubilization of a sample and during separation. However, they should use if there is a requirement. Guanidine is a charged molecule and therefore can participate to the ion exchange process in the same way as NaCl during separation process [13].
Commonly used eluent additives which have been successfully used in ion exchange chromatography can be given as follow;
EDTA; Ethylenediamine tetraacetic acid
Polyols; Glycerol, glucose, and saccharose
Detergents;
Urea and guanidinium chloride
Lipids
Organic solvents
Zwitterions
Sulfhydryl reagents
Ligands
Protease inhibitors [14]
In ion exchange chromatography, pH value is an important parameter for separation and can be controlled and adjusted carefully by means of buffer substances [18]. In order to prevent variation in matrix and protein net charge, maintenance of a constant mobile phase pH during separation is essential to avoid pH changing which can occur when both protein and exchanger ions are released into the mobile phase [11]. By means of buffer substances pH value can be controlled and adjusted. Concentration of H+ and the buffering component influence the protein binding to the stationary phase, chromatographic resolution and structural as well as functional integrity of the protein to be separated. Thus a suitable pH range, in which the stability of sample is guaranteed, has to be identified. Keeping of the sample function is related with the preservation of its three dimensional structure as well as with its biological activity [18]. A number of buffers are suitable for ion-exchange chromatography. A number of important factors influences the selection of mobile phase including buffer charge, buffer strength and buffer pH [11]. Properties of good buffers are high buffering capacity at the working pH, high solubility, high purity and low cost. The buffer salt should also provide a high buffering capacity without contributing much to the conductivity and should not interact with the ion exchanger functional groups as well as with media [11,17]. The buffering component should not interact with the ion exchanger because otherwise local pH shifts can occur during the exchange process which may interfere the elution. Interactions with stationary phase as well as with additives of the mobile phase and with subsequent procedures may be occur with buffer component and selected pH range. Precipitation of the mobile phase components can be observed for example when phosphate buffer and several di- and trivalent metal ions such as Mg+2 and Ca+2 are mixed or when anionic detergents (i.e. cholate) are used under acidic conditions or in the presence of multivalent metal ions. Precipitation of metal oxides and hydroxides can occur under alkaline conditions. Buffer components may also affect enzymatic assays used for screening and analysis of chromatography fractions [14]. The concentration of buffer salts usually ranges from 10 to 50 mM. Commonly used buffers are presented in Table 2 and Table 3 for cation and anion exchange chromatography [17].
Generally, applications of ıon exchange chromatography are performed under slightly acidic or alkali conditions, pH range 6.0-8.5 but there are also more acidic and more alkali buffers. Additionally the buffering component should not act as an eluting ion by binding to the ion exchanger. Anionic buffer component such as phosphate or MOPS in cation exchange chromatography and cationic buffers such as ethanolamine, Tris and Tricine in anion exchange chromatography are recommended. Besides interactions of buffer component with stationary phase, there are also possible interactions with additives of the mobile phase. To achieve sufficient buffer capacity the pKa of the buffer component should be as close to the desired pH value as possible difference no more than ± 0.5 pH units. However there are examples of successful separations at which the buffering capacity is very low [17-18]. It has to be considered that the pKa is a temperature dependent value. Performing on ion exchange separation with the same elution buffer at room temperature or in the cold room can have a remarkable effect on the buffer capacity. For optimal binding of a sample ion to an ion-exchanger the ionic strength and thus also the buffer concentrations has to be low in sample and equilibration buffers [18].
\n\t\t\t\tSubstance\n\t\t\t | \n\t\t\t\n\t\t\t\tpKa\n\t\t\t\t\n\t\t\t | \n\t\t\t\n\t\t\t\tWorking pH\n\t\t\t | \n\t\t
Citric acid | \n\t\t\t3.1 | \n\t\t\t2.6-3.6 | \n\t\t
Lactic acid | \n\t\t\t3.8 | \n\t\t\t3.4-4.3 | \n\t\t
Acetic acid | \n\t\t\t4.74 | \n\t\t\t4.3-5.2 | \n\t\t
2-(N-morpholino)ethanesulfonic acid | \n\t\t\t6.1 | \n\t\t\t5.6-6.6 | \n\t\t
\n\t\t\t\tN-(2-acetamido)-2-iminodiacetic acid | \n\t\t\t6.6 | \n\t\t\t6.1-7.1 | \n\t\t
3-(N-morpholino)propanesulfonic acid | \n\t\t\t7.2 | \n\t\t\t6.7-7.7 | \n\t\t
Phosphate | \n\t\t\t7.2 | \n\t\t\t6.8-7.6 | \n\t\t
\n\t\t\t\tN-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) | \n\t\t\t7.5 | \n\t\t\t7.0-8.0 | \n\t\t
N,N-bis(2-hydroxyethyl)glycine | \n\t\t\t8.3 | \n\t\t\t7.6-9.0 | \n\t\t
Commonly used buffers for cation-exchange chromatography
\n\t\t\t\tSubstance\n\t\t\t | \n\t\t\t\n\t\t\t\tpKa\n\t\t\t\t\n\t\t\t | \n\t\t\t\n\t\t\t\tWorking pH\n\t\t\t | \n\t\t
N-Methyl-piperazine | \n\t\t\t4.75 | \n\t\t\t4.25-5.25 | \n\t\t
Piperazine | \n\t\t\t5.68 | \n\t\t\t5.2-6.2 | \n\t\t
Bis-Tris | \n\t\t\t6.5 | \n\t\t\t6.0-7.0 | \n\t\t
Bis-Tris propane | \n\t\t\t6.8 | \n\t\t\t6.3-7.3 | \n\t\t
\n\t\t\t\tTriethanolamine\n\t\t\t | \n\t\t\t7.8 | \n\t\t\t7.25-8.25 | \n\t\t
Tris | \n\t\t\t8.1 | \n\t\t\t7.6-8.6 | \n\t\t
\n\t\t\t\tN-Methyl-diethanolamine | \n\t\t\t8.5 | \n\t\t\t8.0-9.0 | \n\t\t
Diethanolamine | \n\t\t\t8.9 | \n\t\t\t8.4-9.4 | \n\t\t
Ethanolamine | \n\t\t\t9.5 | \n\t\t\t9.0-10.0 | \n\t\t
1,3-Diaminopropane | \n\t\t\t10.5 | \n\t\t\t10.0-11.0 | \n\t\t
Commonly used buffers for anion-exchange chromatography
Conductivity detector is the most common and useful detector in ion exchange chromatography. However UV and other detectors can also be useful [10]. Conductivity detection gives excellent sensitivity when the conductance of the eluted solute ion is measured in an eluent of low background conductance. Therefore when conductivity detection is used dilute eluents should be preferred and in order for such eluents, to act as effective competing ions, the ion exchange capacity of the column should be low [1].
Although recorders and integrators are used in some older systems, generally in modern ion exchange chromatography results are stored in computer. Retention time and peak areas are the most useful information. Retention times are used to confirm the identity of the unknown peak by comparison with a standard. In order to calculate analyte concentration peak areas are compared with the standards which is in known concentration [10].
Direct detection of anions is possible, providing a detector is available that responds to some property of the sample ions. For example anions that absorb in the UV spectral region can be detected spectrophotometrically. In this case, an eluent anion is selected that does not absorb UV. The eluent used in anion chromatography contains an eluent anion, E-. Anions with little or no absorbance in the UV spectral region can be detected spectrophotometrically by choosing a strongly absorbing eluent anion. An anion with benzene ring would be suitable [10]. Usually Na+ or H+ will be the cation associated with E-. The eluent anion must be compatible with the detection method used. For conductivity the detection E should have either a significantly lower conductivity than the sample ions or be capable of being converted to a non-ionic form by a chemical suppression system. When a spectrophotometric detection is employed, E will often be chosen for its ability to absorb strongly in the UV or visible spectral region. The concentration of E- in the eluent will depend on the properties of the ion exchanger used and on the types of anions to be separated [10].
Ion exchange chromatography can be applied for the separation and purification of many charged or ionizable molecules such as proteins, peptides, enzymes, nucleotides, DNA, antibiotics, vitamins and etc. from natural sources or synthetic origin. Examples in which ion exchange chromatography was used as a liquid chromatograpic technique for separation or purification of bioactive molecules from natural sources can be given as below.
\n\t\t\t\tSample 1:\n\t\t\t | \n\t\t|
\n\t\t\t\tSource:\n\t\t\t | \n\t\t\t\n\t\t\t\tNigella sativa Linn. | \n\t\t
\n\t\t\t\tExtraction procedure:\n\t\t\t | \n\t\t\tWater extract of N. sativa was prepared, dried and powdered. Powder was dissolved in phosphate buffer saline (pH 6.4) and centrifuged at 10.000 rpm for 30 min at 4 ºC. The supernatant was collected as the soluble extract by removing the oily layer and unsoluble pellet. Protein concentration of the soluble extract was determined with Bradford method. Then proteins dialyzed against 0.05 M phosphate buffer (pH 6.4) using 3500 MW cut off dialyzing bags and centrifuged. | \n\t\t
\n\t\t\t\tStationary Phase:\n\t\t\t | \n\t\t\tXK50/30 column (5 x 15 cm) of DEAE sephadex A50. | \n\t\t
\n\t\t\t\tEluent:\n\t\t\t | \n\t\t\t0.05 M phosphate buffer (pH 6.4) containing 0.01 M NaCl. Fractions of each were collected with an increasing concentration of NaCl | \n\t\t
\n\t\t\t\tDetection:\n\t\t\t | \n\t\t\tUV detector at 280 nm | \n\t\t
\n\t\t\t\tAnalyte(s):\n\t\t\t | \n\t\t\tNumber of protein bands ranging from 94-10 kDa molecular mass [19]. | \n\t\t
\n\t\t\t\tSample 2:\n\t\t\t | \n\t\t|
\n\t\t\t\tSource:\n\t\t\t | \n\t\t\t\n\t\t\t\tOlea europea L. | \n\t\t
\n\t\t\t\tExtraction procedure:\n\t\t\t | \n\t\t\tExtract was prepared from the leaves and roots of two years old olive plants with water at room temperature. Internal standard as D-3-O-methylglucopyranose (MeGlu) was used and added in appropriate volume. Extraction was accomplished by shaking for 15 min and finally the suspension was centrifuged at 3000 rpm for 10 min. Before the injection the aqueous phase was filtered and passed on a cartridge OnGuard A (Dionex) to remove anion contaminants. | \n\t\t
\n\t\t\t\tStationary Phase:\n\t\t\t | \n\t\t\tTwo anion exchange columns Dionex CarboPac PA1 plus a guard column and CarboPac MA1 column with a guard column were used for separation procedure (High Performance Anion Exchange Chromatography). | \n\t\t
\n\t\t\t\tEluent:\n\t\t\t | \n\t\t\tEluent was comprised 12 mM NaOH with 1 mM barium acetate. Flow rate was 1 mL/min. | \n\t\t
\n\t\t\t\tDetection:\n\t\t\t | \n\t\t\tPulsed amperometric detection | \n\t\t
\n\t\t\t\tAnalyte(s):\n\t\t\t | \n\t\t\t\n\t\t\t\tmyo-inositol, galactinol, mannitol, galactose, glucose, fructose, sucrose, raffinose and stachyose [20]. | \n\t\t
\n\t\t\t\tSample 3:\n\t\t\t | \n\t\t|
\n\t\t\t\tSource:\n\t\t\t | \n\t\t\tSoybean | \n\t\t
\n\t\t\t\tExtraction procedure:\n\t\t\t | \n\t\t\tSoybeans were defatted with petroleum ether for 30 min and centrifuged repeating the procedure twice. Then proteins were extracted with 0.03 M Tris-HCL buffer containing 0.01 M 2-mercaptoethanol (pH 8) for 1 hour following by centrifugation (16.250 x g for 20 min at 20 ºC). The supernatant was adjusted to pH 6.4 with 2 M HCl and centrifuged (16.250 x g for 20 min at 2-5 ºC). The precipitate was dissolved in Tris-HCl buffer and the process was repeated in order to obtain purified precipitated fraction containing the 11S globulin. The supernatant obtained after the first precipitation of the 11S fraction was adjusted to pH 4.8 with 2M HCl and centrifuged (16.250 x g for 20 min at 2-5 ºC). The supernatant was stored at low temperature and the precipitate was dissolved in Tris-HCl buffer (pH 8). The process was repeated to obtain a purified precipitated fraction containing the 7S globulin. | \n\t\t
\n\t\t\t\tStationary Phase:\n\t\t\t | \n\t\t\tAnion exchange perfusion column POROS HQ/10 packed with cross-linked polystyrene-divinylbenzene beads. | \n\t\t
\n\t\t\t\tEluent:\n\t\t\t | \n\t\t\tThe starting point for the separation of soybean proteins by HPIEC was the use of a binary gradient where mobile phase was a buffer solution at a certain pH (always pHs higher than the isoelectric pH of soybean proteins, pI = 4.8–6.4) and mobile phase B was the same buffer solution containing as well Msodium chloride. The buffer solutions tried were: phosphate ffer (pH 7 and 12), Tris–HCl buffer (pH 8), borate buffer (pH 9), and carbonate buffer (pH 10). In all cases, the buffer concentration was 20 mM. For every buffer, different gradients were tried. The best separation for ybean proteins was obtained with the borate buffer (pH 9) and gradient starting with an isocratic step at 0% B for 2.5 min and from 0 to 70% B in 14 min (gradient slope, 5%B/min). A fine optimization of the selected gradient enabled a reduction of the analysis time keeping the separation. The final gradient was: 0% for 2 min and from 0 to 50% B in 10 min. | \n\t\t
\n\t\t\t\tDetection:\n\t\t\t | \n\t\t\tUV detector at 254 nm | \n\t\t
\n\t\t\t\tAnalyte(s):\n\t\t\t | \n\t\t\t11S globulin or glycinin and 7S globulin [21]. | \n\t\t
\n\t\t\t\tSample 4:\n\t\t\t | \n\t\t|
\n\t\t\t\tSource:\n\t\t\t | \n\t\t\t\n\t\t\t\tCochlospermum tinctorium A. Rich. | \n\t\t
\n\t\t\t\tExtraction procedure:\n\t\t\t | \n\t\t\tThe powdered roots of C. tinctorium were extracted with ethanol (% 96, v/v) using a soxhlet apparatus to remove low molecular weight compounds. Extraction procedures continue until no color could be observed in the ethanol. The residue was extracted with water at 50 ºC, 2 hour for two times. Obtained extract was filtered through gauze and Whatman GF/A glass fiber filter and then concentrated at 40 ºC in vacuum and dialysed at cut-off 3500 Da to give a 50 ºC crude extract. The extracts was kept at -18 ºC or lyophilized. | \n\t\t
\n\t\t\t\tStationary Phase:\n\t\t\t | \n\t\t\tAnion exchange-DEAE-Sepharose column | \n\t\t
\n\t\t\t\tEluent:\n\t\t\t | \n\t\t\tFor obtaining neutral fraction the column was eluted with water firstly. The acidic fractions were obtained by elution of linear NaCl gradient (0-1.4 M) in water. The carbohydrate elution profile was determined using the phenol-sulphiric acid method. Finally two column volumes of a 2 M sodium chloride solution in water were eluted to obtain the most acidic polysaccharide fraction. The relevant fractions based on the carbohydrate profile were collected, dialysed and lyophilized. | \n\t\t
\n\t\t\t\tDetection:\n\t\t\t | \n\t\t\tUV detector, 490 nm | \n\t\t
\n\t\t\t\tAnalyte(s):\n\t\t\t | \n\t\t\tGlucose, galactose, arabinose (in neutral fraction) Uronic acids (Both galacturonic and glucuronic acid), rhamnose, galactose, arabinose and glucose (in acidic fraction) [22]. | \n\t\t
\n\t\t\t\tSample 5:\n\t\t\t | \n\t\t|
\n\t\t\t\tSource:\n\t\t\t | \n\t\t\tHen egg | \n\t\t
\n\t\t\t\tExtraction procedure:\n\t\t\t | \n\t\t\tFresh eggs were collected and the same day extract was obtained. Ovomucin was obtained using isoelectric precipitation of egg white in the presence of 100 mM NaCl solution. The dispersion was kept overnight at 4 ºC and separated by centrifugation at 15.300 x g for 10 min at 4 ºC. The precipitate was further suspended in 500 mM NaCl solution while stirring for 4 h followed by overnight settling at 4 ºC. After centrifugation at 15.300 x g for 10 min at 4 ºC, the precipitate was freeze dried and stored at -20 ºC. The supernatants obtained during the first step (with 100 mM NaCl solution) and the second step (with 500 mM NaCl solution) was further used for ion exchange chromatography to separate other egg white proteins. Separation proteins from 100 mM supernatant were allowed to pass through an anion exchange chromatographic column to separate different fractions. The unbound fractions were then passed through a cation exchange chromatographic column to separate further. | \n\t\t
\n\t\t\t\tStationary Phase:\n\t\t\t | \n\t\t\tHigh-Prep 16/10 column (Q Sepharose FF)-Anion Exchange Chromatography High-Prep 16/10 column (SP Sepharose FF)-Cation Exchange Chromatography | \n\t\t
\n\t\t\t\tEluent:\n\t\t\t | \n\t\t\tThe column was equilibrated with water and the pH was adjusted to 8.0 before injectionAfter sample injection flow-through fraction was collected using water as the eluent, followed by isocratic elution of the sample using 0.14 M NaCl. Finally the bound fraction was eluted using gradient elution (0.14-0.5 M) of NaCl- Anion Exchange Chromatography. The unbound fraction was collected and used as starting material for cation exchange chromatography. The column was equilibrated with 10 mM citrate buffer, which was used as the starting buffer. After sample injection the column was eluted by isocratic elution using 0.14 M NaCl solution followed by gradient elution from 0.14 M to 0.50 M NaCl solution. The fractions were collected and freeze dried-Cation Exchange Chromatography. | \n\t\t
\n\t\t\t\tDetection:\n\t\t\t | \n\t\t\tMS Detector | \n\t\t
\n\t\t\t\tAnalyte(s):\n\t\t\t | \n\t\t\tOvalbumin, ovotransferrin, lysozyme, ovomucin [23]. | \n\t\t
\n\t\t\t\tSample 6:\n\t\t\t | \n\t\t|
\n\t\t\t\tSource\n\t\t\t | \n\t\t\t: Phaseolus vulgaris\n\t\t\t | \n\t\t
\n\t\t\t\tExtraction procedure:\n\t\t\t | \n\t\t\tSeeds were grounded and soaked in 20 mM Tris-HCl buffer (pH 7.6) at 4 ºC for 24 h. The seeds were blended in a blender to extract the proteins followed by centrifugation (30,000g) at 4ºC. Then 450 g/l of ammonium sulphate were added to the supernatant to 70% saturation. The precipitate was removed by centrifugation and the supernatant was extensively dialysed against distiled water. The dialysed protein extract was freeze dried and used for chromatographic separation. | \n\t\t
\n\t\t\t\tStationary Phase:\n\t\t\t | \n\t\t\tQ-Sepharose Column (3 cm x 7 cm), anion-exchange | \n\t\t
\n\t\t\t\tEluent:\n\t\t\t | \n\t\t\tThe column was equilibrated and initially eluted with 20 mM Tris–HCl (pH 7.6). Elution of the bound fraction was carried out by using 1 M NaCl in the equilibration buffer. All chromatographic steps were performed at the flow rate of 100 ml/h. Further separation selected fraction Q1, which was lyophilised and dissolved in 100 mM Tris–HCl (pH 7.6) buffer was performed onto a FPLC Superdex 75 column at a flow rate 0.5 ml min-1. | \n\t\t
\n\t\t\t\tDetection:\n\t\t\t | \n\t\t\tUV detector, 280 nm | \n\t\t
\n\t\t\t\tAnalyte(s):\n\t\t\t | \n\t\t\tA 5447 Da antifungal peptide [24]. | \n\t\t
\n\t\t\t\tSample 7:\n\t\t\t | \n\t\t|
\n\t\t\t\tSource:\n\t\t\t | \n\t\t\tSweet dairy whey | \n\t\t
\n\t\t\t\tExtraction procedure:\n\t\t\t | \n\t\t\tAfter the cheese making process the sweet whey is produced, it is further processed by reverse osmosis to increase the solids content from approximately 5.5% (w/w) to 14.6% (w/w). | \n\t\t
\n\t\t\t\tStationary phase:\n\t\t\t | \n\t\t\tPharmacia\'s Q- and S-Sepharose anion- and cation-exchange resins | \n\t\t
\n\t\t\t\tEluent 1:\n\t\t\t | \n\t\t\tFor the anion-exchange process; it was found that two step changes, simultaneous in pH and salt concentration were necessary to carry out the anion-exchange separation. A 0.01 M sodium acetate buffer, pH 5.8, was used for the starting state or feed loading buffer. After the whey feed was loaded onto the column, one column volume of this buffer was passed through to wash out any material that did not bind to the resin, including the IgG. Next, two column volumes of 0.05 M sodium acetate, pH 5.0, were passed through the column to desorb those proteins whose pI values were above 5.0. This includes the β-lactoglobulin and bovine serum albumin. This was then followed by two column volumes of 0.1 M sodium acetate, pH 4.0, to finally desorb the α-lactalbumin whose pI range is 4.2–4.5, and thus above that of the passing pH wave of 4.0. After this second step change, the cleaning cycle was then implemented to prepare the column for the next run. | \n\t\t
\n\t\t\t\tEluent 2:\n\t\t\t | \n\t\t\tFor the cation-exchange process, it was found that one step change in pH was appropriate to carry out the cation-exchange separation. The buffer used was 0.05 M sodium acetate, pH 5.5, as the starting state or feed loading buffer. One column volume loading of the anion-exchange breakthrough curve fraction was optimum for loading onto the cation-exchange column. After the anion-exchange breakthrough curve fraction was loaded onto the column, one column volume of the initial buffer was passed through to wash out any material that did not bind to the resin. Next a step change in pH was implemented to elute the bound IgG. This was accomplished by passing two column volumes of the buffer, 0.05 M sodium acetate, pH 8.5. As the pH wave of this buffer passed through the cation bed it initiated the elution of the IgG because the upper value of its pI range is 8.3. After this pH step change the cleaning cycle was then implemented. | \n\t\t
\n\t\t\t\tDetection:\n\t\t\t | \n\t\t\tUV Detector | \n\t\t
\n\t\t\t\tAnalyte(s):\n\t\t\t | \n\t\t\tα-lactalbumin, β-lactoglobulin, bovine serum albumin, immunoglobulin G and lactose [25]. | \n\t\t
\n\t\t\t\tSample 8:\n\t\t\t | \n\t\t|
\n\t\t\t\tSource:\n\t\t\t | \n\t\t\t\n\t\t\t\tMorus alba (mulberry) leaves | \n\t\t
\n\t\t\t\tExtraction procedure:\n\t\t\t | \n\t\t\tFresh leaves were homogenized in ice-cold 50 mM Tris–HCl, pH 7.5, containing 0.3 M NaCl, 20 mM diethyldithiocarbamic acid, 5% glycerol, and 2% polyvinylpyrrolidone. The buffer used was 3 ml g-1 of the fresh leaves. The homogenate was filtered through a layer of cheesecloth and stored at 20°C for 24 h. After thawing, it was centrifuged at 8000xg, for 40 min at 4°C. The supernatant was collected and ammonium sulfate was added to 70% saturation. The resulting precipitate was recovered by centrifugation at 8000xg for 40 min, redissolved in tris-buffered saline, TBS (50 mM Tris–HCl, pH 7.5 containing 0.3 M NaCl) and dialysed against the buffer overnight at 4°C. The solution was then centrifuged at 13,000xg for 15 min and the supernatant was collected and stored at -20°C. An aliquot of the dialysed ammonium sulfate fraction containing protein was applied to the affinity chromatography on the N-acetylgalactosamine-agarose column. And then further separation was performed on Sephacryl S-200 column followed by anion exchange chromatography. Further purification was also performed by anion exchange and gel filtration chromatography | \n\t\t
\n\t\t\t\tStationary Phase:\n\t\t\t | \n\t\t\tAnion-exchange chromatography, a DEAE-Sephacel column (2x9 cm) | \n\t\t
\n\t\t\t\tEluent:\n\t\t\t | \n\t\t\tEquilibrated with 20 mM Tris–HCl, pH 7.5 at flow rate 20 ml min-1 and then eluted stepwise with the buffer containing NaCl. | \n\t\t
\n\t\t\t\tDetection:\n\t\t\t | \n\t\t\tUV Detector, 280 nm | \n\t\t
\n\t\t\t\tAnalyte(s):\n\t\t\t | \n\t\t\tLectins, MLL 1 and MLL 2 [26] | \n\t\t
\n\t\t\t\tSample 9:\n\t\t\t | \n\t\t|
\n\t\t\t\tSource:\n\t\t\t | \n\t\t\t\n\t\t\t\tLycium ruthenicum Murr. | \n\t\t
\n\t\t\t\tExtraction:\n\t\t\t | \n\t\t\tFruits of the plant extracted with hot water yielded a crude polysaccharide sample, CLRP. The carbohydrate of CLRP was 66.2% and protein content was 7.3%. CLRP was a black Polysaccharide sample in which the pigment could not be removed by colum chromatography. To avoid the influence of pigment on the structure analysis, decoloration was performed with 30% H2O. After decoloration, the carbohydrate content of decolored CLRP was 93.2% and protein content was 4.4%. Decolored CLRP was purified by anion exchange chromatography, yielding five polysaccharide subfractions LRP1, LRP2, LRP3, LRP4, and LRP5. | \n\t\t
\n\t\t\t\tStationary Phase:\n\t\t\t | \n\t\t\tDEAE-cellulose column | \n\t\t
\n\t\t\t\tEluent:\n\t\t\t | \n\t\t\tDistilled water, 0.05–0.50 mol/L NaHCO3\n\t\t\t\t- solution | \n\t\t
\n\t\t\t\tDetection:\n\t\t\t | \n\t\t\tUV Detector, 280 nm | \n\t\t
\n\t\t\t\tAnalyte(s):\n\t\t\t | \n\t\t\tGlycoconjugate polysaccharide (LRGP1) [27] | \n\t\t
\n\t\t\t\tSample 10:\n\t\t\t | \n\t\t|
\n\t\t\t\tSource:\n\t\t\t | \n\t\t\t\n\t\t\t\tCoprinus comatus\n\t\t\t | \n\t\t
\n\t\t\t\tExtraction:\n\t\t\t | \n\t\t\tStipe powder of C. comatus (100 g) was extracted three times with 1 L 95% ethanol under reflux for 2 h to remove lipid, and the residue was extracted three times with 2 L distilled water for 2 h at 80 °C with intermediate centrifugation (2000 × g, 15 min). After concentrating the collected aqueous supernatants to 400 mL (reduced pressure at 40 °C), a precipitation was performed with 3 volumes of 95% ethanol. The precipitate was washed with ethanol and acetone, and then dried at 40 °C, yielding crude polysaccharide material. Crude polysaccharide material was dissolved in 100 mL 0.2 M sodium phosphate buffer (pH 6.0), and after centrifugation the solution was applied to a DEAE-Sepharose CL-6B column. | \n\t\t
\n\t\t\t\tStationary Phase:\n\t\t\t | \n\t\t\tDEAE-Sepharose CL-6B column (3.5 cm × 30 cm). | \n\t\t
\n\t\t\t\tEluent:\n\t\t\t | \n\t\t\t0.2 M sodium phosphate buffer (pH 6.0), and linear gradient of 0.3–1.5 M NaCl in 0.2 M sodium phosphate buffer (pH 6.0). | \n\t\t
\n\t\t\t\tDetection:\n\t\t\t | \n\t\t\tUV Detector, 490 nm (phenol–H2SO4) and 500 nm (Folin–phenol) | \n\t\t
\n\t\t\t\tAnalyte(s):\n\t\t\t | \n\t\t\tPolysaccharides; disaccharide α,α-trehalose, α-D-glucan, β-D-glucan, α-L-fuco-α-D-galactan [28]. | \n\t\t
\n\t\t\t\tSample 11:\n\t\t\t | \n\t\t|
\n\t\t\t\tSource:\n\t\t\t | \n\t\t\t\n\t\t\t\tPhysalisalkekengi var. francheti\n\t\t\t | \n\t\t
\n\t\t\t\tExtraction:\n\t\t\t | \n\t\t\tThe dried and defatted fruit calyx extracted with different enzyme Neutral proteinase, Papain and alkaline protease, respectively, in their suitable pH and temperature and then each extract was centrifuged at 5000 rpm for 10 min. The supernatant was concentrated and then precipitated by the addition of ethanol in 1:4 (v/v) at room temperature. The precipitate was dissolved in distilled water and the solution was then washed with sevag reagent (isoamyl alcohol and chloroform in 1:4 ratio), which were centrifuged at 5000 rpm for 15 min and the protein was removed. The supernatant was dialyzed against deionized water for 24 h before concentration under vacuum evaporator at 55 °C. The mixture was precipitated by the addition of ethanol in 1:4 (v/v) at room temperature and the precipitate was freeze dried. Total sugars were determined by the phenol–sulfuric acid assay using glucose as standard. | \n\t\t
\n\t\t\t\tStationary Phase:\n\t\t\t | \n\t\t\tDEAE anion-exchange column | \n\t\t
\n\t\t\t\tEluent:\n\t\t\t | \n\t\t\tThe column was eluted first with distilled water, and then with gradient solutions (0.1 M, 0.25 M, 0.5 M NaCl and 0.5 M NaOH), at a flow rate of 0.6 mL/min. The major polysaccharidefractions were collected with a fraction collector and concentrated using a rotary evaporator at 55 °C and residues were loaded onto a Sephadex G-200 gel column (2.5 × 65 cm). The column was eluted with 0.1 M NaCl at a flow rate of 0.3 mL/min. The major fraction was collected and then freeze dried. All of these fractions were assayed for sugar content by the phenol–sulfuric acid method using glucose as standard | \n\t\t
\n\t\t\t\tDetection:\n\t\t\t | \n\t\t\tUV Detector, 490 nm | \n\t\t
\n\t\t\t\tAnalyte(s):\n\t\t\t | \n\t\t\tPolysaccharides [29]. | \n\t\t
\n\t\t\t\tSample 12:\n\t\t\t | \n\t\t|
\n\t\t\t\tSource:\n\t\t\t | \n\t\t\t\n\t\t\t\tOrnithogalum caudatum Ait. | \n\t\t
\n\t\t\t\tExtraction:\n\t\t\t | \n\t\t\tThe whole dried plant was soaked with 95% ethanol to remove the pigments, defats and inactivates enzymes, and refluxed by hot distilled water for 4 h at 90 °C. The aqueous extract was concentrated to 30% of the original volume under reduced pressure in a rotary evaporator, and proteins were removed with Sevag method. The obtained solution was precipitated with 40% ethanol. The supernatant was added by ethanol up to 60%, and kept at 4 °C overnight. The polysaccharide pellets were obtained by centrifugation at 4000 rpm for 15 min, and completely dissolved in appropriate volume of distilled water followed by intensive dialysis for 2 days against distilled water (cut-off M\n\t\t\t\tw 3500 Da). The retentate portion was then concentrated, and centrifuged to remove insoluble material. Finally the supernatant was lyophilized to give crude extract. The crude extract was dissolved in 0.2 mol/L tris (hydroxymethyl) aminomethane hydrochloride buffer solution, and filtered through a filter paper. The solution was passed through an anion-exchange chromatography column. After ion exchange chromatography other chromatographic methods was used for further separations. | \n\t\t
\n\t\t\t\tStationary Phase:\n\t\t\t | \n\t\t\tDEAE-Sepharose fast flow anion-exchange chromatography column (10 × 300 mm) | \n\t\t
\n\t\t\t\tEluent:\n\t\t\t | \n\t\t\tThe polysaccharides were eluted with Tris–HCl buffer solution, followed with gradient elution of 0.1–0.8 mol/L NaCl at a flow rate of 0.8 ml/min. | \n\t\t
\n\t\t\t\tDetection:\n\t\t\t | \n\t\t\tUV Detector, 486 nm (phenol–sulfuric acid method) | \n\t\t
\n\t\t\t\tAnalyte(s):\n\t\t\t | \n\t\t\tWater soluble polysaccharides [30]. | \n\t\t
\n\t\t\t\tSample 13\n\t\t\t | \n\t\t|
\n\t\t\t\tSource:\n\t\t\t | \n\t\t\t\n\t\t\t\tPaecilomyces variotii\n\t\t\t | \n\t\t
\n\t\t\t\tExtraction:\n\t\t\t | \n\t\t\tAfter fermentation process, ammonium sulphate was added to the supernatant to give a final concentration of 80% saturation. The ammonium sulphate was added with constant stirring at 4ºC and the mixture stood overnight at 4ºC. The precipitated proteins were separated by centrifugation at 10000 rpm at 5ºC for 30 min. The separated proteins were then re-suspended in a minimum amount of distilled water and the solution dialyzed (using cellulose dialysis tubing) for 24 hrs against distilled water and concentrated by freeze-drying. The partially purified enzyme was dissolved in acetate buffer (20 mM - pH 6.0) and passed through a column | \n\t\t
\n\t\t\t\tStationary Phase:\n\t\t\t | \n\t\t\tDiethylaminoethyl (DEAE) Sepharose column (0.7 x 2.5 cm) | \n\t\t
\n\t\t\t\tEluent:\n\t\t\t | \n\t\t\tAcetate buffer (20 mM - pH 6.0)equilibrated with the same buffer. The solution was passed through the column at a flow rate of 1 mL.mim-1 with acetate buffer (20 mM - pH 6.0), followed by a linear gradient from 0-1M NaCl in the acetate buffer. The eluted fractions were collected in an automated fraction collector (Pharmacia Biotech) and the absorbance of the fractions was measured at 280 nm. The major peak fractions were then assayed for tannase activity, and only the fractions possessing tannase activity were pooled. | \n\t\t
\n\t\t\t\tDetection:\n\t\t\t | \n\t\t\tUV Detector, 280 nm | \n\t\t
\n\t\t\t\tAnalyte:\n\t\t\t | \n\t\t\tTannase [31] | \n\t\t
\n\t\t\t\tSample 14\n\t\t\t | \n\t\t|
\n\t\t\t\tSource:\n\t\t\t | \n\t\t\t\n\t\t\t\tCastanospermum australe\n\t\t\t | \n\t\t
\n\t\t\t\tExtraction:\n\t\t\t | \n\t\t\t50% MeOH extract of seeds | \n\t\t
\n\t\t\t\tStationary Phase:\n\t\t\t | \n\t\t\t(1) Amberlite IR-120B (500 mL H+ form), (2) Dowex 1-X2 column (3.8×90 cm, OH−form), (3) Amberlite CG-50 column (3.8×90 cm, NH4 + form), (4) Dowex 1-X2 column (3.8×90 cm, OH− form) (Repeated separation on different ion exchange columns). | \n\t\t
\n\t\t\t\tEluent:\n\t\t\t | \n\t\t\t0.5 M NH4OH, H2O | \n\t\t
\n\t\t\t\tDetection:\n\t\t\t | \n\t\t\tUV Detection by HPTLC | \n\t\t
\n\t\t\t\tAnalyte(s):\n\t\t\t | \n\t\t\tPyrolizidine alkaloids; fagomine; 6-epi-castanospermine; castanospermine; australine; 3-epi-fagomine; 2,3-diepi-australine; 2,3,7-triepi-australine; 3-epi-australine; 2R-hydroxymethyl-3S-hydroxypyrrolidine; castanospermine-8-O--D-glucopyranoside; 1- epi-australine-2-O--D-glucopyranoside and 1-epi-australine [32]. | \n\t\t
Since the isolation of pharmacologically active substances which are responsible for the activity became possible at the beginning of the 19th century drug discovery researches have increased dramatically [33]. Therefore within the last decade there has also been increasing interest in the liquid chromatographic processes because of the growing pharmaceutical industry and needs from the pharmaceutical and specialty chemical industries for highly specific and efficient separation methods. Several different types of liquid chromatography techniques are utilized for isolation of bioactive molecules from different sources [25]. Ion exchange chromatography is probably the most powerful and classic type of liquid chromatography. It is still widely used today for the analysis and separation of molecules which are differently charged or ionizable such as proteins, enzymes, peptides, amino acids, nucleic acids, carbohydrates, polysaccharides, lectins by itself or in combination with other chromatographic techniques [34]. Additionally ion exchange chromatography can be applied for separation and purification of organic molecules from natural sources which are protonated bases such as alkaloids, or deprotonated acids such as fatty acids or amino acid derivatives [35]. Ion exchange chromatography has many advantages. This method is widely applicable to the analysis of a large number of molecules with high capacity. The technique is easily transferred to the manufacturing scales with low cost. High levels of purification of the desired molecule can be achieved by ion exchange step. Follow-up of the nonsolvent extractable natural products can be realized by this technique [17,35]. Consequently ion exchange chromatography, which has been used in the separation of ionic molecules for more than half a century is still used as an useful and popular method for isolation of natural products in modern drug discovery and it continue to expand with development of new technologies.
Stereotactic body radiotherapy (SBRT) was developed using the concepts of stereotactic radiosurgery (SRS). SRS was conceived by neurosurgeons and physicists in Sweden to allow the delivery of radiation to precise targets in the brain while minimizing injury to adjacent areas. The procedure delivers a high dose of radiation to the target accurately focused using multimodality imaging, such as computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography/CT (PET/CT). The total dose is divided into several smaller doses of radiation, administered on separate days of treatment, typically in a single fraction or a few fractions. SRS treats tumors by destroying and distorting the DNA of these cells, in the same way as other forms of radiotherapy. As a result, these cells lose their ability to reproduce and die. Applied to the treatment of body tumors, the technique is called SBRT [1, 2, 3, 4].
SBRT is also known as stereotactic ablative radiotherapy (SABR). SBRT ablates tumors by delivering precise and intensive radiation, guaranteeing minimal normal tissue complications. The characteristics of SBRT are summarized as follows: (1) a limited number of high dose-per-fraction treatments with a biologically equivalent dose (BED) of at least 75–100 as a minimum or even higher; (2) fields only slightly larger than gross tumor volume (GTV) with high accuracy even for moving targets, including the entire target with margins of 0.5–1.0 cm (i.e., exact delivery to tumor targets, sparing normal tissue); (3) dosimetry constructed to be very conformal, with sharp gradients from high- to low-dose areas; and (4) secure patient fixation during treatment and accurate duplication of patient position between simulation and treatment [2, 5, 6, 7, 8].
Because of the high dose in a single fraction or fewer than five fractions, organs at risk (OARs) can be greatly affected by slight positional errors. Therefore, positional errors should be minimized. The margins of expansion can be reduced through the immobilization and control of respiratory motion of patients. Various commercial treatment delivery units in conjunction with the immobilization and respiratory motion control systems are available for the delivery of SBRT.
SBRT is currently both in use and being investigated for use in treating malignant or benign small- to medium-sized tumors in the body and at common disease sites, including the head and neck, lung, liver, abdomen, spine, and prostate. In particular, up to 70% of patients with malignancies are found to have skeletal involvement on postmortem examination, with the spine being the most common location [9]. For the treatment of spinal tumors, an extremely rapid dose falloff between the vertebral body and the spinal cord should be achieved [10, 11]. Implementation of correct beam-shaping and image-guided techniques has improved SBRT safety margins as well as accuracy and efficiency while accurately meeting 3D tumor contours. Spinal SBRT demands the highest accuracy in dose placement. In addition to patient fixation and multi-image guidance, a sophisticated treatment planning system that accurately models highly modulated small field beams is an indispensable factor in achieving high accuracy of radiation delivery.
To achieve this high accuracy, appropriate treatment planning technique should be used. Therefore, we will discuss various planning techniques for spinal SBRT in this chapter.
The spine is a frequent site of metastases from primary cancer of the prostate, lung, breast, and kidney. After the lung and liver, the skeletal system is the most frequent site of metastases [12, 13], and 30% of all patients with cancer develop bone metastases [12, 14, 15]. In particular, bone metastasis occurs in 85% of patients with breast, prostate, and bronchial carcinoma [12, 16]. Approximately 50% of all bone metastases occur in the spinal cord. Of these, 60–80% are located in the thoracic spine, followed by 15–30% in the lumbar spine and less than 10% in the cervical spine [12, 13].
If left untreated, spinal metastases can cause axial pain, vertebral body fractures, radiculopathy, and the debilitating complications of metastatic epidural spinal cord compression (MESCC) [9]. The major complications of spinal metastases include neurologic dysfunction [12, 17, 18] and potential hypercalcemia, reduced activity, and bone fractures, resulting in a reduced quality of life [12, 16].
In general, primary spinal tumors are treated surgically, with the goal of maximal tumor removal. Numerous important blood vessels and adjacent organs surround the vertebrae. In particular, the spinal cord located in the vertebrae is a part of the central nervous system, which includes sensory and motor nerves. Complete resection of a tumor while preserving the nerve function of the spinal cord is difficult. In addition, vertebral instability due to tumor destruction or complete resection of the tumor must be considered, and fusion or fixation is often required for stability of the vertebrae. Depending on the malignancy of the tumor or the difficulty of complete resection, the patient may be treated with radiotherapy.
Traditional radiotherapy methods of treating spinal tumors use large field radiation to treat the entire pathological vertebra and to treat one or two vertebral bodies, generally above and below the disease. This practice prevents missing the tumor owing to the limitations of diagnostic imaging and localization. In addition, the irradiation field of this technique is large but safe in the volume of the normal tissues irradiated because of the low biological effectiveness.
Large field radiation for spinal metastases has been the standard approach with outcomes of ~30% complete pain response and ~70% any response. The main limitation of the dose prescribed by traditional radiation techniques was the spinal cord. Overdosing radiation to the spinal cord has the devastating consequence of radiation-induced myelopathy that can leave the patient paralyzed. In addition to radiation myelopathy, possible toxicities include vertebral compression fractures and pain flares. Owing to the limitations of technology to prevent overdosing, clinical trials of high-dose effects on spinal metastasis have not been possible [19].
To overcome the limitations of conventional radiotherapy for the spine, hypofractionated treatment has been proposed, to deliver a high dose per fraction (typically 10–20 Gy/fraction), in contrast to the conventional fractionated treatment (2 Gy/fraction). The cumulative BED is significantly higher than that received in conventional treatment. Accurate delivery is of utmost importance owing to the high fractional dose and a small number of fractions. The delivery of an ablative dose to the target and rapid falloff doses away from the target enables minimization of the treatment toxicity to a tolerable level [20, 21]. In addition, there are other characteristics that distinguish SBRT from conventional radiotherapy, such as the number of beams used for treatment, the frequent use of non-coplanar beam arrangements, small or no beam margins on the penumbra, and the use of inhomogeneous dose distributions and dose-painting techniques ‘including IMRT’. All of these technology improvements result in the highly conformal dose distribution that characterizes the SBRT technique [2].
Hypofractionated spinal SBRT has been shown to effectively and rapidly alleviate pain and improve neurological function in patients with or without epidural cord compression. SBRT allows minimal radiation exposure outside the target; the most significant problem associated with this procedure is related to spinal cord dose tolerance. Depending on the vertebral level of spinal metastasis, adjacent organs should be considered OARs. The tolerance of OARs to radiation from conventional fractionated radiotherapy is based on the entire organ or on a considerably large irradiated volume. SBRT delivers a highly conformal, hypofractionated radiation dose to a small target with minimal exposure of the surrounding areas to radiation [22].
A new radiotherapy technology that allows for intensity-modulated radiotherapy (IMRT) has emerged with spinal SBRT. IMRT is a technique designed to deliver a high biologically effective dose only to tumors within the vertebra for the purpose of tumor regression through permanent local control. The technique allows radiation beams to avoid the spinal cord, and even though a high dose is delivered to tumors, the dose received by the spinal cord is below the toxic threshold dose [23]. More details will be discussed in Section 3.
Table 1 lists maximum dose limits to a point or volume within several critical organs recommended for SBRT in one fraction (refer to TG-101 for multiple-fraction dose constraints [2]). The recommended dose constraints are shown in max critical volume and the maximum dose to the given volume for each organ. These limitations have been determined based on the widely accepted radiosurgery norms currently in practice. Regardless of these limitations, the participating centers are encouraged to adhere to the prudent treatment planning principle to avoid unnecessary radiation exposure to critical normal structures [24].
Serial tissue | Max critical volume | Max dose in critical volume (Gy) | End point (≥ Grade 3) |
---|---|---|---|
Spinal cord | <0.035 cc | 14 Gy | Myelitis |
<0.35 cc | 10 Gy | ||
<1.2 cc (SBRT only) | 7 Gy (SBRT only) | ||
Cauda equina | <0.035 cc | 16 Gy | Neuritis |
<5 cc | 14 Gy | ||
Sacral plexus | <0.035 cc | 18 Gy | Neuropathy |
<5 cc | 14.4 Gy | ||
Esophagus* | <0.035 cc | 16 Gy | Stenosis/fistula |
<5 cc | 11.9 Gy | ||
Ipsilateral brachial plexus | <0.035 cc | 17.5 Gy | Neuropathy |
<3 cc | 14 Gy | ||
Heart/pericardium | <0.035 cc | 22 Gy | Pericarditis |
<15 cc | 16 Gy | ||
Great vessels* | <0.035 cc | 37 Gy | Aneurysm |
<10 cc | 31 Gy | ||
Trachea* and larynx | <0.035 cc | 20.2 Gy | Stenosis/fistula |
<4 cc | 10.5 Gy | ||
Skin | <0.035 cc | 26 Gy | Ulceration |
<10 cc | 23 Gy | ||
Stomach | <0.035 cc | 16 Gy | Ulceration/fistula |
<10 cc | 11.2 Gy | ||
Duodenum* | <0.035 cc | 16 Gy | Ulceration |
<5 cc | 11.2 Gy | ||
Jejunum/ileum* | <0.035 cc | 15.4 Gy | Enteritis/obstruction |
<5 cc | 11.9 Gy | ||
Colon* | <0.035 cc | 18.4 Gy | Colitis/fistula |
<20 cc | 14.3 Gy | ||
Rectum* | <0.035 cc | 18.4 Gy | Proctitis/fistula |
<20 cc | 14.3 Gy | ||
Renal hilum/vascular trunk | <2/3 volume | 10.6 Gy | Malignant hypertension |
Parallel tissue | Critical volume (cc) | Max dose in critical volume (Gy) | End point (≥Grade 3) |
Lung (right and left) | 1000 cc | 7.4 Gy | Pneumonitis |
Renal cortex (right and left) | 200 cc | 8.4 Gy | Basic renal function |
One fraction dose constraints of several critical organs from RTOG 0613 [24].
Avoid circumferential irradiation.
Various commercial treatment delivery units can be used to deliver SBRT [1, 5, 7], as shown in Figure 1. They all have the capability of image-guided radiotherapy, enabling tumor or target localization prior to treatment delivery and allowing treatment setup uncertainty to be significantly reduced. All delivery units, with the exception of proton therapy, used as photon-based SBRT, are linear accelerators (LINACs). There are several types of image-guidance equipment: 2D imaging types, including room-mounted or gantry-mounted orthogonal kilovoltage (kV) radiographs and fluoroscopy, and 3D imaging types including kV or megavoltage (MV) cone-beam CT (CBCT) and CT-on-rails in room.
Commercial treatment delivery units. From left to right are versa HD (Elekta AB), Radixact (Accuray Inc.), and CyberKnife M6 (Accuray Inc.).
In addition to general LINACs, there are many types of treatment systems. The CyberKnife (CK, Accuray Inc., Sunnyvale, CA, USA) unit has a six-axis robotic manipulator that enables delivery of the beam to the target from many different directions in order to minimize radiation exposure to nearby organs. A pair of orthogonally positioned imaging systems enables monitoring of the target motion, with automatic correction. CK is a commonly used modality for SBRT owing to its highly conformal dose distributions, steep gradient, and near real-time image-guidance system. The helical tomotherapy (HT, Accuray) unit is a special device performing continuous 360° rotations using a binary multi-leaf collimator (MLC), with the treatment couch moving continuously during the treatment [1].
Each treatment delivery system has strengths and weaknesses. An appropriate treatment delivery system and corresponding optimal planning technique should be used for successful and safe treatment.
SBRT is a high-precision radiotherapy technique that utilizes the high doses of radiation in a single fraction or a few fractions, as mentioned in the above sections. In principle, three-dimensional conformal radiotherapy (3D-CRT) planning can be applied to SBRT. When the beams at multiple angles are concentrated at the center of small lesions, a high-dose heterogeneity that contributes to a steep dose gradient at the target edge appears and may be desirable in terms of normal tissue sparing and dose escalation to the GTV [1].
To treat a spinal tumor, conventionally fractionated 3D-CRT modifies the beam shape to match the projection of target volume at each gantry angle using an MLC. The accuracy of the shape of the beam projected onto the target depends on the width of leaves. MLC leaf widths of 2.5–10 mm have been reported for use in SBRT planning [25, 26].
However, delivery to the target is limited by tolerance of normal tissues, particularly the spinal cord, so it is necessary to irradiate the target with lower dose. In suboptimal cases, several side effects can occur, such as paraplegia, pain, increased steroid use, and reduced survival rate.
The development of IMRT was a major improvement over 3D-CRT for SBRT [27]. IMRT allows for the radiation dose to conform more precisely to the shape of the tumor by modulating the intensity of the radiation beam and allows higher radiation doses to be focused to regions within the tumor, sparing the surrounding normal critical structures. In particular, when treating spinal tumors, intensity modulation allows production of a concave-shaped dose distribution with the exception of the spinal cord.
The IMRT technique uses computerized inverse planning. Conformal radiotherapy is forward planning and depends on the skills of the treatment planner to determine the number, shape, and orientation of the beams. Inverse planning, in contrast, specifies the plan outcome in terms of the tumor dose and normal structure dose limits. The computer system then adjusts the beam intensities to identify a configuration best matched to the desired plan [28].
During the procedure, each beam is divided into several beam elements (beamlets) of a few millimeters, and the relative weight is optimized so that the desired dose distribution appears. The optimization process involves inverse planning in which beamlet weights or intensities are adjusted to satisfy predefined dose criteria for the composite plan. When optimization is complete, an optimized fluence map generates a sequence of MLC leaves for each beam. The field at one gantry angle is subdivided into a set of subfields irradiated at a uniform beam intensity level. The subfields are shaped by the MLC, and the intensity-modulated field is obtained by summing several subfields.
The two most common methods of IMRT delivery are segmental (step-and-shoot) and dynamic (sliding window). The difference between the two is the motion of MLC at a given gantry angle. In segmental MLC delivery, the beam is turned off while the leaves move until the next subfield is prepared. The advantage of the segmental MLC method is that it is easy to plan and no additional dose can occur while the MLC is moving to create the next subfield. On the other hand, the dose delivery is slow owing to the delay in turning the beam on and off, resulting in an increase in treatment time. In the dynamic MLC delivery, the MLC leaves are moving during irradiation. Each pair of leaves sweeps across target volumes under computer control. Dynamic MLC delivery offers better dose homogeneity for target volume and shorter treatment time in comparison to the segmental MLC; however, the larger total irradiated dose is a disadvantage.
Compared to 3D-CRT, the dose distribution can be made even more sophisticated because target coverage and avoidance of critical structures located adjacent to the target volume are better. The more sophisticated implementation of SBRT has become possible with the IMRT technique. The technique mentioned in this section (Section 3.1) was the IMRT technique with a fixed gantry, and IMRT with a rotating gantry will be discussed in the following section (Section 3.2).
Intensity-modulated arc therapy (IMAT) is a combined technique of IMRT and rotational treatment. When performed for a C-shaped target with a sensitive structure in the concavity of the “C,” like a spinal tumor, the rotational treatment has a dosimetric advantage. The result of simulation that supports this is that when all the planning parameters except the beam angle number are constant, the dose becomes more homogeneous in the tumor and decreased in the critical structures as the number of angles increased [29].
IMAT uses rotational cone beams of varying aperture shapes and varying dose weightings to achieve intensity modulation. However, the speed of rotation cannot have frequent and drastic variations owing to the weight of the LINAC gantry; therefore, the variations in dose weighting are primarily achieved through varying the machine dose weight. MLC moves dynamically to shape each subfield while the gantry is rotating and the beam is on continuously [30]. Arcs are approximated as multiple-shaped fields in a regular angular interval. One subfield is delivered at each arc. The next new arc is started to deliver the next subfield and so on until all the planned arcs and their subfields have been delivered. That is, overlapping arcs create intensity modulation.
To create more effective treatment plans, various techniques have been purposed within IMAT. Volumetric-modulated arc therapy (VMAT) and modulated arc therapy (mARC) are examples of such techniques. VMAT is a single or multi-arc form of IMRT technique that changes the dose rate and gantry speed while the gantry is rotating. Currently there are several VMAT systems available under various names (RapidArc, Varian Medical Systems, Palo Alto, CA, USA; SmartArc, Philips Radiation Oncology Systems, Fitchburg, WI, USA; and Elekta VMAT, Elekta, Stockholm, Sweden) [31]. The mARC technique as an alternative to VMAT is a rotational IMRT irradiation with burst mode delivery. Both the dose rate and gantry speed are modulated to allow for delivery of the correct dose per IMRT segment, and an MLC velocity servo is required to continuously adjust the leaf velocity to facilitate accurate, and timely, leaf positioning [32].
The technique is similar to HT, which is an IMRT technique that rotates in a helical form and will be discussed in Section 3.3. As compared with HT, IMAT has certain advantages: (1) IMAT eliminates the need for transferring the patient during treatment and avoids abutment issues as seen with serial HT, (2) IMAT retains the ability to use non-coplanar beams and arcs, and (3) IMAT uses a conventional LINAC; thus, complex rotational IMRT treatments and simple palliative treatments can be delivered with the same treatment unit [30].
The main advantages of rotational therapy compared to fixed-gantry IMRT are improved conformity of the dose distribution in the high-dose regions, as well as possible reduction of the treatment time. The short treatment time can lead to improved patient comfort and reduce the risk of movement. Moreover, shorter treatment times can be biologically beneficial. Radiation survival is not only a function of the total dose delivered but also depends on the duration of radiation delivery [33, 34]. IMAT offers the efficient use of monitor units (MUs). The number of MUs per treatment is correlated with the amount of scatter dose and leakage radiation, which could be important in view of the induction of secondary malignancies [35]. The decrease in MUs achieved with IMAT partly addresses this issue, which is one of the major concerns with IMRT [36].
However, the complex nature of IMAT planning has been one of the primary barriers to routine clinical implementation. From one angle to the next in each VMAT arc, leaf motion between adjacent angles is limited by leaf travel speed and gantry rotation speed. Therefore, the technique has disadvantages such as difficulty and complexity of planning.
HT is a radiotherapy modality that combines helical CT scanning with an MV linear accelerator. A 6 MV LINAC rotates on a ring gantry at a source-axis distance (SAD) of 85 cm, and the beam passes through a primary collimator into a fan-beam shape. During treatment, the ring gantry continuously rotates, while the couch is continuously translated through the rotating beam plane. The dose is thus delivered in a helical fashion. The ring gantry also contains a detector system that is mounted opposite the accelerator and is used to collect data for megavoltage CT (MVCT) acquisition. A beam stopper is used to reduce room-shielding requirements [37].
The MVCT in HT is used as a tool to enhance image-guided daily treatment setup and positioning of the patient. Because SBRT usually requires a longer treatment period owing to the use of high-dose hypofraction, the patient must be fixed in place to limit the patient’s movement during treatment. However, patients with vertebral metastases, in particular, often move involuntarily during treatment owing to back pain that cannot be controlled. Therefore, it is important to ensure the accuracy of high-dose delivery and to avoid side effects of OARs on intrafractional movement. A daily MVCT image scan is generated prior to treatment to ensure accurate delivery of each treatment according to the patient’s anatomy on a particular day. This MVCT is integrated with the kilovoltage CT (kVCT) imaging plan to provide a reference for patient setup and positioning [38].
The fan-beam has an extension of 40 cm in the lateral direction and smaller or equal to 5 cm (typically 1.0, 2.5, and 5.0 cm) in the longitudinal direction at the isocenter. With the use of a compressed air-driven multi-leaf (64 leaves) binary collimator (MLC), radiation beams are shaped, and their intensities are modulated. The leaves are mounted on two opposite blocks, and each individual leaf is driven from open to closed state. The intensity modulation is achieved by controlling the length of time each leaf is open. Each leaf has a width of 6.25 mm (40 cm divided by 64 leaves) and rapid transitioning (about 20 ms); thus it can produce a sufficiently accurate shape even within a short rotation period. Therefore, HT offers a very useful treatment modality of spinal SBRT by implementing image-guided radiation therapy (IGRT) and IMRT techniques.
For the treatment planning of each rotation, a rotation is divided into 51 projections (360°/7° = 51). For each projection, each MLC leaf has a unique opening time as shown in Figure 2 [39]. Unlike the usual LINAC radiotherapy, there are additional parameters: slice width, pitch factor, and modulation factor. These parameters influence both treatment time and quality of the treatment plan.
Illustration of the helical tomotherapy delivery. Copyright © Journal of Medical Physics.
Slice width (or field width) is the longitudinal extent (i.e., in the y-direction) of the treatment field. For planning purposes, a nominal 1.0, 2.5, or 5.0 cm is selected. Pitch is defined as distance traveled by the couch per gantry rotation, divided by the slice width. With a lower pitch value, there is greater overlap between spirals. This factor influences the treatment time. Modulation factor is defined as the maximum leaf opening time divided by the average opening time of all leaves. This value can range from 1.0 to 10 (typically using from 1.5 to 3.5). For a complex treatment requiring a lot of MLC motion, a high modulation factor is selected.
One of the most important differences between the HT system and other radiotherapy systems is that the HT system does not have a flattening filter. The main advantages of an absent flattening filter are an increased dose rate, reduced scatter, reduced leakage, and reduced out-of-field doses [40, 41]. The main reason for allowing the nonuniform profile is that HT is a dedicated IMRT system, without the need for a flat dose profile. If it is still desired, the MLC can be used to modulate the treatment field to produce a flat dose distribution [42].
In treating spinal tumors, the major requirement is minimization of the dose to the spinal cord. The dose gradient should be increased to improve the conformity while allowing increased heterogeneity in the tumor volume coverage. In addition, the slice width and pitch parameters are considered to increase cord avoidance and target coverage.
CK is one of the representative delivery units of SBRT. As mentioned briefly in the above section, CK has uniquely different features compared with the common medical LINACs. The compact LINAC mounted on a computer-controlled six-axis robotic manipulator delivers radiation beams anywhere in the body with submillimeter accuracy. The integrated orthogonally positioned kV X-ray imaging system is utilized to monitor the patient position throughout the course of radiotherapy. Patients are positioned automatically or manually by a therapist by matching fiducial markers or bony anatomy from X-ray images to digital reconstructed radiographs generated by CT simulation [43].
The robotic manipulator with six degrees of freedom can deliver the beam anywhere in space. Accordingly, the beam position and orientation can be adjusted by the robot to accommodate changes in target position and orientation during treatment without the need to move the patient.
The beam field size is controlled through various collimation types: 12 fixed cone collimators or an Iris variable collimator (Accuray) consisting of 12 tungsten leaves that produce beam diameters ranging from 5 to 60 mm (defined at 800 mm distance from the X-ray source) [44]. Furthermore, to compensate for the limit caused by the fixed field size, an MLC has recently been introduced for the CK [45]. The new MLC system consists of 41 leaf pairs, each with a width of 2.5 mm. The maximum field size is 12 × 10.25 cm. This new system allows the fields to be shaped matching the tumor shape and allows reduction of treatment time. In particular, using the MLC offers a dosimetric advantage for targets near OARs, as shown in Figure 3 [46].
Dose-volume parameters in circular collimator and multi-leaf collimator (MLC) plans for 1–7 cm brain target volumes. (White bars indicate multi-leaf collimator (MLC) and gray indicate circular collimator.) Copyright © 2017, Oxford University Press.
The unit delivers multiple isocentric or non-isocentric photon beams to a desired target from many different angles through a robotic arm, as well as optic image guidance for motion management. The isocentric treatment planning is similar to that of the Gammaknife (Elekta) and conventional LINACs, which have a fixed mechanical center of the gantry and collimator. The location of the isocenter is not limited, providing a great advantage over many other delivery units. However, this advantage can be overcome by using inverse planning; the final target dose distributions can be manipulated to a certain level by modifying the order of the targets as well as the contours and dose limits assigned to the target and critical organs.
In non-isocentric treatment planning, radiation beams are delivered to a specific portion of the tumor without couch repositioning. This technique makes the high-dose isodose lines match the target shape and avoid nearby critical organs. Therefore, non-isocentric planning is very useful for treatment of irregularly shaped targets. CK, which is available with both plans, is advantageous for combining the rapid dose falloff of isocentric plans with the dose conformity of non-isocentric plans [1].
In spinal SBRT, the target volume includes the involved vertebral body and both left and right pedicles and the grossly visible tumor, if a paraspinal or epidural lesion is present. The target volume is generally delineated with no margin. However, depending on the treatment system, a beam aperture margin of 2–3 mm beyond the target volume is allowed to ensure adequate dose coverage of the target. This margin can be reduced to 0–1 mm in the area of the spinal cord to meet spinal cord dose constraints. The target volume may be selected at the discretion of the treating radiation oncologist based on the extent of tumor involvement. In any circumstance in which there is an epidural or paraspinal soft tissue tumor component, the visible epidural or paraspinal tumors are included in the target volume [24].
Normal tissue contouring is required starting at 10 cm above the target volume to 10 cm below the target. The treatment plan should be established according to the recommended maximum dose limit for several critical organs, as shown in Table 1. Among the dose-limiting critical OARs, the spinal cord is a key concern. Because of the nature of radiosurgery with a rapid dose falloff, there is a radiation dose gradient within the diameter of the spinal cord. Therefore, a partial spinal cord volume defined as from 5 to 6 mm above to 5–6 mm below the target volume is used. The partial or absolute volume spinal cord constraints are applied to each treated spine level when the patient has multiple spine levels treated. Any spinal cord dose exceeding this constraint is not acceptable and is a major deviation [24].
Successful treatment planning requires 90% coverage of the target volume by the prescribed dose. Typically, the 80–90% isodose line is used as the prescription line, although the prescription isodose line may be different depending on the delivery system. Coverage of <90% of the target volume is an acceptable variation, and any coverage of <80% of the target volume is an unacceptable deviation. The treatment plan is acceptable as long as ≥90% of the target volume receives the prescribed dose. It should be noted, however, that owing to the irregular shape of the target volume and the location of the spinal cord, hot spots may be created in the immediate vicinity outside of the target volume [24].
Because of the characteristics of the spinal SBRT, in the case of a beam with a small size, the higher the beam energy, the larger the beam penumbra as a result of lateral electron transport in the medium. The commonly available 5 mm MLC leaf width has been found to be adequate for most applications, with negligible improvements using the 3 mm leaf width MLC for all but the smallest lesions (<3 cm in diameter). A 6 MV photon beam, available on most modern treatment machines, provides a reasonable compromise between the beam penetration and penumbra characteristics. Additionally, beam arrays should be placed mostly in the posterior direction to avoid entrance of the radiation beam through the lungs. In the case of arc rotation techniques, every effort should be used to limit the passage of radiation through the lungs [2].
In Section 3, several spinal SBRT planning techniques were discussed. Because the planning technique should be selected depending on the patient’s condition or situation, numerous studies have been performed to compare various planning techniques for treating spinal tumors. To evaluate the results of each plan for spinal SBRT, the following quantitative parameters were used [22, 47, 48, 49, 50].
Conformity index (CI): a measure of the dose coverage to the planned target volume (PTV).
Dice similarity coefficient (DSC): a spatial overlap index and a reproducibility validation metric [51].
Homogeneity index (HI): a measure of uniformity of the dose within the target volume.
PTV coverage: 100% of the PTV receiving the prescribed dose [52].
Spinal cord dose: maximum dose to the spinal cord.
High-dose spillage: The cumulative volume of all tissue outside the PTV receiving a dose >105% of prescription dose should be no more than 15% of the PTV volume [53].
Intermediate-dose spillage (R50% and D2cm): the falloff gradient located outside of the PTV.
R50%: volume that received 50% of the prescribed dose/PTV volume
D2cm: maximum dose in terms of the percentage of the prescribed dose at 2 cm beyond the PTV in any direction
Equivalent uniform dose (EUD): the absorbed dose that, if homogeneously delivered to a tumor, causes the same expected number of clonogens to survive as does the actual nonhomogeneous absorbed dose distribution.
Biological effective dose (BED): the dose producing equivalent biological effect regardless of dose uniformity or fractionations.
Gamma index: the standard method for planar dose verification in IMRT QA; calculates the quantity γ for each point of interest using preselected dose difference (DD) and distance to agreement (DTA) criteria and then uses the γ value to determine the outcome (pass-fail) of the IMRT QA [53].
In addition, plans were evaluated by the treatment delivery time (beam irradiated time) or the target point dose for the phantom measured in the ion chamber.
Zach et al. compared VMAT to static beam IMRT for spinal SBRT. The plans were compared for conformity, homogeneity, treatment delivery time, spinal cord dose, and Dmax of the spinal cord and V 10 Gy, which is the volume of the spinal cord exposed to at least 10 Gy. The authors also compared the monitor units required in each plan to compute the net irradiated time.
All evaluated parameters were shown to favor the VMAT plans over the IMRT plans. Dmin for PTV in the IMRT was significantly lower than that in the VMAT plan. The DSC and treatment time were found to be significantly better for the VMAT plans than for the IMRT plans. A reduction of almost 50% in the net treatment time was calculated. The authors reported that VMAT provides better conformity, homogeneity, and spinal cord dose. They also suggested that the shorter treatment time is a major advantage and not only provides convenience for patients experiencing pain but also contributes to the precision of this high-dose radiotherapy [47].
In another study, Choi et al. compared the treatment planning performance of RapidArc (i.e., VMAT) and CK for spinal SBRT. The optimized dose priorities for both plans were similar for all patients. The highest priority was to provide sufficient dose coverage to the PTV while limiting the maximum dose to the spinal cord. Plan quality was evaluated with respect to PTV coverage, CI, high-dose spillage, intermediate-dose spillage, and maximum dose to the spinal cord, which are criteria recommended by the RTOG 0631 spine and 0915 lung SBRT protocols.
The mean CI ± standard deviation (SD) values of the PTV were 1.11 ± 0.03 and 1.17 ± 0.10 for RapidArc and CK, respectively. On average, the maximum dose delivered to the spinal cord in CK plans was approximately 11.6% higher than that in RapidArc plans. High-dose spillages were 0.86 and 2.26% for RapidArc and CK, respectively. Intermediate-dose spillage characterized by D2cm was lower for RapidArc than for CK; however, R50% was not statistically different between the plans. Although both systems can create highly conformal volumetric dose distributions, the study of Choi et al. shows that RapidArc was associated with lower high- and intermediate-dose spillages than was CK. The authors also suggested that RapidArc plans for spinal SBRT may be superior to CK plans [48].
Sahgal et al. compared the treatment planning quality of the CK and Novalis (BrainLAB AG, Heimstetten, Germany) systems for vertebral body SBRT. Physical parameters and biological modeling parameters such as PTV dose coverage, dose conformity, EUD, integral BED, and a generalized BED were used to compare the treatment plans.
In the study, both the CK and Novalis treatment plans fulfilled the specified requirements with comparable PTV dose coverage and dose conformity. For the target volume, CK plans produced significantly higher values of all calculated parameters to the PTV. For OARs, CK plans produced a somewhat lower dose to small volumes (0.1–1 cm3) of the spinal cord and esophagus but exposed larger volumes of these structures to a low dose as compared to the Novalis plans.
The authors reported that restricting the dose to a small volume of the spinal cord and esophagus resulted in a modest decrease in the dose to 1 cm3 volume of these structures for CK planning but at the expense of a larger volume of these structures exposed to low-dose levels [49].
In another study, Kim et al. compared the planning characteristics for hypofractionated spinal SBRT administered using three treatment techniques (IMRT, mARC, and HT). The factors evaluated for spinal SBRT planning were dose coverage, cord avoidance, target conformity, homogeneity, and dose spillage.
Target dose coverage was 82.74 ± 3.35, 80.92 ± 0.81, and 85.01 ± 7.27% for IMRT, mARC, and HT, respectively. The authors reported that HT was therefore a powerful technique with respect to target coverage. The spinal cord dose for HT (mean, 1763.96 cGy; SD, 164.48) was significantly different from those for mARC (mean, 1991.75 cGy; SD, 248.00) and IMRT (mean, 2053.24 cGy; SD, 164.48). In addition, the partial spinal cord volume at 2000 cGy for HT (mean, 0.12 cc; SD, 0.01) was significantly different from those for IMRT and mARC (0.50 ± 0.10 cc and 0.56 ± 0.25 cc, respectively). The CIs were 1.30 ± 0.12, 1.08 ± 0.05, and 1.36 ± 0.23 for IMRT, mARC, and HT planning, respectively. mARC showed the highest conformity. Regarding HI, HT (mean, 1763.96 cGy; SD, 164.48) differed statistically from both mARC (mean, 1991.75 cGy; SD, 248.00) and IMRT (mean, 2053.24 cGy; SD, 164.48) with respect to the spinal cord dose.
HT used a narrow field fan-beam and exhibited remarkable improvement of target coverage and cord dose, offering an important benefit to spinal SBRT. mARC had the highest target conformity and showed more favorable high- and intermediate-dose spillage than did HT and IMRT. These three planning techniques have different advantages. The authors suggested utilizing different planning techniques according to the cases. In the case of spinal SBRT, HT should be used for cord avoidance. In some cases, such as for a short treatment duration when the patient is considered to be in poor general condition, mARC can be used [22].
Gallo et al. performed end-to-end (E2E) testing for a set of representative spinal targets planned and delivered using four different treatment planning systems and delivery systems, specifically HT, Vero, TrueBeam with flattening filter free (FFF) and flattened, and CK, to evaluate the various capabilities of each. An anthropomorphic E2E SBRT phantom was simulated and treated on each system to evaluate agreement between measured and planned doses. The phantom accepted 0.007 cm3 ion chambers in the thoracic region and radiochromic film in the lumbar region.
Ion chamber measurements in the thoracic targets resulted in an overall average difference of 1.5% with planned doses. Specifically, measurements agreed with the treatment planning system to within 2.2, 3.2, 1.4, 3.1, and 3.0% for all three measureable cases on HT, Vero, TrueBeam (FFF), TrueBeam (flattened), and CK, respectively. Film measurements for the lumbar targets resulted in average global gamma index passing rates of 100 at 3%/3 mm, 96.9 at 2%/2 mm, and 61.8 at 1%/1 mm, with a 10% minimum threshold for all plans on all platforms. Local gamma analysis was also performed with similar results. While gamma passing rates were consistently accurate across all platforms through 2%/2 mm, treatment beam-on delivery times varied greatly among the platforms, with TrueBeam (FFF) the shortest, averaging 4.4 min, TrueBeam using flattened beam at 9.5 min, HT at 30.5 min, Vero at 19 min, and CK at 46.0 min.
In the study, despite the complexity of the representative targets and their proximity to the spinal cord, all treatment platforms were able to create plans that meet all RTOG 0631 dose constraints and produced exceptional agreement between calculated and measured doses. However, there were differences in the plan characteristics and significant differences in the beam-on delivery time between platforms. Thus, the authors stated that clinical judgment is required in each particular case to determine the most appropriate treatment planning/delivery platform [50].
This chapter has described various planning techniques for spinal SBRT and summarized the studies comparing these techniques. The spine is a frequent site of tumor metastasis, but there are many important vessels and adjacent organs in the vicinity of the vertebrae. In particular, the spinal cord within the spine is part of the central nervous system. Radiotherapy is performed depending on the malignancy of the tumor or the difficulty of complete resection, considering potential spinal instability caused by the tumor destruction or complete resection. However, the major limitation of traditional radiotherapy is the tolerance dose of the spinal cord. If the spinal cord is irradiated with an overdose, toxicities such as radiation-induced myelopathy, vertebral compression fracture, or pain flare may occur. To overcome the limitation of conventional radiotherapy, SBRT has been proposed. The technique of SBRT delivers a higher BED, within the range of what is considered locally curative. A conformal high-dose beam in a few fractions should be used, and an intensity modulation technique is required for the sparing of normal organs surrounding the spinal lesion. Various planning technologies based on intensity modulation technology are available, including IMRT with fixed gantry, IMAT, HT, and CK. Different planning techniques have their distinct features and advantages. Therefore, it is important to use appropriate treatment planning depending on the patient’s condition and situation.
This research was supported by Advanced Institute for Radiation Fusion Medical Technology (AIRFMT) at the Catholic University of Korea.
The authors report no conflicts of interest.
IntechOpen publishes different types of publications
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