Kinetic of adjustment of enzyme catalytic concentrations in the extracellular space of the man, the dog and the rat. Approach to a quantitative diagnostic enzymology, V. Communication.

The high degree of constancy of enzyme catalytic activity in the plasma of a given individual is regulated by a complex system of flux equilibria consisting of eight basic processes. Some of these processes are of primarily theoretic importance. Enzymes from all tissues of the body, including the liver, are released via a continuous physiological process into the interstitial space and get into the intravascular space by way of lymphatic transport. The release of enzymes from tissues directly into the intravascular space is of secondary importance as is the exchange of enzyme molecules across capillary membranes from the intravascular to the interstitial space and vice versa. In contrast, enzymes from circulating blood cells are transported directly into the intravascular space. Enzymes are removed from the intravascular space at rates which vary greatly between both enzymes and species. In a review of the literature, half-lives of diagnostically important enzymes in plasma of man, dogs and rats were given and the striking differences in the results for a given enzyme are discussed from a methodological point of view. In a mathematical analysis, data for lymphatic transport of enzymes from dogs and rats (Lindena et al. (1986) this J. 24, 19-33) and of enzyme efflux from in vivo ageing erythrocytes (Lindena et al. (1986) this J. 24, 49-59) into the plasma are related to the elimination rate constants of enzymes from the plasma. The contribution of lymphatically transported enzymes to the basal catalytic activity in plasma (Lindena & Trautschold (1986) this J. 24, 11-18) amounts to 55-80% for lactate dehydrogenase and malate dehydrogenase, 80-90% for adenylate kinase and phosphohexose isomerase, 90-95% for aspartate aminotransferase and aldolase and 99% for creatine kinase. A model of Ca2+ -mediated vesicular transport of enzymes out of ageing erythrocytes is proposed. The importance of lymphatically transported enzymes to total plasma catalytic activity in dogs and rats argues for a similar contribution of lymph transport in man.

The decline of catalytic enzyme activity concentration of in vivo ageing erythrocytes of the man, the dog and the rat. Approach to a quantitative diagnostic enzymology, IV. Communication.

Human, dog and rat erythrocytes were separated by centrifugation on a discontinuous buffered Percoll gradient into fractions of progressively increasing mean cell age to measure the in vivo decline in catalytic activity of eleven enzymes during the erythrocyte lifespan. Erythrocyte enzymes decline exponentially at different rates and also differ between the species. The maximal and minimal catalytic activities (erythrocyte catalytic activity at the beginning and at the end of the appropriate erythrocyte life-span for a given species) and the intracellular half-life of enzymes estimated. To test the hypothesis that circulating erythrocytes make a significant contribution to the normal catalytic activity in plasma it was assumed as a working hypothesis that the measured loss of catalytic activity in ageing erythrocytes is equivalent to the amount of the enzymes released in catalytically active form into plasma. This contribution was calculated.

Catalytic enzyme activity concentration in plasma of man, sheep, dog, cat, rabbit, guinea pig, rat and mouse. Approach to a quantitative diagnostic enzymology, I. Communication.

For the most convenient laboratory animals in experimental medicine such as the sheep, dog, cat, rabbit, guinea pig, rat and mouse, the catalytic concentrations of seventeen enzymes and the protein and albumin concentrations in plasma were determined. The corresponding data for man were taken from the literature. The experimental conditions were selected so as to minimize the influence on catalytic activity, variance and statistical distribution pattern of such factors as handling procedures, the choice of blood drawing technique, the choice of anaesthetic agent for distinct species and the preparation procedure of plasma from the blood specimen. Because these considerations have been largely neglected to date in experimental enzymology, normal values for most enzymes require revision and were in some instances established for the first time in certain species. Our use of assay conditions, which were identical for all animal species, satisfies current requirements in clinical chemistry with respect to standardization and optimization of enzyme catalytic activity concentration measurement, and allowed comparison of these enzymes. The catalytic activities established this way will be the basis for a later comparative study of their kinetics of adjustment in plasma.

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 release from the perfused rat heart. The functions of the cytoskeleton under cell-pathological conditions.

The mechanism of enzyme release from Langendorff-perfused rat hearts was studied under the injury conditions of the Ca2+ paradox and 2,4-dinitrophenol poisoning. During perfusion with Krebs-Ringer buffer or in buffered sucrose sarcoplasmic enzymes were massively released when Ca2+ was reintroduced to the perfusion medium (Ca2+ paradox). Mitochondrial matrix enzymes were released to a very small extent. Only the cytoplasmic isoenzyme of the bilocular enzyme malate dehydrogenase was released. The release kinetics of various enzymes with greatly differing molecular weights showed no significant differences. Qualitatively the same results were obtained under 2,4-dinitrophenol poisoned conditions in Ca2+ -free sucrose media. Sarcoplasmic enzymes were massively released, mitochondrial enzymes did not appear in the perfusate. 2,4-Dinitrophenol poisoning alone was not sufficient to cause enzyme release. An additional swelling under these conditions was necessary. ATP from the extracellular space was able to enhance the enzyme release, which was brought about by 2,4-dinitrophenol and cell swelling. A hypothesis is presented that enzyme release is produced by initiating a membrane blebbing process. An elevated intracellular Ca2+ concentration is a necessary prerequisite. In the presence of ATP, active membrane blebbing is caused by contractions of the membrane-anchored cytoskeleton. In the absence of ATP passive membrane blebbing is induced by cell swelling, provided that the cytoskeleton has been crosslinked by Ca2+.

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.

High-performance liquid chromatography of iodine-labeled insulin and glucagon derivatives with on-line gamma-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.

Biological, analytical and experimental components of variance in a long-term study of plasma constituents in rat.

Statistical analysis of variance was applied to data from determinations of 14 plasma constituents in 25 rats in order to evaluate the analytical, experimental and biological (inter-and intraindividual) component of variance. Blood was taken seven times in intervals of 8-10 days, the last one by catheter technique and the other by heart puncture. The analytical portion of variance was determined by the concurrent analysis of a pool plasma standard. The experimental component of variance was evaluated by the comparison of the variation of the catheter values with that of the pooled data from heart puncture. The coefficient of variation for the latter may be grouped into three categories: less than 10% for protein, Na+, K+, Ca2+; 10-20% for urea, phosphate and the enzymes as alanine aminotransferase, choline esterase, alkaline phosphatase and leucine arylamidase and 20-65% for the other enzymes lactate dehydrogenase, malate dehydrogenase, aspartate aminotransferase and creatine kinase. The results from the samples taken by catheter technique generally revealed the lower values for the mean as well as for the variance. It became evident that the procedure of heart puncture is afflicted with the most aggravating interference factors, thus accounting for most of the experimental component of variance. The observed differences between the single blood drawings, the non-Gaussian distribution for several constituents, and the interactions between the components of variance do not always fit for the statistical concept of additivity of the single components.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme activities in thoracic duct lymph and plasma of anaesthetized, conscious resting and exercising dogs.

The significance of changes in lymph flow for the extracellular distribution and transport of cellular enzymes and for the level of enzyme activities in plasma was investigated. Specimens of thoracic duct lymph were obtained from an extracorporal lymph shunt in anaesthetized, conscious resting and treadmill exercising dogs (6 km X h-1 for 1 h) The activity of 10 enzymes and of protein content in lymph and plasma were studied, as well as lymph flow, lymphatic transport, and the lymph-plasma ratio of these compounds. Lactate, pH, and blood gases were monitored in venous blood. Lymph flow of 0.80 ml X min-1 in anaesthetized dogs more than doubled (to 1.86 ml X min-1) when the animals were conscious and resting. In anaesthetized dogs lymph enzyme activity was higher only for enzymes of predominately hepatic origin, such as choline esterase (CHE) and alanine aminoferase (ALAT), and was lower for aspartate aminotransferase (ASAT) and aldolase (ALD). In conscious dogs, due to activation of the skeletal muscle "tissue pump", lymphatic transport of enzymes with rather high activity in skeletal muscle, and of protein, is significantly enhanced. Enzyme activities in plasma, however, did not differ between the groups. Lymph-plasma activity ratios higher than one were found for lactate dehydrogenase (LDH), malate dehydrogenase (MDH), ASAT, creatine kinase (CK), ALD, and phosphohexose isomerase (PHI). Exercise stimulated lymph flow up to 4.9 ml X min-1, and increased the lymphatic activities of those enzymes with a lymph-plasma ratio higher than unity, these enzymes increasing in the plasma due to the highly increased lymphatic transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Ductus thoracicus lymph in mice. 1. A technique for cervical approach.

A method is described for the collection of lymph from the thoracic duct in anaesthetized mice by a silastic catheter inserted into the duct proximal to the jugulo-subclavian junction. The operative procedure takes about 5 min, and there is less operative trauma to the animals than by the previously used abdominal approach. Lymph flow amounts to 0.396 ml/h and cell content to 38.6 X 10(6)/ml.

Ductus thoracicus lymph in mice. 2. Enzyme activities and protein content.

Lymph was collected in anaesthetized mice by a newly developed cervical approach (see preceding paper). Short term drainage, which should not alter the colloid-osmotic forces, meets the physiological requirements for determination of protein content and enzyme activities in lymph and plasma. For cellular enzymes such as creatine kinase, lactate dehydrogenase, and aspartate aminotransferase, lymph plasma ratios higher than 1 are reported. Besides the well known lymphatic transport route for protein and albumin, the highly significant lymphatic transport of cellular enzymes to the actual intravasal enzyme activity is emphasized.

[Effect of total hormone concentration on the determination of amount of binding in the hormone/receptor interaction between erythrocytes and insulin]. / Zum Einfluss der totalen Hormonkonzentration auf die Ermittlung von Bindungskenngrössen bei der Hormon/Receptor-Wechselwirkung zwischen Erythrocyten und Insulin.

The interaction of erythrocytes and [125I]insulin/insulin were studied up to a total insulin concentration of 409 mumol/l. Assuming a single class receptor model the evaluation of receptor affinity Ka and concentration R0 may be performed either by non-linear regression analyses with iteration procedures of R0, Ka and U (nonspecific binding), or by a linear regression analysis of the initial part of the Scatchard plot. Nonlinear fitting of data to a two class receptor model gives results that are reliable only for the high affinity receptor site. The non-definable step of R0 determination leads to uncertainties in results determined by the negative cooperativity model. From the results of this investigation and from considerations of signal modulation by receptor occupancy, some recommendations have been formulated for the evaluation of binding parameters; these should contribute to an improvement in the comparability of studies on the interactions of erythrocytes and insulin.

Enzymes in lymph: a review.

Lymph is the minute net volume of contending hydrostatic and osmotic capillary forces. It is built up extravasculary in tissues and reaches the intravasal space by definite lymph collecting vessels, which enter the venous system at the angulus venosus at the root of the neck. Sampling methods for lymph from individual tissues or from lymph collecting vessels of man and various animals are cited and the preparation of lymph for enzymatic analysis is described. The extracellular distribution and transport of enzymes is important for diagnostic enzymology, because enzymes released from cells in continuous physiological processes, or after injury to the tissue, reach the intravasal space mainly via the lymphatics. This is evident from the high lymph-plasma ratios of diagnostically important enzymes. The type of enzyme transport (lymphatic or by direct interstitial-venous entry) depends on the heterogeneity of the capillary barrier characteristic of the different organs. The permeability is extremely high in liver, i.e. enzymes in hepatic lymph originate mainly from blood, which they have reached through the large openings in the sinusoidal endothelial lining; in contrast the permeability is extremely low in skeletal muscle, where lymphatic transport therefore predominates. The phenomenon of increased enzyme activities in plasma after physical exertion is explained by alterations of lymph flow. A table gives a survey of enzyme activities, lymph-plasma quotients, and lymph flow from lymph vessels of various tissues as well as from the lymph collecting vessels of man and animals, with comments on the signficance for diagnostic enzymology.

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.

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.

Insulin binding to erythrocytes after acute 16-methyleneprednisolone ingestion.

The binding of [125I]insulin to erythrocytes, glucose and insulin were determined before and 1, 7 and 35 days after ingestion of 2 X 60-methyleneprednisolone. None of two groups of volunteers (7 males, 4 females showed clear alterations of the insulin binding parameters (Ka and R0), or of the fasting cortisol, glucose and insulin concentrations. These results exclude the possibility that the diabetogenic effect of glucocorticoides is accompanied by an alteration of the insulin receptor characteristics of erythrocytes.

Degradation of glucagon receptors by dispase treatment of isolated intact rat hepatocytes.

Collagenase-isolated rat hepatocytes were treated with dispase II, a neutral proteolytic enzyme which is often used for the disintegration of neonatal cells. The treatment of hepatocytes with dispase II caused a significant reduction of glucagon binding to the intact cells. The deleterious effect of this enzyme on the specific glucagon binding site is accompanied by a reduction of the maximum intracellular cyclic AMP production.

Insulin binding to erythrocytes from pregnant, postpartum, follicular and luteal states.

Specific binding of [125I] insulin to isolate erythrocytes from four groups of women was investigated: (A) pregnant subjects between weeks 38 and 40 of pregnancy (n = 18), (B) postpartum subjects within 6 days after delivery (n = 20), (C) normal women during the follicular phase of the menstrual cycle (n = 12) and (D) normal women during the luteal phase of the menstrual cycle (N = 11). Specific [125I] insulin binding (fraction), fasting plasma glucose concentrations (mmol/l) and the corresponding insulin concentrations (mU/l) were 0.074 +/- 0.012 / 4.00 +/- 0.58 / 29.4 +/- 21.4 for group A, 0.065 +/- 0.016 / 4.40 +/- 0.75 / 41.5 +/- 26.2 for group B, 0.052 +/- 0.008 / 4.58 +/- 0.62 / 6.7 +/- 4.0 for group C and 0.054 +/- 0.011 / 4.49 +/- 0.63 / 8.3 +/- 5.9 for group D. By using a modified Scatchard analysis, statistically significant differences were observed between the receptor affinities of the groups A and D, B and D, A and C. The receptor affinities and concentrations were not significantly different between the follicular and the luteal phases. From the data, no inverse correlation between the plasma insulin concentration and receptor binding was seen, i.e. the phenomenon of downregulation of insulin receptor concentration with hyperinsulinaemia seemed not to apply to erythrocytes.

Effect of transient hypoxia in skeletal muscle on enzyme activities in lymph and plasma.

The effect of hypoxia lasting one hour on the hind leg muscle of anaesthetised dogs was investigated. Ten enzyme activities in plasma and leg lymph, and the lymphatic transport of these enzymes were investigated. Enzymes with high activity in muscle, like creatine kinase, lactate dehydrogenase, malate dehydrogenase and adenylate kinase only show an increase in the plasma, if lymph--propulsed by passive motion of the hind leg--can reach the intravascular space. This effect is independent of transient hypoxia. Depending on the level of enzyme activity in the muscle, the activity in leg lymph is up to 6-fold higher than in plasma. Enzymes from muscle have to be transported into the blood by lymph flow and not via a direct interstitial-venous entry. The results are discussed especially with respect to enzyme activity changes in plasma during physical exercise.

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.