Chromogenic endotoxin assay in plasma. Selection of plasma pretreatment and production of standard curves

The aim of this study was to define the optimal conditions for the plasma pretreatment and to improve the production of standard curves for plasma endotoxin determination by a chromogenic substrate assay. Endotoxin standard from E. coli O 111:B 4 (0-50 ng/l) was added to pyrogen-free water or to plasma samples from 12 healthy subjects and 24 alcoholics, before pretreatment by heating (75 degrees C, 5 minutes) or with perchloric acid (0.32 mol/l). When endotoxin standard curves were determined using a microprocessor-controlled reader, the slopes of the curves obtained with plasma differed from those with pyrogen-free water. The slope of the standard curve prepared with plasma samples from different patients exhibited marked interindividual variations. Compared with the heating method, the perchloric acid method gave more variable results and a lower recovery of added endotoxin, especially in plasma from alcoholics. The results permit the following conclusion: 1. For plasma endotoxin determination, a standard curve should be prepared for each individual plasma sample. 2. The endotoxin standard should be added before pretreatment of the plasma. 3. Pretreatment of the plasma by heating at 75 degrees C for 5 minutes provides more reliable results than pretreatment with perchloric acid.     

Benzalkonium interference with test methods for potassium and sodium

Six automated instruments that measure sodium and potassium were tested for interference from two compounds used in catheters. Tridodecylmethylammonium heparin did not interfere with any of the methods. However, benzalkonium heparin falsely increased sodium measurement with the Kodak Ektachem, and falsely increased potassium measurements with three instruments (Beckman Astra, Baxter Paramax, and the Instrumentation Laboratory Monarch) in which ion-selective electrodes measure potassium in diluted serum. Three instruments in which ion-selective electrodes measure serum directly--Du Pont Dimension, Abbott Spectrum, and Kodak Ektachem--experienced no interference with potassium measurements. Interference of benzalkonium with potassium measurements may result from its interaction with the electrode membranes, which is accentuated in diluted serum.     

Chromogenic assay for functional determination of TFP

Due to the manifold functions of tissue factor (TF), also tissue factor pathway inhibitor (TFPI), its endogenous antagonist, is of interest. For the determination of TFPI, either its antigen concentration or its activity can be measured. METHOD: A chromogenic assay for the functional determination of TFPI is presented and modified for its application in the routine laboratory. Its suitability for daily use was evaluated by testing more than 7000 plasma samples from a heparin study in healthy volunteers. In addition, our data confirm that the method records the activity of both free- and lipoprotein-associated TFPI. CONCLUSION: The chromogenic assay proved to be rapid and easy to perform and features a high reproducibility. Due to excellent correlation of the results with the total TFPI antigen concentrations, the method represents a more rapid and economic alternative to the determination of total TFPI antigen by ELISA in plasma samples after heparin application.     

Evaluation of an automated spectrophotometric assay for reactive oxygen metabolites in serum.

The in vivo assessment of free radicals concentration is hampered by their instability and extremely short half-life. The Diacron Reactive Oxygen Metabolites (D-ROM) test is a recently introduced method to evaluate the peroxidation of organic compounds. Since the manual performance of the test provides excessive analytical imprecision, the aim of this study was to evaluate the automation of this test. Within- and between-run imprecision and interference were assessed according to the guidelines proposed by the NCCLS. The reactive oxygen metabolites' (ROM) stability was evaluated in different physical conditions. For within-run and between-run imprecision the coefficients of variation were consistently lower than 5%. The maximum allowable concentration was 28.2 mmol/l, 0.068 mmol/l and 171 mmol/l for triglycerides, haemoglobin and bilirubin, respectively. Serum storage at -20 degrees C provided adequate ROM stability for up to 3 months, whereas storage at 4 degrees C yielded non-reproducible results. In conclusion, our data provide evidence that the D-ROM assay has both an acceptable stability and an adequate imprecision. The automated assay may be regarded as a fast and reproducible method for the quantitative evaluation of oxidative stress. Since it is easily performed, the method is suitable for routine in clinical laboratories and may provide an accurate estimation of oxidative stress in vivo.     

An enzymatic assay for available zinc in plasma and serum

A convenient enzymatic assay for available zinc in plasma and serum was developed. The assay is based upon the reactivation of the zinc metalloenzyme alkaline phosphatase which had been previously inactivated by nitrilotriacetic acid. The enzymatic assay was applied to serum samples from human subjects in a study of zinc utilization in pregnancy and to plasma samples from rats in a zinc deficiency study. Most plasma samples with low zinc levels (less than 70 μg/dl) reactivated the enzyme to less than 30% of control activity. Most samples with normal zinc levels (70–120 μg/dl) reactivated the enzyme 30–65%. Samples with elevated zinc levels (> 120 μg/dl) reactivated the enzyme over 70%. The assay is quick, convenient, and in principle measures functionally available zinc.     

Chromogenic methods in coagulation diagnostics

Chromogenic peptide substrates were developed more than 30 years ago. Although the use of chromogenic substrate methods in coagulation and fibrinolysis diagnostics was not as rapidly implemented as initially believed, they are now well established for several analytes such as antithrombin, FVIII, protein C, plasminogen, plasmin inhibitor, heparins, and pentasaccharides. The advent of direct thrombin and factor Xa inhibitors has stimulated the development of new, specific chromogenic methods and these may find their way into routine use if these new drug candidates will prove to be valid replacements for coumarin derivatives. A large number of chromogenic research methods for other analytes were developed, too. The current interest in global chromogenic methods for thrombin generation and the protein C pathway may turn out as clinically important and thus enter into routine use.     

Neutral ionophore-based selective electrode for assaying the activity of magnesium in undiluted blood serum

The concentration of free, ionized magnesium in undiluted blood serum can be determined potentiometrically with a new magnesium-selective carrier in polymeric membrane electrodes (by weight 66% plasticizer, approximately 33% polyvinyl chloride and approximately 1% ionophore). However, discrimination of calcium by the new ionophore-added membrane is not sufficient to keep calcium interference to less than 1%. To correct for the interference by calcium, we determine the calcium activity of the serum sample with a calcium-selective electrode and calibrate the magnesium-selective electrode with standard solutions containing calcium in the concentrations found. In this way we determined the free ionized magnesium in five plasma samples from five different persons and found it to be within the range expected for magnesium in undiluted serum.     

An enzymatic assay for lysophosphatidylcholine concentration in human serum and plasma

OBJECTIVES: Several methods for measuring lysophosphatidylcholine (LPC) concentrations have been reported. However, these methods are not practical because they are either too complicated and/or too time-consuming for LPC determinations in human serum and plasma. DESIGN AND METHODS: We have developed a new enzymatic LPC assay, which uses lysophospholipase, glycerophosphorylcholine phosphodiesterase and choline oxidase, and which determines the quantities of hydrogen peroxide generated in the presence of peroxidase using an oxidative chromogenic reagent and 4-aminoantipyrine. RESULTS: Various samples were mixed with LPC assay reagents, and their changes in absorbance were measured. The present method produced a linear calibration line between LPC concentration and absorbance change. It also measured only LPC, and not other phospholipids such as phosphatidylcholine, sphingomyelin and lysophosphatidic acid. The within-run and between-run coefficients of variation were 0.3-0.7% and 0.7%, respectively. The recovery of exogenous LPC added to control serum was 99.5-102.1%. The correlation coefficient obtained in a comparison with a method for analyzing fatty acids was 0.9122. CONCLUSIONS: The present method is simple, specific for LPC, and can be applied with an automatic analyzer. It may also be useful for further studies of the biological functions of LPC as well as clinical applications in various disorders.     

酶法测定血清钾、钠的干扰分析

目的对酶法测定血清钾、钠的部分干扰因素进行分析.方法配制含不同浓度胆红素及甘油三酯的血清标本,分别上机测定钾、钠浓度.控制试剂的不同复溶时间对标本钾钠重复进行测定.结果总胆红素浓度在135.8μmol/L以上时测得的钠显著降低(P＜0.05).胆红素浓度在267.5μmol/L组才使钾结果降低(P＜0.05).各脂浊标本组与基础组比较,甘油三酯浓度在8.21mmol/L以下对钾、钠的测定影响较小(P＞0.05).当天复溶测得的结果比前一天的变异大(当天:钾CV:11.25%,钠CV:8.57%,前一天:钾3.17%钠1.69%).结论对胆红素超过263.5umol/L时钾,131.8umol/L时钠和/或严重脂浊标本的钾、钠测定改用离子选择电极法.