I. Trautschold

Lymphatic transport of cellular enzymes from muscle into the intravascular compartment.

In an experimental study, employing anaesthetized dogs, it was investigated whether cellular enzymes from peripheral skeletal muscle get into the circulating blood by diffusion across capillary membranes or by lymphatic transport. In the experimental group 1, the animals were anaesthetized only. The plasma activities of the four enzymes measured--lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, creatine kinase--did not show any mentionable change during a time period of 6 h. In group 2 one hind limb of each animal was moved passively for 1 h. Alanine aminotransferase remained unchanged in plasma, the activities of the three other enzymes increased significantly. In group 3 one hind limb was made hypoxic by clamping the femoral blood vessels for 1 h. No activity changes were observed. When the period of hypoxia was followed by a 1-hour period of passive movement in group 4, the alterations in plasma activities were almost identical to those observed in group 2. In group 5 the experimental procedure was as in group 4, in addition the lymph from the thoracic duct was quantitatively withdrawn. The enzyme activities in plasma revealed a tendency to decrease rather than increase. Lymph flow increased significantly as well as the lymphatic activities of those enzymes which have high intracellular activities in muscle. The results prove, that enzymes from muscle are transported from the interstitial into the intravascular compartment mainly by lymphatic transport. Indications were found that the interruption of blood flow in one hind limb did not result in an enzyme release from muscle cells. It is discussed how changes in lymph flow, occurring during physical exercise for example, affect enzyme activities in plasma.

Catalytic Enzyme Activity Concentration in Tissues of Man, Dog, Rabbit, Guinea Pig, Rat and Mouse. Approach to a Quantitative Diagnostic Enzymology, III. Communication

The catalytic activity of up to fifteen enzymes was investigated in the liver, heart, skeletal muscle, kidney (medulla, cortex), brain, lung, duodenum, spleen and pancreas from man and animals. Human specimens were obtained from autopsies and immediately post-mortem from dogs, rabbits, guinea pigs, rats and mice. The differences between our results and previous reports of considerably lower activities for structural enzymes (e.g. creatine kinase) and for enzymes partly of mitochondrial origin (e.g. glutamate dehydrogenase, aspartate aminotransferase, malate dehydrogenase), is attributed to our use of a detergent extraction technique. The superiority of the detergent technique with regard to enzyme yield is exemplified by a comparison of various methods of extraction in rat liver, heart and skeletal muscle. Use of standardized assays allows a qualitative inter-species comparison of results. The influence of autolysis on catalytic activity of human autopsies is considered of minor importance.

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.

A quantitative analysis of glucagon binding to isolated intact neonatal and adult rat hepatocytes on the basis of two different binding models

The interaction of glucagon with specific receptors has been studied in isolated intact neonatal and adult rat hepatocytes. The hormone binding measured directly with 125I-labelled glucagon was saturable and reversible. The 125I-labelled glucagon binding was inhibited by unlabelled homologous hormone at concentrations ranging from 0.5 nM to 50 microM. Two different binding models were assumed to analyse the binding data by a nonlinear least-squares procedure: (I) a single class of independent sites and (II) two classes of independent sites. The comparison of the fitted theoretical curves reveals that both binding models are in fact compatible with these data. Adult hepatocytes have a considerably higher affinity for glucagon than neonatal hepatocytes; the binding capacity of neonatal liver cells from 1-7-days-old rats proved to be markedly reduced compared with the cells from adult rats. The glucagon-induced intracellular cyclic AMP production was measured at various hormone concentrations under conditions identical to those for the determination of extracellular hormone binding. The correlation of both parameters indicates a direct connection between receptor-occupancy and adenylate cyclase stimulation of both parameters indicates a direct connection between receptor-occupancy and adenylate cyclase stimulation. These results suggest that a decreased receptor concentration in neonatal hepatocytes is responsible for the decreased cyclic AMP production.

High-performance liquid chromatography of iodine-labeled insulin and glucagon derivatives with on-line γ-detection

Insulin and glucagon were labeled with iodine. The reaction products were analyzed by high-performance liquid chromatography. It is shown that the pH of the reaction medium has a large effect on the position and the degree of iodine substitution as well as on the oxidation of the Met-containing glucagon and, furthermore, that the molar ratio of iodine to polypeptide hormone used during the labeling procedure affects not only the amount of iodine incorporated but also the distribution of iodinated products. The results show that certain iodinated derivatives are separated from each other and from the respective unlabeled polypeptide and thus can be obtained in a pure state.

Enzyme activities in blood cells of man and dogs after separation on a discontinuous Percoll gradient.

From human and dog peripheral blood, thrombocytes (TRC), mononuclear cells (MNC), polymorphonuclear cells (PMNC), and red blood cells (RBC), were harvested after separation on a discontinuous Percoll gradient in a two-step centrifugation procedure, and 12 enzyme activities were determined in these highly purified cells. The enzyme activities measured are generally severalfold higher than previously reported, a fact which is attributed to the gentle and time-shortening isolation in Percoll, the cell disruption technique using detergent and the enzyme test conditions.

Catalytic Enzyme Activity Concentration in Thoracic Duct, Liver, and Intestinal Lymph of the Dog, the Rabbit, the Rat and the Mouse Approach to a Quantitative Diagnostic Enzymology, II. Communication

In the mixed body lymph of the thoracic duct and in the defined organ lymph of the liver and the intestine, the catalytic activity concentrations of up to sixteen enzymes and the concentrations of albumin and protein were determined, as well as the transport rate of these substances and their lymph/plasma ratio. Thoracic duct lymph specimens were obtained from an extracorporeal lymph shunt in anaesthetized and conscious dogs and from short-term fistulas in anaesthetized rabbits, rats and mice. Additionally, rabbits and rats underwent passive motion of the hind limbs in another experimental trial. Thoracic duct flow in anaesthetized dogs is only half that seen in conscious dogs, due to bypassed muscular lymph. A similar flow change is seen during passive motion of hind limbs in anaesthetized rabbits and rats. From a literature review of flow in the four main lymphatics of the body, it is concluded that the thoracic duct flow should account for 50-70% of total body lymph flow. In the anaesthetized state, flow is mainly of visceral origin. In the conscious state and during passive motion the increased flow is of muscular origin. In the latter case, the catalytic activities of enzymes like lactate dehydrogenase, malate dehydrogenase, creatine kinase, aldolase and phosphohexose isomerase, increase in lymph as does their lymph/plasma ratio. These enzymes have high catalytic activities in muscle. Their transport into the blood increases 2-3-fold, due to a doubling of lymph flow. Reported data for anaesthetized and immobile animals therefore far underestimate the significance of thoracic duct enzyme transport. Liver lymph was obtained from anaesthetized dogs and rabbits. Our finding that lymph catalytic activity for several enzymes is higher than in plasma is not compatible with the proposed delivery of plasma proteins directly into the sinusoidal space without prior mixing with the Space of Disse. Enzymes in liver lymph should derive from parenchymal and endothelial lining cells. Their site of delivery from the hepatocyte seems different from that of proteins. Liver lymph is an important transport route of enzymes into the blood. Intestinal lymph was sampled from anaesthetized dogs, rabbits and rats. It was shown that most enzymes from the intestine are primarily released into the interstitial space and from there are transported via the lymph into the blood.

Enzyme activities in rabbit, guinea pig, rat and mouse blood cells after separation on a discontinuous Percoll gradient.

A technique is described which allows a fractionation of rabbit, guinea pig, rat and mouse blood cells using a discontinuous Percoll gradient. With only two steps of centrifugation a simultaneous isolation of thrombocytes, mononuclear cells, polymorphonuclear cells, and erythrocytes in nearly pure form is performed in a very short time, starting from only 1 ml of blood. In these morphologically almost homogeneous cell fractions the activities of 12 enzymes were determined. The enzyme pattern, which with respect to the number of enzymes, specific cell populations and species was not yet investigated to such an extent, revealed a manifold higher enzyme content in the few cases, in which comparative studies were made. This can be attributed mainly to the completeness of the cell disruption technique using a detergent and certainly also to the use of Percoll as a preferable gradient material.

Dichtegradienten-Zentrifugation menschlicher Blutplättchen. Enzymmuster und osmotische Resistenz einzelner Fraktionen

ZusammenfassungEs wird eine Methode zur Fraktionierung menschlicher Blutplättchen durch Zentrifugation auf einem mit Ficoll erstellten diskontinuierlichen Dichtegradienten beschrieben. Bei den gewählten Versuchsbedingungen verteilen sich normale Plättchen nahezu gleichmäßig auf die vier isolierten Fraktionen. In der Gesamtpopulation, die als Ausgangsmaterial für die Fraktionierung diente, sowie in den Einzelfraktionen wurden das Extraktprotein und die Aktivitäten folgender Enzyme bestimmt: Lactatdehydrogenase (LDH), Malatdehydrogenase (MDH), Glucose-6-Phosphatdehydrogenase (G-6-PDH), Aspartataminotransferase (GOT), Pyruvatkinase (PK) und Glucosephosphat-Isomerase (PGI). Sämtliche Enzymaktivitäten sind in den Einzelfraktionen höher als in der Gesamtpopulation. Innerhalb der Fraktionen nahmen die Aktivitäten der LDH, G-6-PDH, PK und PGI von der spezifisch schwersten Fraktion zu der leichtesten hin um 27 bis 48% ab. Da die Alterung von Plättchen in vitro mit einer Verschiebung des Verteilungsmusters in Richtung auf die spezifisch leichten Fraktionen verbunden ist, wird die Abnahme der Enzymaktivitäten auf die physiologische Alterung der Plättchen zurückgeführt. Die Aktivitäten der bilokulären Enzyme MDH und GOT sind in den Einzelfraktionen nahezu gleich hoch. Die Konzentration an Extraktprotein nimmt von der spezifisch schwersten Fraktion zur leichtesten hin um ca. 80% ab. Die Abnahme der Dichte geht parallel mit einer Abnahme der osmotischen Resistenz der Zellen aus den Einzelfraktionen.SummaryHuman blood platelets were separated into four fractions of different specific gravities by centrifugation in Ficoll density gradients. The total populations and the different fractions were examined on their content of proteins and the activities of the following enzymes: Lactatedehydrogenase (LDH), Malatedehydrogenase (MDH), Glucose-6-phosphatedehydrogenase (G-6-PDH), Aspartateaminotransferase (GOT), Pyruvatekinase (PK), Phosphoglucoseisomerase (PGI). Enzyme activities in the different fractions are constantly higher than in the total population. The activities of LDH, G-6-PDH, PK and PGI show a decrease by 27 to 48% from the heavy to the light fractions. This decrease is attributed to the ageing of platelets in vivo because there is evidence that the heavy fractions consist of young platelets, whereas the light fractions consist of older cells. The concentration of extractable proteins decreases by 80% from the heavy to the light fractions. The heavy young cells are more resistent to osmotic shock than the light old cells.

Analysis of iodine-labelled glucagon derivatives by high-performance liquid chromatography

Abstract A reversed-phase high-performance liquid chromatographic system is presented for the separation of iodinated glucagon from the corresponding unlabelled and oxidized hormone, obtained by iodination of glucagon using the hydrogen perioxide—lactoperoxidase or the chloramine-T method. As the iodinated derivatives are well separated from the iodinated and oxidized as well as from the unlabelled glucagon molecules, this separation technique has been used both for optimizing the iodination procedures and for the rapid, efficient purification of mono- and diiodinated glucagon derivatives.