Christopher J. Holloway

Principles of isotachophoresis

ZusammenfassungObwohl die theoretischen Grundlagen des Prinzips der Isotachophorese bereits seit 1897 existieren, fand diese Technik ihre eigentliche praktische Realisierung für die analytische und präparative Anwendung erst in den letzten Jahren.Die Isotachophorese ist zur Zeit zwar die neueste elektrophoretische Technik, deren breiter Einsatz jedoch noch aussteht. In den letzten 10 Jahren sind einige Beiträge erschienen, welche die Prinzipien und Theorie behandeln. Besonders hervorzuheben ist die Gruppe um Everaerts, Eindhoven, welche die praktische Anwendung der Capillar-Isotachophorese als analytische Methode der 60er Jahre ermöglichte.Vielfach wurden Anwender dieser Methode bei der Erarbeitung der Prinzipien der Isotachophorese durch deren relativ komplizierte mathematische Grundlagen abgeschreckt. Andererseits sind nach unserer Meinung in den bisher erschienenen Übersichten die grundlegenden Prinzipien nicht genügend berücksichtigt worden.Dieser Beitrag soll versuchen, als Kompromiß das Gebiet einerseits nicht zu detailliert und theoretisch abzuhandeln und andererseits die wichtigsten Aspekte in kurzer Form, jedoch weitgehend vollständig darzustellen. Dazu wurden theoretische und Anwendungs-orientierte Beiträge gleichermaßen berücksichtigt.Heute verstehen wir unter Isotachophorese im wesentlichen die Capillartechnik; wir beschränken uns daher bei der apparativen Beschreibung auf solche Systeme. Wie auch andere elektrophoretische Methoden, kann die Isotachophorese in einer Gelphase wie Polyacrylamid oder Agarose durchgeführt werden, wobei diese Anwendung dann mehr präparativen als analytischen Zwecken dient.Es scheint uns nicht überflüssig, die Isotachophorese kurz zu definieren: Die Wanderung gleichsinnig geladener Teilchen kann dann als „isotachophoretisch“ bezeichnet werden, wenn sich Ionen aus einem Gemisch in diskreten Zonen mit definierten Konzentrationen hinter einer vorauslaufenden Zone mit Ionen höherer Mobilität (Leit-Ionen) und vor einer entsprechenden Zone mit Ionen geringerer Mobilität (Terminator-Ionen) anordnen. In diesem „Pseudo-Steady State“ wandern im elektrischen Feld alle Zonengrenzen mit identischer Geschwindigkeit.Zu den Merkmalen der Isotachophorese-Trenntechnik gehört die Verwendung eines im gesamten System vorhandenen puffernden Gegenions, das den pH-Wert festlegt und damit die effektive Mobilität und Reihenfolge der Zonen im „steady-state stack“. In diesem diskontinuierlichen Elektrolytsystem ist der von der Disc-Elektrophorese bekannte Konzentrierungseffekt nach Ornstein und Davis erkennbar. Dieser Teil des Trennverfahrens ist in der Tat „isotachophoretisch“. In der Disc-Elektrophorese kommt jedoch das eigentliche analytische Ergebnis erst durch den zweiten Schritt, der „zonenelektrophoretisch“ ist, zustande. Hierbei wird die Isotachophorese nur zur Konzentrierung der Proteinzonen verwendet, um die Auflösung der zonenelektrophoretischen Trennung zu verbessern.Abschließend sollte darauf hingewiesen werden, daß für die Isotachophorese immer noch zahlreiche Begriffe existieren, die in der Literatur zur Verwirrung führen können. Beispiel hierfür sind: „displacement electrophoresis“, „ionic migration technique“, „cons electrophoresis“, „omegaphoresis“, „transphoresis“, „steady-state stacking“.Immer häufiger jedoch bürgert sich, entsprechend der Wanderungscharakteristik, die Bezeichung „Isotachophorese“ ein, die von allen führenden Gruppen auf diesem Gebiet übernommen wurde.SummaryAlthough the theoretical groundwork for isotachophoresis was laid down as long ago as 1897, it is only really in recent years that this technique has been exploited for analytical or preparative purposes. Isotachophoresis is not only the newest, but also generally speaking the least well understood of the modern electrophoretic methods. In the past 10 years, several articles have appeared which describe the principles and theory of isotachophoresis. Notable are the works of the Everaerts group in Eindhoven, who brought about the practical realisation of isotachophoresis as an analytical procedure in capillary tubes in the late 1960s. However, the mathematics involved in the theory of isotachophoresis may be off-putting to many scientists of other disciplines who are attempting to gain a basic understanding of the principles. Many shorter reviews by other authors during the past decade have not provided sufficient information for a clear picture of the principles. In the present article, we have attempted to find a suitable compromise between extensive and detailed, but too theoretical treatments, and brief description which leave too much unsaid.It is true to say that isotachophoresis has become associated with the technique in capillary tubes, and in our discussion of instruments, we restrict ourselves to such a system. However, in common with all other electrophoretic techniques, isotachophoresis can be performed in a supporting phase, such as polyacrylamide or agarose gel, whereby these procedures have more applicability from the preparative rather than from the analytical point-of-view.The literature list at the end of this article provides some key references for those interested in following-up particular aspects of isotachophoresis, ranging from a more detailed theoretical background, to applications in various fields.It is perhaps useful to provide a short definition of isotachophoresis at this point. An isotachophoretic migration is one in which a mixture of ions of similar charge quality separate into discrete zones of regulated concentration behind an ionic zone of higher mobility than all ions in the mixture (leading ion) and in front of an ion of lower effective mobility than all ions in the mixture (trailing or terminating ion). In the pseudo-steady state, all zone boundaries migrate with identical velocity through the electric field. A characteristic of isotachophoretic separations is the fact that a common buffering counter ion is employed, which defines the pH and hence the effective mobility and migrating order of the zones in the steady state stack. Those conversant with electrophoretic methods will recognise in this discontinuous electrolyte system the concentrating stage of the disc electrophoresis of Ornstein and Davis. This procedure is in fact isotachophoresis. In disc electrophoresis, however, the actual analysis is derived from the second stage, which is zone electrophoresis. Isotachophoresis is used here simply to concentrate the protein zones to improve the resolution of the zone electrophoretic separation.Finally, it is worth pointing out that isotachophoresis is known by several names, which provides a certain degree of confusion for literature searches. Examples are displacement electrophoresis, ionic migration technique, cons electrophoresis, omegaphoresis, transphoresis, steady-state stacking. Although articles still appear using these nomenclatures, the name isotachophoresis, derived from the equal migrating velocity of all zones in the system, is now generally accepted, at least by the major contributors to research in this field.

Agarose-Encapsulated Adsorbents I. Concept and General Properties

The development of a new material for use in haemoperfusion systems, agarose-encapsulated adsorbents, is reported. In contrast to conventional „coating“ methods, the required adsorbing phase, such as active charcoal or resin, is actually trapped within the matrix of the agarose gel, which is formed into smooth, soft, but resilient beads of virtually any desired diameter, conveniently in the range 3–5 mm. It is our belief, derived from the unusual properties of this system which have so far emerged, that the technique of agarose-encapsulation could provide a further step forward in the field of extracorporeal detoxification. The decisive advantage of the new material is undoubtedly the possible use of adsorbent phases in powder form encapsulated in the beads, which affords a vastly improved degree of adsorption without a negative effect on haemocompatibility. An approach to a semi-selective system is offered by the use of combinations of adsorbers in the beads.

The Biochemistry of Hepatic Detoxification

The biochemistry of hepatic detoxification mechanisms is reviewed, and the application of the enzyme systems which are responsible for such processes in a device for extracorporeal detoxification in liver failure is discussed.

Detoxification of Phenols by Sulphate Conjugation: An Alternative to Glucuronidation in an Enzymatic Liver Support System

The relevance of phenolic compounds in acute liver failure is well established. There are two conjugating processes in the liver which are normally responsible for the detoxification of this class of substance, namely the glucuronyl and sulphate transferases, of which the latter play a subordinate role in vivo. In an artificial liver support device, however, sulphate conjugation presents several advantages over glucuronidation. The apparent superiority of glucuronidation is simply attributable to the availability of the donor molecule, a situation which is reversible in an artificial system, where the quantity of donor molecule can be controlled. Some characteristics of sulphate transferase are discussed in this contribution, together with some suggestions for the application of this enzyme in an extracorporeal detoxification device.

Enzymatic Methylation of Mercaptans: Applicability in an Enzyme Reactor Liver Support System

Mercaptans are recognised endogenous toxins, which are found in increased concentration in the body fluids and tissues of patients with acute liver failure. A possible detoxification route for these compounds is via methylation since the thioether products are several orders of magnitude less toxic than the mercaptans themselves. The enzyme system responsible for this is the so-called alkane thiol S-methyl-transferase; the methyl donor is S-adenosylmethionine. In common with glucuronyltransferase, the methyltransferase is located in the endoplasmic reticulum and can only be extected from this organelle via detergent solubilisation. In this contribution, the method of isolation of alkane thiol S-methyltransferase is reported, and some of its properties are discussed. It is apparent from this work that this enzyme system would be extremely useful in any artificial liver support system involving enzymes.