Application of the CEN/ISO Standard for Phytase Activity Measurements to a New Phytase Product: Determination of a Robust Conversion Factor by an Interlaboratory Study for Screening Feed Samples.

Background: Phytase-based preparations are important feed additives currently authorised in the European Union (EU). The European Standard (EN) and International Organization for Standardization (ISO) standard 30024 describes a harmonized method for the determination of phytase activity and is fit-for-purpose for official control of a group of phytase products. However, it is not suitable for the determination of the phytase activity of a new feed additive encoded as 4a16 in the EU Register of Feed Additives, to which a slightly different phytase activity definition has been attributed. Objective: To establish a robust conversion factor to support official control laboratories that apply the EN ISO method when monitoring feed products containing 4a16. Methods: The phytase activity of test materials was determined by the participants using the EN ISO and/or the "applicant" methods. Results: Robust relative SDs for repeatability and for reproducibility of the methods applied for the determination of the phytase activity in the materials containing the 4a16 feed additive ranged from 2.6 to 22% (EN ISO method) and from 2.4 to 39% (applicant method). Conclusions: The data obtained confirmed the performance characteristics published for other phytase-based feeds in the related standard methods. These results allowed us to estimate a factor of 2.68 to convert phytase activities measured with the EN ISO method into the enzyme activity measured with the applicant method. Highlights: The obtained conversion factor will allow EU official laboratories to screen feed samples supplemented with the 4a16 phytase by applying EN ISO Standard 30024.

Practical application of the sigma-metric run size nomogram for multistage bracketed statistical quality control analysis of eight enzymes.

BACKGROUND: A sigma-metric run size nomogram is used to recommend quality control (QC) strategies to reduce patient risks. Herein, we aimed to evaluate the sigma performance of 8 enzymes and apply multistage bracketed statistical QC (SQC). METHODS: Sigma performance of alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatine kinase (CK), amylase (AMY), and lipase (LIP) were determined. Daily workload of each test was estimated and expected reporting QC intervals were designed. Per the nomogram, "start-up" and "monitor" QC rules were determined from sigma performance. SQC was finally applied, followed by quality improvement. RESULTS: Sigma metrics were as follows: 5.26 (ALT), 4.80(AST), 5.25(GGT), 3.36(ALP), 4.71(LDH), 15.45(CK), 10.77(AMY), and 3.70 (LIP). "Start-up" rules were MR N2, MR N4, MR N2, MR N4, MR N4, 1:2.5 s N1, 1:3 s N1, and MR N4, and "monitor" QC rules were 1:2.5 s N1, 1:3 s N2, 1:2.5 s N1, MR N4, 1:3 s N2, 1:3 s N1, 1:3 s N1, MR N2 for 8 enzymes, respectively. CONCLUSION: Multistage bracketed SQC is determined by sigma performance. Risk monitoring is significant during assaying to reduce patient risks and improve quality.

Phase-plane geometries in coupled enzyme assays.

The determination of a substrate or enzyme activity by coupling one enzymatic reaction with another easily detectable (indicator) reaction is a common practice in the biochemical sciences. Usually, the kinetics of enzyme reactions is simplified with singular perturbation analysis to derive rate or time course expressions valid under the quasi-steady-state and reactant stationary state assumptions. In this paper, the dynamical behavior of coupled enzyme catalyzed reaction mechanisms is studied by analysis of the phase-plane. We analyze two types of time-dependent slow manifolds - Sisyphus and Laelaps manifolds - that occur in the asymptotically autonomous vector fields that arise from enzyme coupled reactions. Projection onto slow manifolds yields various reduced models, and we present a geometric interpretation of the slow/fast dynamics that occur in the phase-planes of these reactions.

Enthalpy-entropy compensation and the isokinetic temperature in enzyme catalysis.

Enthalpy-entropy compensation supposes that differences in activation enthalpy ΔH++ for different reactions (or, typically in biochemistry, the same reaction catalysed by enzymes obtained from different species) may be compensated for by differences in activation entropy ΔS++. At the isokinetic temperature the compensation is exact, so that all samples have the same activity. These ideas have been controversial for several decades, but examples are still frequently reported as evidence of a real phenomenon, nearly all of the reports ignoring or discounting the possibility of a statistical artefact. Even for measurements in pure chemistry artefacts occur often, and they are almost inescapable in enzyme kinetics and other fields that involve biological macromolecules, on account of limited stability and the fact that kinetic equations are normally valid only over a restricted range of temperature. Here I review the current status and correct an error in a recent book chapter.

Incidence, laboratory detection and prognostic relevance of hypoxic hepatitis in cardiogenic shock.

BACKGROUND: Despite the improvement of therapeutic options for patients in acute myocardial infarction (AMI), cardiogenic shock (CS) remains a complication with high mortality rates. Organ failure centrally determines the prognosis of these high-risk patients. Aim of the current study was to assess the incidence of hypoxic hepatitis (HH) in CS, its laboratory detection evaluating novel and established biomarkers and to estimate the prognostic relevance of HH in current clinical practice. METHODS: In 172 patients with CS complicating AMI, blood samples were collected at admission and after 1 day as prespecified subanalysis of the intra-aortic balloon pumping IABP-SHOCK II trial. Classic parameters of HH were measured in addition to argininosuccinate synthase 1 and sulfotransferase isoform SULT2A1 was determined as new biomarker using standard enzyme-linked immunosorbent assay kits. All-cause mortality at 30 days was used for outcome assessment. RESULTS: The overall mortality rate was 40%. The incidence of HH with an increase of aminotransferase levels to be 20 times above the upper normal level was 18%. Patients with HH had a distinctly higher 30-day mortality rate compared to patients without HH (68 vs. 34%; p < 0.001). After multivariable adjustment aspartate-aminotransferase (ASAT) remained an independent predictor of 30-day mortality together with serum lactate and serum creatinine, while the new biomarkers failed to predict outcome. Comparing different liver markers using receiver operating characteristic analysis, ASAT showed the highest area under the curve for the prediction of outcome. CONCLUSIONS: HH occurs frequently in CS and is associated with particular poor outcome. As conventional biomarker, ASAT is the strongest laboratory predictor of outcome. ClinicalTrials.gov Identifier: NCT00491036.

Haemoglobin variants may cause significant differences in haemoglobin A1c as measured by high-performance liquid chromatography and enzymatic methods in diabetic patients: a cross-sectional study.

Background We aimed to determine whether the discrepancy between haemoglobin A1c values determined by high-performance liquid chromatography and enzymatic haemoglobin A1c measurements in diabetic patients was clinically relevant. Methods We randomly recruited 1421 outpatients undergoing diabetic treatment and follow-up who underwent at least three haemoglobin A1c measurements between April 2014 and March 2015 at our clinic. In 6369 samples, haemoglobin A1c was simultaneously measured by HA-8160 and MetaboLead (enzymatic assay), and the values were compared. Results haemoglobin A1c measurements by high-performance liquid chromatography and enzymatic assay were strongly correlated (correlation coefficient: 0.9828, linear approximation curve y = 0.9986x - 0.2507). Mean haemoglobin A1c (6.8 ± 1.0%) measured by high-performance liquid chromatography was significantly higher than that measured by enzymatic assay (6.5 ± 1.0%, P < 0.0001). During the sample processing, four (0.3%) subjects presented consistently lower haemoglobin A1c values (<0.7%) by high-performance liquid chromatography than those from enzymatic assay. Of these, three had Hb Toranomon [ß112 (G14) Cys→Trp]. The fourth had Hb Ube-2 [α68 (E17) Asn→Asp]. One other subject presented consistently higher haemoglobin A1c values (>1%) by high-performance liquid chromatography than those from enzymatic assay and was diagnosed with a -77 (T > C) mutation in the δ-globin gene. These unrelated asymptomatic subjects had normal erythrocyte profiles, without anaemia. Conclusions We showed that haemoglobin A1c values measured by high-performance liquid chromatography were significantly higher than those measured by enzymatic assay in diabetic subjects. However, when an oversized deviation (>0.7%) between glycaemic control status and haemoglobin A1c is apparent, clinicians should check the methods used to measure haemoglobin A1c and consider the possible presence of a haemoglobin variant.

Post-standardization of routine creatinine assays: are they suitable for clinical applications.

Introduction Reliable serum creatinine measurements are of vital importance for the correct classification of chronic kidney disease and early identification of kidney injury. The National Kidney Disease Education Programme working group and other groups have defined clinically acceptable analytical limits for creatinine methods. The aim of this study was to re-evaluate the performance of routine creatinine methods in the light of these defined limits so as to assess their suitability for clinical practice. Method In collaboration with the Dutch External Quality Assurance scheme, six frozen commutable samples, with a creatinine concentration ranging from 80 to 239 µmol/L and traceable to isotope dilution mass spectrometry, were circulated to 91 laboratories in four European countries for creatinine measurement and estimated glomerular filtration rate calculation. Two out of the six samples were spiked with glucose to give high and low final concentrations of glucose. Results Results from 89 laboratories were analysed for bias, imprecision (%CV) for each creatinine assay and total error for estimated glomerular filtration rate. The participating laboratories used analytical instruments from four manufacturers; Abbott, Beckman, Roche and Siemens. All enzymatic methods in this study complied with the National Kidney Disease Education Programme working group recommended limits of bias of 5% above a creatinine concentration of 100 µmol/L. They also did not show any evidence of interference from glucose. In addition, they also showed compliance with the clinically recommended %CV of ≤4% across the analytical range. In contrast, the Jaffe methods showed variable performance with regard to the interference of glucose and unsatisfactory bias and precision. Conclusion Jaffe-based creatinine methods still exhibit considerable analytical variability in terms of bias, imprecision and lack of specificity, and this variability brings into question their clinical utility. We believe that clinical laboratories and manufacturers should work together to phase out the use of relatively non-specific Jaffe methods and replace them with more specific methods that are enzyme based.

Biological variation of thiopurine methyltransferase enzyme activity: when has a significant change taken place?

BACKGROUND: Thiopurine methyltransferase (TPMT) enzyme activity is measured before initiating thiopurine therapy to reduce the risk of severe drug-associated myelotoxicity in patients with low enzyme activity. TPMT activity may vary over time in relation to drug treatment and patient clinical condition. What constitutes a significant change in TPMT activity can be derived from biological variation and analytical imprecision. METHODS: A large national laboratory database was used to identify patients with three or more TPMT activity measurements. Variance of TPMT activity was analysed by determining the total coefficient of variation (CVTOT) of repeated measurements and by correlation with parameters including gender and follow-up time. Between-run analytical imprecision (CVa) was determined by replicate analysis (n = 314). RESULTS: Of 7383 patients with TPMT measurements, 136 were identified as having three or more measurements over time (range 3-14). Median CVTOT for individual patient results was 14.5% (range 2.5-36.7%). Analytical imprecision (CVa) was 10.3%. A reference change value (or critical difference) with 95% probability was calculated as 42%. Therefore, a change in measured TPMT activity above 42% should lead to considering sources of variation other than biological variation and analytical imprecision. CONCLUSIONS: TPMT enzyme activity needs to change by at least 42% to determine that a true change has taken place beyond biological variation and analytical imprecision. A single measurement of TPMT activity is sufficient for most clinical purposes.

Comparison between IDMS-traceable Jaffe and enzymatic creatinine assays for estimation of glomerular filtration rate by the CKD-EPI equation in healthy and diabetic subjects.

OBJECTIVES: The aim of this paper was to compare the agreement between creatinine measured by Jaffe and enzymatic methods and their putative influence on eGFR as calculated by the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equation in healthy and diabetic individuals. DESIGN AND METHODS: Cross-sectional study conducted in 123 adult southern Brazilians with GFR>60 mL/min/1.73 m² (53 patients with type 2 diabetes, 70 healthy volunteers). Mean age was 49±16 years (range of 19-86). Most were female (55%) and white (83%). Creatinine was measured by a traceable Jaffe method (Modular P, Roche Diagnostic) and by an enzymatic method (CREA plus, Roche/Hitachi 917). GFR was measured by the 5¹Cr-EDTA single-injection method. RESULTS: Serum creatinine measured by the Jaffe and enzymatic methods was similar in healthy subjects (0.79±0.16 vs. 0.79±0.15 mg/dL, respectively, P=0.76), and diabetic patients (0.96±0.22 vs. 0.92±0.29 mg/dL, respectively, P=0.17). However, the correlation between the two methods was higher in the healthy group (r=0.90 vs. 0.76, P<0.001). The difference between Jaffe creatinine and enzymatic creatinine was <10% in 63% of cases in the healthy group and 40% of cases in the diabetes group (P=0.018). In the subset of patients with diabetes, eGFR based on enzymatic assay results showed better agreement with measured GFR than did eGFR based on Jaffe results. CONCLUSION: Jaffe and enzymatic creatinine methods show adequate agreement in healthy subjects, but in the presence of diabetes, the enzymatic method performed slightly better.

What have we learnt about high-density lipoprotein cholesterol measurements during 32 years? Experiences in Finland 1980-2012.

BACKGROUND: High-density lipoprotein cholesterol (HDL-C) is important in risk assessment for cardiovascular disease or metabolic syndrome; however, different direct HDL-C assays may lead to erroneous risk estimates and potentially misclassify people. METHODS: Data for 30-year HDL-C trends in Finland were obtained from the national FINRISK surveys during 1982-2012 (n=45766) taking into account biases from three external quality assessment programs (EQA). We also compared two different direct HDL-C and turbidimetric apolipoprotein A-I methods using 413 fresh serum samples. RESULTS: HDL-C concentrations in the Finnish population were on average 1.33 (±0.04) mmol/l for men and 1.62 (±0.05) mmol/l for women after bias-correction. Positive HDL-C trends were observed for both sexes with original data, but trends disappeared after bias-correction. Comparison of two direct HDL-C methods demonstrated concentration-dependent difference. When HDL-C concentrations were <1.0 mmol/l, the mean bias was -12.0% (95% CI -13.5 to -10.0) whereas HDL-C concentrations >1.55 mmol/l showed mean bias of 9.0% (95% CI 7.0-10.5). CONCLUSIONS: Accurate reporting of HDL-C concentrations at the population level requires proper and regular attendance to reliable EQA programs. We found evidence for a concentration-dependent difference between some direct HDL-C methods, which may cause misclassification of people in cardiovascular risk assessment.