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.     

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.     

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 preanalytical stage of under measuring of concentration of catalytic activity of enzymes: the characteristics and tasks of standardization

The article explains the importance of standardization, the development and maintenance of rules and recommendations regulating the conditions and order of implementation of particular parts of preanalytical stage. The order of these conditions is noted including the rules stated and published in such normative documents as national standards GOST R 15 189-2009, GOST R 53079.4-2008, GOST R ISO 6710-209 and in the recommendations of foreign National societies ofclini-cal chemistry and laboratory medicine. These requirements concern all the analytes, enzymes included. The cited data have a practical significance for acquisition of reliable results in everyday functioning of laboratories. Enough to mention data concerning the anticoagulants influence on catalytic concentration of enzymes, the most often measured concentrations of alpha-amylase, lipase, amynotransferase, alkaline and acid phosphatase, creatine kinase, lactate dehydrogenase, choline esterase, hemolysis impact, the increased concentration of bilirubin and hyperlipemia in samples and significance of measurement of indices of serum and plasma as well. The possible mechanisms of impact of these interferents on the results of measurement of catalytic concentration of enzymes are discussed.     

Comparative activities of clavulanic acid, sulbactam, and tazobactam against clinically important beta-lactamases.

Clavulanic acid, sulbactam, and tazobactam are inhibitors of a variety of plasmid-mediated beta-lactamases. However, inhibition data for these three inhibitors with a wide range of different plasmid-mediated beta-lactamases have not yet been compared under the same experimental conditions. A number of groups have inferred that clavulanic acid inhibits extended-spectrum TEM and SHV beta-lactamases, but inhibition data have rarely been published. In this study, the 50% inhibitory concentrations of these three beta-lactamase inhibitors for 35 plasmid-mediated beta-lactamases have been determined. Of these 35 beta-lactamases, 20 were extended-spectrum TEM- or SHV-derived beta-lactamases. The other 15 enzymes were conventional-spectrum beta-lactamases such as TEM-1 and SHV-1. Clavulanic acid was a more potent inhibitor than sulbactam for 32 of the 35 plasmid-mediated beta-lactamases tested. In particular, clavulanic acid was 60 and 580 times more potent than sulbactam against TEM-1 and SHV-1, respectively, currently the two most clinically prevalent gram-negative plasmid-mediated beta-lactamases. Statistical analysis of the data of the 50% inhibitory concentrations showed that clavulanic acid was 20 times more active overall than sulbactam against the conventional-spectrum enzymes. In addition, clavulanic acid was 14 times more potent than sulbactam at inhibiting the extended-spectrum enzymes. Tazobactam also showed significantly greater activity than sulbactam against the two groups of beta-lactamases. There were no significant differences between the overall activities of tazobactam and clavulanic acid against the extended-spectrum TEM and SHV enzymes and conventional-spectrum enzymes, although differences in their inhibition profiles were observed.     

Some glycolytic enzymes in normal cerebrospinal fluid, brain tissue and blood plasma of infants.

The activities of several glycolytic enzymes (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase) as well as glycerol-1-phosphate dehydrogenase and (Mg2+)ATPase in normal cerebrospinal fluid (CSF) and blood plasma samples, from 12 healthy infants, aged 2-18 months, and in supernatants from brain tissue slices, taken during neurosurgical operations from infants of the same range of age were estimated. The values obtained confirm the high activity of the above enzymes found in animal brains, and indicate an independence of these activities in blood plasma and CSF. The origin of the activities of the investigated enzymes in CSF seems to be mainly, if not, exclusively, from brain tissue. This might be useful for detection of brain tissue damage as was earlier proven with LDH activity in CSF.     

The importance of glutathione in human disease.

Reduced glutathione (GSH) is the most prevalent non-protein thiol in animal cells. Its de novo and salvage synthesis serves to maintain a reduced cellular environment and the tripeptide is a co-factor for many cytoplasmic enzymes and may also act as an important post-translational modification in a number of cellular proteins. The cysteine thiol acts as a nucleophile in reactions with both exogenous and endogenous electrophilic species. As a consequence, reactive oxygen species (ROS) are frequently targeted by GSH in both spontaneous and catalytic reactions. Since ROS have defined roles in cell signaling events as well as in human disease pathologies, an imbalance in expression of GSH and associated enzymes has been implicated in a variety of circumstances. Cause and effect links between GSH metabolism and diseases such as cancer, neurodegenerative diseases, cystic fibrosis (CF), HIV, and aging have been shown. Polymorphic expression of enzymes involved in GSH homeostasis influences susceptibility and progression of these conditions. This review provides an overview of the biological importance of GSH at the level of the cell and organism.     

The intensity of processes of lipoperoxidation and antioxidant defense in patients with recurrent viral herpes infection

The article discusses some parameters of free radical metabolism of lymphocytes of blood plasma in 65 patients with recurrent virus herpes infection in course of disease. It is established that in patients occurs increasing of both of level of malonic dialdehyde and functional activity of neutrophils in blood plasma. On the background of these alterations, compensatory increase of activity of such antioxidant enzymes as ceruloplasmin and catalase is noted. The intensity of stationary derangement of free radical oxidation and antioxidant defense under recurrent virus herpes infection depended on the period of disease and severity of pathologic process.     

alpha 2-Macroglobulin in serum and plasma: reference values with carbobenzoxy-valyl-glycyl-arginine-p-nitroanilide, a chromogenic substrate

alpha 2-Macroglobulin can be determined using the amidolytic activity of the alpha 2-macroglobulin-trypsin-complex. Reference values are reported using a commercially available kit for continuous assay at 25 degrees C. Reference ranges obtained in a population of 235 children and adults aged between 1 day and 61 years were found to be age-dependent. Reference ranges established for the different age groups of children were grouped together in order to obtain useful guide values. The following values were obtained for serum: 1 to 10 days: 5-11 kU/l 11 days to 18 years: 8-16 kU/l The reference values (5th to 95th percentile) determined for juveniles and adults were as follows: 19 to 25 years: 4.1-12.2 kU/l (serum); 4.0-11.2 kU/l (citrated plasma) greater than or equal to 25 to 61 years: 4.1-8.1 kU/l (serum); 3.7-7.4 kU/l (citrated plasma) No variation according to sex was observed, nor was any difference noted between preprandial and postprandial catalytic concentrations, or between values in smokers and non-smokers or between values in the group of women taking hormonal contraceptives and the group not taking such drugs. If the dilution of the blood sample with citrate solution is taken into account when calculating the catalytic concentrations in citrated plasma, the values in plasma are higher than those in serum (median 11%). Measurement of the alpha 2-macroglobulin-trypsin complex in plasma is assumed to give a more realistic reflection of the in vivo activity than measurement in serum. Comparison with two immunological methods (radial immunodiffusion and kinetic nephelometry) revealed that there is a linear relationship between the measurement of the catalytic concentration of the alpha 2-macroglobulin-trypsin complex and the immunological concentration of alpha 2-macroglobulin. Therefore mutual conversion is possible for the described concentration range.     

六种临床诊断酶学测定参考方法的应用及国际比对结果分析

目的 应用国际临床化学联合会(IFCC)公布的参考方法对血清丙氨酸氨基转移酶(ALT)、(天)门冬氨酸氨基转移酶(AST)、乳酸脱氢酶(LDH)、肌酸激酶(CK)、γ-谷氨酰基转移酶(GGT)及淀粉酶(AMY)6个酶进行测定,并通过参加参考实验室国际比对计划评估参考方法的准确性.方法 按照IFCC有关酶学活性测定(37℃)参考方法的标准操作程序(SOP),分别使用PE和Agilent的两套测定系统进行6个酶的测定.通过测定质控血清监测两套测定系统的稳定状态、有证参考物(CRM)并参加酶学国际比对(ring trial),验证参考方法的准确性.结果 两套系统测定室内质控血清的测值稳定,6种酶天间测定变异系数为0.5%～1.9%;测定结果一致,两者结果偏移小于2.1%;CRM测定结果在允许的范围内,初步验证了参考方法的准确性;高低两个浓度样本,两个独立系统的国际比对结果中有4个酶(ALT、AST、GGT、AMY)的测定值位于所有参考实验室测定的(x)±s范围内,LDH与CK测值位于(x)±2s之间;用稳健统计学方法评价,除了LDH的样本A测值属于离散值以外,其他5个酶ALT、AST、CK、GGT、AMY及LDH样本B的比对结果未发现离群.结论 应用IFCC参考方法采用两套测定系统对6个酶进行的测定结果稳定、准确,具有等效性.     

Glutathione peroxidase activity, lipid peroxides and selenium status in blood in patients with Down's syndrome

The concentrations of selenium and lipid peroxides and the catalytic activity of glutathione peroxidase were measured in the blood of 6 children (6-16 years of age) and 8 adults (17-27 years old) with Down's syndrome (trisomy 21). The values were compared with those for a control group of age-matched normal people. The selenium concentration in whole blood, erythrocytes and plasma was significantly lower in trisomy 21 patients than in normal subjects (p less than 0.001) in both age groups. No statistically significant differences were observed in selenium concentration in whole blood, erythrocytes and plasma between children and adults in the Down's syndrome group. Glutathione peroxidase catalytic activity in erythrocytes was significantly higher in Down's syndrome children than in healthy children (p less than 0.001). Plasma glutathione peroxidase catalytic activity in both investigated age groups was statistically considerably lower in the Down's syndrome patient group. The concentration of lipid peroxides, expressed as the malondialdehyde concentration, is lower in Down's syndrome patients. No correlation between selenium concentration, glutathione peroxidase catalytic activity and amount of lipid peroxides was found in the trisomy 21 patient group.     

Metabolism of intravenously administered dipeptides in rats: effects on amino acid pools, glucose concentration and insulin and glucagon secretion.

1. Studies were performed to investigate the metabolic fate of dipeptides when administered intravenously in rats. Glycyl-leucine, glycylglycine or glycylsarcosine was injected into the jugular vein. The plasma disappearance rate after the peak plasma concentrations was most rapid for glycyl-leucine and least rapid for glycylsarcosine. 2. During urine collection for 40 min, trace amounts of glycyl-leucine and glycylglycine and 13% of the injected glycylsarcosine were excreted. 3. Neither glycylglycine nor glycyl-leucine was detected in the liver, muscle, intestinal mucosa or renal cortex, but concentrations of glycine or leucine, or both, in these tissues were increased after each injection. In contrast, glycylsarcosine was recovered in all these tissues with concentrations in the renal cortex being far greater than in any other tissue, but sarcosine was found only in the renal cortex and intestinal mucosa. 4. The changes in plasma concentrations of free amino acids, glucose and glucagon, and tissue concentrations of free amino acids, were similar after the intravenous administration of glycyl-leucine and an equimolar mixture of free glycine and leucine. However, the amount of insulin secreted during the 40 min after glycyl-leucine injection was 1-6 times that produced after the injection of the corresponding amino acid mixture. 5. Results show that, within the present experimental conditions, the intravenous administration of dipeptides is as effective as that of the corresponding free amino acids in enriching the tissue pools of amino acids. It is suggested that efficient hydrolysis by cellular enzymes prohibits accumulation of intact dipeptides in body tissues.     

Optimization of methods for aspartate aminotransferase and alanine aminotransferase.

Conditions for accurate measurement of catalytic activity of aspartate aminotransferase and alanine aminotransferase in human serum have been reinvestigated. The basic variables (kind of buffer, buffer concentration, pH, ion effects, and the influence of pyridoxal-5-phosphate) can now be considered optimized. On this basis, the kinetic parameters of both aminotransferases were determined, i.e., Michaelis and inhibitor constants for substrates and reaction products. With a mathematical approach for two-substrate enzyme reactions the substrate concentrations were calculated from the viewpoints "most economical," "most convenient," and "lowest variability." Also the conditions for the indicator reactions have been newly defined with respect to a kinetic model. All calculated data were rechecked experimentally and it can be shown that both approaches fully agree. Furthermore, we show that the mathematical approach allows more precise recommendations for optimized methods. For technical reasons, the catalytic activity of aspartate aminotransferase in human serum can only be measured as a 0.96 fraction of its theoretical maximum velocity, the catalytic activity of alanine aminotransferase as a 0.91 fraction. The assay conditions for a Reference Method are finally described and recommendations are made for optimized routine methods for determination of the catalytic activity of these transferases in human serum.     

The inhibition of glutathione S-transferases: mechanisms, toxic consequences and therapeutic benefits.

Inhibition of the enzymes belonging to the family of glutathione S-transferases is important from several points of view. These involve applications in studies of the catalytic mechanism, e.g. studying the topology and binding characteristics of the active site. Also, from a therapeutic standpoint, inhibition of glutathione S-transferases steadily becomes more interesting, since these enzymes appear to be involved in drug resistance, and in the biosynthesis of a number of important arachidonic acid metabolites such as prostaglandins and leukotrienes. Modulation of the glutathione S-transferase activity could be used to regulate the concentrations of these compounds, Thirdly, unwanted inhibition by xenobiotics makes a cell more vulnerable for alkylating agents and can thus have toxic consequences. This review describes the state of the art, dealing with the various types of inhibiton employed (reversible, irreversible or nonsubstrate ligands). Furthermore, isoenzyme selectivity, organ distribution and interindividual differences are discussed.     

"Lysosomal" enzyme activities in red blood cells of normal individuals and patients with homozygous beta-thalassaemia.

Four hydrolases, beta-galactosidase, beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase were examined in red blood cells (RBC) of normal donors and patients with homozygous beta-thalassaemia. Highly sensitive fluorimetric substrates were used to determine the specific activities of these enzymes. In order to avoid contamination by lysosomal activities derived from white blood cells (WBC), the mature RBV were separated from other blood elements by cellulose chromatography. The hydrolase activities in normal RBC were detected only in their plasma membranes and were found to be considerably lower than in WBC or platelets. In thalassaemic RBC, hydrolase activities were present in both plasma membranes and in the soluble fraction. The normoblast fraction contributed most of the hydrolase activity found in these preparations, suggesting the presence of lysosomal particles in thalassaemic RBC. No differences in the enzymatic activities were found when purified membranes of mature RBC from thalassemic and normal preparations were compared. The origin and roles of these hydrolytic enzymes in normal and thalassaemic RBC membranes are not known.     

Pathogenic factors involved in renovascular hypertension. State of the art.

The complex hormonal action of angiotensin II in the long-term control of blood pressure or sodium metabolism, or in renal hypertension, is not completely understood. Structure-activity relations with analogues of angiotensin II gave information about the functions responsible for pressor and myotropic response in the molecule that led to the synthesis of competitive antagonists of this hormone. These antagonists, however, show variable agonist/antagonist ratios in different species or different tissues of the same species. This fact necessitates further work to induce tissue specificity. Although des-Asp1-angiotensin II ("angiotensin III") has been recognized as a hormone, its exact role in the biosynthesis of aldosterone is yet to be discovered. The antagonists such as des-Asp1-[Ile8]-angiotensin II or des-Asp1-[Thr8]-angiotensin II have provided important leads in this direction. Many of the biologic effects of angiotensin I have been attributed to its conversion to angiotensin II by the converting enzyme. Recent investigations indicate that angiotensin I itself may play a direct role; however, most of these studies were carried out by inhibiting the converting enzyme activity with peptides obtained from the venom of Bothrops jararaca. Since these peptides also potentiate bradykinin action, the observed biologic activities could be caused by either angiotensin I or bradykinin. Bsides, converting enzyme is no longer thought to be a single enzyme and its nature varies from species to species and from tissue to tissue in the same species. Renin inhibitors related to renin substrate or pepstatin are not freely soluble in plasma and are not effective under physiologic conditions. This points to the importance of renin inhibitors isolated from kidney or other natural sources. Thus, although the renin-angiotensin system appears to be an integral part of the problem of hypertension, characterization of various converting enzymes, roles of extrarenal renin, isorenin, tonin, and brain-renin, and the involvement of other humoral, neurogenic, and immunogenic factors should be pieced together to get a clear picture of the hypertension problem.     

Cardioprotection against ischaemia/reperfusion by vitamins C and E plus n-3 fatty acids: molecular mechanisms and potential clinical applications

The role of oxidative stress in ischaemic heart disease has been thoroughly investigated in humans. Increased levels of ROS (reactive oxygen species) and RNS (reactive nitrogen species) have been demonstrated during ischaemia and post-ischaemic reperfusion in humans. Depending on their concentrations, these reactive species can act either as benevolent molecules that promote cell survival (at low-to-moderate concentrations) or can induce irreversible cellular damage and death (at high concentrations). Although high ROS levels can induce NF-κB (nuclear factor κB) activation, inflammation, apoptosis or necrosis, low-to-moderate levels can enhance the antioxidant response, via Nrf2 (nuclear factor-erythroid 2-related factor 2) activation. However, a clear definition of these concentration thresholds remains to be established. Although a number of experimental studies have demonstrated that oxidative stress plays a major role in heart ischaemia/reperfusion pathophysiology, controlled clinical trials have failed to prove the efficacy of antioxidants in acute or long-term treatments of ischaemic heart disease. Oral doses of vitamin C are not sufficient to promote ROS scavenging and only down-regulate their production via NADPH oxidase, a biological effect shared by vitamin E to abrogate oxidative stress. However, infusion of vitamin C at doses high enough to achieve plasma levels of 10?mmol/l should prevent superoxide production and the pathophysiological cascade of deleterious heart effects. In turn, n-3 PUFA (polyunsaturated fatty acid) exposure leads to enhanced activity of antioxidant enzymes. In the present review, we present evidence to support the molecular basis for a novel pharmacological strategy using these antioxidant vitamins plus n-3 PUFAs for cardioprotection in clinical settings, such as post-operative atrial fibrillation, percutaneous coronary intervention following acute myocardial infarction and other events that are associated with ischaemia/reperfusion.