Clinical Enzymology Research Articles

"CARBGAME" (CARd & Board GAmes in Medical Education) as an innovative gamification tool for learning clinical enzymology in biochemistry for first year medical students.

Gamification is emerging as an active learning innovation in medical education to enhance student engagement and promote life-long learning in a unique and collaborative environment. Clinical enzymology in biochemistry is one of the core topics in the medical curriculum. However, students face challenges in comprehension and retention of information. Hence, CARd & Board GAmes in Medical Education (CARBGAME)was introduced and evaluated for its effectiveness in enhancing learning, application, and retention of knowledge in clinical enzymology via gamification context. This mixed-method study involved 150 first-year undergraduate medical students. Before the game, students completed a pre-test in clinical enzymology. Later they were divided into 25 small groups to compete in the board game designed for enzymology in biochemistry. The students took turns throwing the dice and answering the questions on the game board to continue moving forward. The first team to reach 100 and solve the case-based question was deemed the winner. Following the board game, the students took up the post-test to compare the educational impact of the innovation. Also, the subsequent internal assessment scores were compared with previous batch who were not implemented with this intervention. Then students evaluated the effectiveness of CARBGAME-Clinical Enzymology using a 32-item questionnaire on 5-point Likert scale. The feedback obtained on a 10-point rating scale and for qualitative analysis, students' and faculty perceptions were recorded in small groups. CARBGAME received overwhelmingly positive feedback from both students and faculty. It was perceived well by students for being fun, relevant, consistent, motivating, collaborative, and promoting experiential learning. The game's low-stakes approach, effective feedback, and sense of accomplishment were highly appreciated, making it a valuable tool for education. A significant improvement in knowledge was recorded, from a mean score of 8.37 ± 1.126 on a 20-point scoring scale before the game to 16.53 ± 1.219 after with a p-value of 0.0001. The comparison of the internal assessment scores between the intervention and non-intervention group of students also showed a significant improvement among those implemented with CARBGAME (p < 0.0001). The CARBGAME innovation has achieved the intended outcome of promoting active learning and enhanced performance in clinical enzymology. Highly positive responses from faculty and students also indicate the exigent need to introduce innovative components like games into curricula to achieve student engagement and promote a meaningful learning experience.

Clinical enzymology of the dog and cat.

Clinical enzymology studies the enzyme activity in serum or other body fluids for the diagnosis, prognosis or monitoring of a variety of diseases. Clinical enzymology has greatly benefited from advances in technology and is now an integral part of laboratory analysis. However, to maximise the clinical benefits of serum enzyme measurement, clinicians and clinical pathologists must have a good understanding of the pathophysiology behind serum enzyme alterations. They must also be aware of the preanalytical and analytical factors that can affect the accuracy of serum enzyme activity measurement. This review article first covers the basic concepts of clinical enzymology and the general mechanisms related to serum enzyme alterations. Then, the review discusses the potential effects of various preanalytical and analytical factorson enzyme activity measurement. Lastly, it explores the pathophysiology and clinical use of various serum enzymes in canine and feline medicine. The present review article aims to be a comprehensive one-stop source for clinical pathologists and small animal practitioners.

Progress and impact of enzyme measurement standardization.

International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has established reference measurement procedures (RMPs) for the most popular enzymes. Manufacturers should assign values to commercial calibrators traceable to these RMPs to achieve equivalent results in clinical samples, independent of reagent kits, instruments, and laboratory where the measurement is carried out. The situation is, however, far from acceptable. Some manufacturers continue to market assays giving results that are not traceable to internationally accepted RMPs. Meanwhile, end-users often do not abandon assays with demonstrated insufficient quality. Of the enzyme measurements, creatine kinase (CK) is satisfactorily standardized and a substantial improvement in performance of marketed γ-glutamyltranspeptidase (GGT) assays has been demonstrated. Conversely, aminotransferase measurements often exceed the desirable analytical performance because of the lack of pyridoxal-5-phosphate addition in the commercial reagents. Measurements of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and α-amylase (AMY) still show major disagreement, suggesting the need for improvement in implementing traceability to higher-order references. This is mainly the result of using assays with different analytical selectivities for these enzymes. The definition by laboratory professionals of the clinically acceptable measurement uncertainty for each enzyme together with the adoption by EQAS of commutable materials and use of an evaluation approach based on trueness represent the way forward for reaching standardization in clinical enzymology.

Photoluminescent Enzymatic Sensor Based on Nanoporous Anodic Alumina

Herein, we present a smart enzymatic sensor based on nanoporous anodic alumina (NAA) and its photoluminescence (PL) in the UV-visible range. The as-produced structure of NAA is functionalized and activated in order to perform the enzyme immobilization in a controlled manner. The whole process is monitored through the PL spectrum and each stage is characterized by an exclusive barcode, which is associated with the PL oscillations. This characteristic property allows us to calculate the change in the effective optical thickness that takes place after each stage. This makes it possible to accurately detect and quantify the immobilized enzyme within the NAA structure. Finally, the NAA geometry (i.e., the pore length and its diameter) is optimized to improve the enzyme immobilization and its detection inside the pores. This enzymatic sensor can give quick and accurate measurements of enzyme levels, what is crucial in clinical enzymology to prevent and detect diseases at their primary stage.

Standardization in clinical enzymology: a challenge for the theory of metrological traceability

The goal of standardization for measurement of the catalytic concentration of enzymes is to achieve comparable results in human samples, independent of the reagent kits, instruments, and laboratory where the assay is performed. To pursue this objective, the IFCC has established reference systems for the most important clinical enzymes. These systems are based on the following requirements: a) reference methods, well described and evaluated extensively; b) suitable reference materials; and c) reference laboratories operating in a highly controlled manner. When these reference systems are used appropriately, the diagnostic industry can assign traceable values to commercial calibrators. Clinical laboratories that use procedures with validated calibrators to measure human specimens can now obtain values that are traceable to higher-order reference procedures. These reference systems constitute the structure of the traceability chain to which the routine methods can be linked via an appropriate calibration process, provided that they have a comparable specificity (i.e., they are measuring the same catalytic quantity).

Real-time mass spectrometry in enzymology

IntroductionResearch on enzymatic (catalysis) reactions is of largeinterest since it is known that many proteins have abiological function. Thus the word “enzymology” encom-passes the branch of biochemistry dealing with the chemicalnature and biological activity of enzymes [1], and thusresearch on their function and activity, and on inhibition oftheir reactions. In recent times the word has been used in avariety of other research fields, for example clinical(medical) enzymology, cryoenzymology, engineering enzy-mology, micellar enzymology, metal enzymology, soil en-zymology, and others [2]. Since “functional proteomics”became a very productive research field [3–5], enzymologyhas found new popularity, especially due to advances inmass spectrometric detection.In the last twenty years atmospheric pressure ionization–mass spectrometry (API–MS) has achieved acceptance as animportant analytical tool in biochemical research. Theeffective coupling of liquid chromatography with MS laidthefoundationforasensitive,specific,andspeedytechniquestudyingmoleculesfromcomplexbiologicalsources.Thus,inthe last five years over a dozen ABC Trend articles haveappeared on mass spectrometry, including technical develop-ments and biological approaches [6–18]. In summary, all ofthese predict MS has a successful future as a detector.The current state of enzymatic real-time MSGenerally, functions of proteins, their activity and regula-tion can be studied in vivo and in vitro. In vitro assaysrequire enzymes which are active in an artificial environ-ment and which can be isolated, expressed, or synthesizedin manageable amounts. Although in vitro assays often lackphysiological conditions, the results obtained give rudi-mentary information about proteins and their catalyticpotential. Furthermore, in contrast with in vivo assays,single enzymes can be handled and regulated more easily.A typical enzymatic in vitro assay consists of:1. starting the reaction by mixing the reaction compounds;2. taking aliquots after different reaction times;3. interrupting the reaction in each aliquot;4. separating the reaction compounds; and5. detecting the compounds for quantification.Classical methods use spectroscopic and/or radioisotopicdetection for step 5. However, disadvantages of bothtechniques are the need for substance labeling and, after-wards, very difficult identity differentiation between theindividual non-labeled reaction components. With massspectrometry (API–MS) the labels become redundant andseveral compound ions can be identified. This also enablesresearch on enzymatic reactions involving various types ofmolecule (e.g. hydrophobic substrates, semi-polar inter-mediates, and/or polar products).From the very first day, API–MS was applied in hyphen-ation with liquid chromatography (LC) for compound quanti-ficationandidentification.Gener ally,thisprinciplewasutilizedon the basis of LC–UV–visible and LC–fluorescence. Todaythis method is mainly used for protein identification viapeptides obtained from proteases like trypsin [4, 5] but, never-

Clinical enzymology: An autobiographical history

In this paper, I review the origins of Clinical Enzymology with special emphasis on the years between 1960 and 1980 when the greatest advances in the subject took place. These are described in relation to my own research interests and contributions, focusing upon subclinical hepatic damage caused by viral infection and by alcohol; myocardial infarction; detection of cervical carcinoma by vaginal fluid analysis; evaluation of pancreatic function; and the clinical implications of microsomal enzyme induction. Reasons are proposed for the failure of enzymology to attain the status of an autonomous medical specialty, in contrast to the success of similar fields of knowledge that grew up in the same era.

Enzimologia Clínica Veterinária. Parte I: fatores que interferem na interpretação dos resultados

A enzimologia clínica veterinária tem assumido um importante papel nos tempos atuais, principalmente no que se refere ao auxílio diagnóstico de diversas enfermidades. Este trabalho teve por objetivo apresentar uma revisão sobre os principais fatores que interferem na determinação das enzimas no soro e no plasma e que podem acarretar uma interpretação inadequada dos resultados

Mass spectrometry in coupling with affinity capture–release and isotope‐coded affinity tags for quantitative protein analysis

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) and isotope-coded affinity tags (ICAT) are two recently introduced techniques for the quantitation of protein activity and content with applications to clinical enzymology and functional proteomics, respectively. One common feature of these methods is that they use biotinylated tags that function as molecular handles for highly selective and reversible affinity capture of conjugates from complex biological mixtures such as cell homogenates and sub-cellular organelles. ACESIMS uses synthetic substrate conjugates specifically to target cellular enzymes that, when deficient, are the cause of genetic diseases. Multiplex determination of enzyme activities is used for the diagnosis of lysosomal storage diseases. The ICAT method relies on selective conjugation of cysteine thiol groups in proteins, followed by enzymatic digestion and quantitative analysis of peptide conjugates by mass spectrometry. Another common feature of the ACESIMS and ICAT approaches is that both use conjugates labeled with stable heavy isotopes as internal standards for quantitation. Selected applications of the ACESIMS and ICAT techniques are presented that include molecular-level diagnosis of genetic diseases in children and quantitative determination of protein expression in cells.

Direct profiling of multiple enzyme activities in human cell lysates by affinity chromatography/electrospray ionization mass spectrometry: application to clinical enzymology.

We describe a new method for enzyme analysis using affinity capture followed by electrospray ionization mass spectrometry (ACESIMS) for the quantitative determination of the initial velocities of four heparin-modifying enzymes. These enzymes, when defective in affected children, lead to the lysosomal storage disease known as Sanfilippo syndrome. The method relies on substrates and internal standards conjugated to the molecular handle biotin via a heavy isotope-encodable, mass-adjustable linker. Reaction velocities of the Sanfilippo enzymes in a crude lysate prepared from as little as 2500 human skin fibroblasts can be determined. In addition, the ACESIMS method is widely applicable to the simultaneous analysis of multiple enzymes in a complex biological sample by a single analytical technique and will thus serve as a useful tool in basic and clinical biomedical research.

Establishing a Reference System in Clinical Enzymology

The goal of standardization for measurements of catalytic concentrations of enzymes is to achieve comparable results in human samples, independent of the reagent kits, instruments and laboratory where the procedure is carried out. To pursue this objective, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has launched a project to establish a reference system in clinical enzymology. This system is based on three hinges: a) extensively evaluated and carefully described reference procedures, b) certified reference materials and c) a network of reference laboratories operating in a highly controlled manner. The original IFCC-recommended procedures for alanine aminotransferase, aspartate aminotransferase, creatine kinase, gamma-glutamyltransferase, lactate dehydrogenase and alpha-amylase have been slightly modified to optimize them at 37 degrees C, with the definition of detailed operating procedures. A group of laboratories perform these procedures manually, with self-made reagents on carefully calibrated instruments. Partially purified and stabilized materials, prepared in the past by the Community Bureau of Reference, have been re-certified by these laboratories for alanine aminotransferase, creatine kinase, gamma-glutamyltransferase and lactate dehydrogenase activities. Using these materials and the manufacturer's standing procedures, industry can assign traceable values to commercial calibrators. Thus, clinical laboratories, which will use routine procedures with these validated calibrators to measure human specimens, can finally obtain values which are traceable to reference procedures.

Tietz Textbook of Clinical Chemistry

Part I: Laboratory Principles. General Laboratory Techniques and Procedures. Specimen Collection and Processing, Sources of Biological Variation. Part II: Analytical Techniques and Instrumentation. Spectrophotometric Techniques. Fluorometry, Nephelometry, And Turbidimetry. Basic Principles of Radioactivity and Its Measurement. Electrochemistry. Electrophoresis. Chromatography/Mass Spectrometry. Principlesof Immunochemical Techniques. Automation in the Clinical Laboratory. Part III: Chemometrics. Statistical Procedures. Selection and Interpretation of Laboratory Procedures. Selection and Evaluation of Methods. Establishment and Use of Reference Values. Part IV: Laboratory Management. Clinical Laboratory Informatics. Laboratory Management. Quality Management. Part V: Analytes. Nucleic Acid Biochemistry and Diagnostic Applications. Amino Acids. Proteins. Cytokines. Clinical Enzymology. Tumour Markers. Carbohydrates. Lipids, Lipoproteins, And Apolipoproteins. Therapeutic Drug Monitoring. Clinical Toxicology. Toxic Metals. Vitamins. Trace Elements. Electrolytes and Blood Gases. Part VI: Pathophysiology. Physiology and Disorders of Wates, Electrolyte, And Acid-Base Metabolism. Liver Function. Cardiac Function. Renal Function and Nitrogen Metabolics. Gastric, Pancreatic, And Intestinal Function. Organ Transplantation. Nutritional Assessment, Therapy, And Monitoring. Mineral and Bone Metabolism. General Endocrine Function. Pituitary Function. Thyroid Function. Function of the Adrenal Cortex. Cathecol Amines and Serotonin. Reproductive Endocrine Function. Biochemical Aspects of Hematology. Porphyrins and Disorders of Porphyrin Metabolism. Clinical Chemistry of Pregnancy. Lysosomal Storage Disease. Appendix.

Preparation of enzyme calibration materials

Standardisation in clinical enzymology needs not only reference methods but also reference materials. While single-enzyme reference enzymes have been developed, a multienzyme certified reference material (MECRM) available in high amount remains to be produced. To transfer trueness from the value of the reference system to patients' results, validated enzyme calibrators (EC) are also needed. Both the MECRM and the ECs must exhibit the same catalytic properties as the corresponding enzymes in human plasma. Moreover, commutability of these materials with patients' samples must be experimentally tested for one or a set of methods defined by an analytical specificity equal to that of the reference method. Various experimental studies have shown that the commutability of an enzyme material depends on the source of enzyme and its purification process, the matrix (including cofactors, effectors, additives, stabilisers…) and the mode of processing of the final material. To promote intermethod calibration in clinical enzymology, a collaborative programme between the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), Institute for Reference Materials and Measurements (IRMM, Geel, Belgium) and IFCC corporate members is in progress for the development of a MECRM containing amylase, ALT, AST, ALP, CK, GGT, LDH, and lipase and exhibiting a wide and defined commutability.

Citation Classics in Lipid Measurement and Applications

In 1998 the AACC celebrates its 50th anniversary. Thus, we are honored to call attention to the outstanding professional contributions of the journal Clinical Chemistry over the years by pointing out the citation classics that have been published in its pages. By the end of 1995, 31 articles in Clinical Chemistry had been cited more than 300 times (1). Thirty of these articles were still being cited in 1995, and some are still cited to the present day. These 31 papers are distributed in multiple areas of biological sciences, and in particular: lipids, 8 papers (2)(3)(4)(5)(6)(7)(8)(9); endocrinology, 2 papers; clinical enzymology, 4 papers; intermediary metabolism, 6 papers; renal function, 2 papers; cancer, 1 paper; and calcium metabolism, 3 papers. Publications on methodology research and laboratory applications of lipid, lipoprotein, and apolipoprotein measurements in clinical investigations and epidemiologic trials have had a major impact on research of lipid metabolism and cardiovascular medicine (2)(3)(4)(5)(6)(7)(8)(9). The combined efforts of epidemiologists, clinicians, and laboratorians have produced classic publications on the understanding of lipid metabolic disorders, have stimulated development of new novel equipment and methodology, and have helped to establish the development of beneficial preventive and control measures for diseases of the heart and blood vessels. The cited lipid papers published in Clinical Chemistry included mainly the original papers on measurement of lipoproteins, enzymatic measurements of cholesterol and triglycerides, and immunoassays of apolipoproteins. The method used most widely today for estimation of the concentration of LDL-cholesterol (LDL-C) in both clinical and research laboratories arose from the publication of Friedewald et al. (2) that showed that the serum concentration of LDL-C can be estimated from available established measurements …

Interassay calibration as a major contribution to the comparability of results in clinical enzymology

Objective: Factors contributing to the applicability of interassay calibration of methods measuring enzyme catalytic activities are described. Also discussed are the properties essential for such a material. Similarity of specificity for the methods to be calibrated as well as commutability between the material(s) intended to be used as calibrator are the main criteria to be satisfied. Result: Several examples demonstrated that interassay calibration is feasible but a multi-enzyme calibrator with a wide commutability for the most popular methods remains to be developed. This is the project of the IFCC Working Group on Calibrators in Clinical Enzymology (WG-CCE). Several experimental data are also presented that indicate that the temperature at which the reaction is carried out is not a limiting factor in the implementation of interassay calibration in clinical enzymology.

Validation of an enzyme calibrator-an IFCC guideline

Objectives: The objective of this guideline is to improve standardization in clinical enzymology in order to improve intermethod comparability of patients’ results. Design and Methods: The reference system, combination of the reference method and the reference material, is used to produce a reference value for a given catalytic activity. Sets of methods are formed of methods exhibiting the same analytical specificity. Materials intended to be used as enzyme calibrators are experimentally checked for their commutability. Results: The transfer of accuracy from the reference value to patients’ results is dependent on methods (analytical specificity) and on materials (experimentally assessed commutability). The feasibility of this approach was demonstrated with materials of high level for several enzymes and for each of them for several routine methods. Conclusion: Expected advantages of this approach in clinical enzymology are presented.

They Use Enzymes for Everything!

This year marks the 50th anniversary of the American Association for Clinical Chemistry. In tribute to this semicentenary, this journal will reflect on its published achievements over the past five decades.1 Our Editor has recently enumerated some of the publications that have had an impact on the field, as demonstrated by a solid history of citation (1). This editorial, the first in a projected series of four to appear this year, will reflect on those papers that have advanced the area of enzymes and protein markers–now a regular feature in these pages. Of the papers that have achieved citation fame (1), approximately 20% deal with clinical enzymology (2)(3)(4)(5)(6)(7). Yet another 20% focus on the use of enzymes as analytical tools, e.g., those listed in references 8–10 . In the first volume of this journal (1954), enzyme measurements as markers of human disease were the focal points of only two papers. The topics were amylase and arginase. None characterized the use of enzymes as analytical reagents, reflecting clinical laboratory practice of the day. Today, in most laboratories, of the two dozen “routine” chemistry analytes, nearly two-thirds are often determined by enzymatic analyses. This is largely the result of the explosion in the number of studies of enzymes as catalysts, markers of disease, and tools for the laboratorian over the 1960s and the subsequent two decades. This growth phase in enzymology was concurrent with the growth of the practice of clinical chemistry and of the AACC. Without these advances, modern clinical laboratory analyses would not be possible. The detection of proteins in serum by their catalytic activity as a reporter of tissue damage is a cornerstone of medical laboratory analyses. The uses of a wide variety of enzymes from a still …

Importance of the definition of catalytic properties for the commutability of an enzyme reference material: example of lipase

Assays for pancreatic lipase activity in serum need the use of calibrators, but there is a lack of a reference method and of a reference material. Under routine conditions, comparability of lipase catalytic activities was found to be poor when lipase was determined in 50 patients’ specimens by a turbidimetric (Boehringer) and a colorimetric (Sigma) assay. Mean values of the results differed by a ratio of 2.39. Catalytic properties of three materials, two commercial calibrators and a home-purified preparation of human pancreatic lipase (HPL), have been compared in titrimetry at constant pH. Optimal common conditions were defined for the titration of lipase activity in the three materials. When using these titres for each calibrator, comparability was greatly improved (ratio = 1.25). This result indicates that a significant part of between-method discrepancy was due to the lack of a reference method for the titration of lipase calibrators. The inter-method behavior of each material was compared to that of patients’ specimens. Using HPL as calibrator, the comparability of patients’ results was still dramatically improved (ratio = 1.01). This study shows the importance of verifying the similarity of catalytic properties of an enzyme reference material to those of the corresponding enzyme in human specimens, in order to ensure its commutability. The use of validated commutable calibrators, traceable to certified reference materials, is an opportunity to improve the inter-method comparability of results in clinical enzymology.

JSCC Guidelines (1994) on Standardization in Clinical Enzymology

JSCC Guidelines (1994) on Standardization in Clinical Enzymology

Clinical enzymology for the assessment of organ damage: Interspecific differences

Enzyme activities in serum/plasma or urine are the most widely used markers of organ damage as well in human or animal clinics as in experimental toxicology. Significant interspecific differences can be observed for enzyme distribution in various species, extracellular metabolism, preanalytical and analytical conditions. Thus, to avoid any misinterpretation, all these criteria have to be documented and/or validated before use.