Comparability of a new turbidimetric digoxin test with other immunochemical tests and with HPLC--a multicenter evaluation.

A new turbidimetric inhibition immunoassay for digoxin (Tina-quant [a] Digoxin, Boehringer Mannheim) was evaluated in seven laboratories. It can be performed without sample pretreatment with ready-to-use reagents on nondedicated analyzers in combination with routine clinical chemistry. The studies revealed a good analytical performance: lower limit of detection 0.12 microg/L (3 SD from mean of blank); linearity up to 7.5 microg/L; median between-run CVs 8.1% (0.6 microg/L), 2.8% (1.5 microg/L), 1.9% (3 microg/L); mean analytical recovery in control sera 98-102%; slopes from 0.97 to 1.09 and intercepts from -0.28 to 0.10 microg/L in comparison with four immunoassays; and a high resistance to common interferents. The test was more resistant to digoxin-like immunoreactive factor (DLIF) interference than other methods, showing cross-reactivity only in some intensive care patient samples. Among 192 patients in whom DLIF is expected (e.g., pregnant women, patients with renal failure, newborns), 90% of results were < or =0.26 microg/L digoxin. Cortisol showed no cross-reactivity and digoxigenin had a low reactivity. An interlaboratory survey revealed a good comparability of the Tina-quant [a] test with the median of all methods (slope 0.99, intercept -0.06 microg/L). An HPLC method for digoxin based on isocratic separation of samples on an RP-18 column followed by detection by an immunoassay yielded a reasonable comparability with the immunochemical tests with noncritical samples. Divergent results of immunoassays caused by DLIFs or different cross-reactivities with digoxin metabolites or derivatives can be explained by the use of this HPLC method.

Stepwise binary gradient high-performance liquid chromatographic system for routine drug monitoring.

Drug therapy is usually optimized by concentration measurement in patient serum. High-performance liquid chromatography (HPLC) is one of the most important analytical techniques used for therapeutic drug monitoring (TDM) of drugs for which no immunoassay kits are available. HPLC has been frequently used for screening purposes in toxicology, too. The Merck Tox Screening System (MTSS) has been developed for the identification of substances by a combination of gradient HPLC with diode-array detection and identification with a database system. For routine TDM an isocratic HPLC system is more suitable because of shorter analysis time, better reproducibility of retention index and better precision of results. Therefore we defined a set of methods in steps of 10% of the two MTSS eluents. Three examples are shown: Amiodarone, Indometacine and Thiopental. New applications to test for other substances can be transferred to an isocratic system after a complete MTSS gradient run.

[Immune paralysis in acute pancreatitis--HLA-DR antigen expression on CD14+DR+ monocytes]. / Immunparalyse bei akuter pankreatitis--HLA-DR-antigen-expression auf monozyten CD14+DR+.

UNLABELLED: Determination of the prognosis in acute cases of pancreatitis, particularly in its serious and necrotizing form, still presents problems. Patients require intensive care and suffer from severe septic complications that do not correlate with pancreatic enzyme levels (amylase lipase). METHOD: Thirty-one patients with acute pancreatitis were examined: group 1 -- necrotizing pancreatitis (lethal outcome n = 7); group 2 -- necrotizing pancreatitis (surviving n = 12); group 3 edematous pancreatitis (surviving n = 12). For 11 consecutive days after admission to a clinical ward, flow cytometric check-ups were carried out daily on all patients. The antigen-presenting system HLA-DR antigen expression on monocytes and C-reactive protein were examined. RESULTS: When groups 1 and 2 were compared with group 3, HLA-DR values on monocytes were significantly different following the third day after admission (P < 0.01). Comparison of groups 1 and 2 were significant from the third day of observation (P < 0.001). During all 11 days of observation, patients in group 1 remained in immune paralysis (HLA-DR expression on monocytes CD14+DR+20% antigen density). All of these patients had infected necroses. Patients in group 2 overcame their immune paralysis. HLA-DR depression of monocytes and a long-standing high C-reactive protein level are almost certain predictors of a fatal outcome in cases with severe pancreatitis. A routine passage cytometric check/FACS to determine the activity of monocytes (HLA-DR) is of prognostic significance.

Comparison of serum phospholipase A2, polymorphonuclear granulocyte elastase, C-reactive protein and serum amyloid A with the APACHE II score in the prognosis of multiple injured patients.

This prospective study of 35 multitraumatized intensive care unit patients requiring mechanical ventilation examined the relative utility of four biochemical parameters with a physiological scoring system for predicting lethal outcome. Levels of serum phospholipase A2 (PLA2), serum amyloid A (SAA), polymorphonuclear granulocyte elastase (PMN elastase), and C-reactive protein (CRP) were determined at short intervals during the patient's hospitalization. The first specimen was obtained at the time of admission, and subsequent specimens were drawn at 8 h intervals for the first 48 h and then twice daily until death or convalescence. Calculations of the APACHE II score used the most deranged variables during the first 24 h of admission to assess patient outcome. Additional calculations of the APACHE II score at the time of each blood draw served as an indicator of patient status. The results indicate that during the first 24 h after admission none of the four examined biochemical parameters gives reliable information about the outcome. The APACHE II score provided the earliest indicator of patient outcome (83% sensitivity, 65% specificity). PMN elastase provided useful information first at 32 h (83% sensitivity, 45% specificity) and better at 132 h (86% sensitivity, 86% specificity). CRP was of intermediate use in predicting outcome initially at 72 h (83% sensitivity, 50% specificity) and later at 132 h (86% sensitivity, 93% specificity). PLA2 and SAA were not useful as early indicators of lethal outcome.

Fast HPLC determination of serum free fatty acids in the picomole range.

We developed a method for determining individual free fatty acids in serum by using a modified one-step Dole extraction, derivatization, and a new high-performance liquid chromatographic (HPLC) separation. Sample handling is minimized to a single transfer of the fatty acids (upper layer of the Dole extract), which are readily derivatized at 85 degrees C with p-bromophenacyl bromide without significant hydrolysis of esterified fatty acids. The derivatization mixture is directly injected into the HPLC apparatus. The new method, which uses C6 (3-microns particle) column material and an isocratic acetonitrile-water eluent, separates nearly to baseline 12 of the physiologically most abundant long-chain fatty acids (C12-C22) in < 20 min with a detection limit of approximately 2 pmol. It is therefore suitable for routine analysis even with basic HPLC equipment and can easily analyze a series of 10-20 samples in about 2 h including extraction until first results are available. The method is also applicable to other matrices than serum, e.g., for determination of precursor fatty acids such as arachidonic acid in platelets or of fatty acid patterns liberated by lipases or phospholipases A1/A2 in test systems.

Characteristics and clinical application of a radiometric Escherichia coli-based phospholipase A2 assay modified for serum analysis.

Determination of activities of phospholipase A2 (PLA2) in human sera was based on the hydrolysis of phospholipids from [1-14C]oleic acid-labeled Escherichia coli biomembranes. The E. coli membranes served as substrate specifically for the PLA2 of human serum and were essentially resistant to other lipases in human sera, i.e., lipoprotein lipases, hepatic triacylglycerolipase, or pancreatic lipase in acute pancreatitis. Exchange of phospholipids between the serum and the biomembrane compartment aggravates the determination of PLA2 activity in human serum, which is naturally rich in phospholipids. In our modified E. coli assay, which overcomes these difficulties, the main substrate components phosphatidylethanolamine (70%) and cardiolipin (25%) were > 90% labeled in the sn-2 position. Fatty acids released by PLA2 activity were eluted from an aminopropyl solid-phase column directly into scintillation vials, where the radioactivity was counted. The ratio of [1-14C]oleic acid to released total fatty acids was used to calculate true enzymatic activity. The linear assay range extended from 0 to 3.6 U/L (0-60 nkat/L), with a detection limit of < 0.03 U/L (< 0.5 nkat/L). Within-assay imprecision (CV) was < 6% and between-assay is < 10% over the whole activity range. The normal range for men was 0-0.44 U/L (0-7.33 nkat/L) and for women 0.044-1.11 U/L (0.73-18.4 nkat/L). Patients with septicemia, pancreatitis, acute respiratory distress syndrome, or other severe diseases had PLA2 values up to 540 U/L (9000 nkat/L).

[Studies of lipid and lipoprotein metabolism in man after surgical interventions]. / Untersuchungen zum Lipid- und Lipoproteinstoffwechsel beim Menschen nach operativen Eingriffen.

The pattern of serum lipids and lipoproteins was investigated before and after surgery in 77 patients with respect to the "acute-phase reaction". Special attention was paid to the severity of the surgical trauma and the time course of postoperative alterations. Therefore, 12 different serum parameters were measured in 18 patients just before surgery and on days 1, 3, 5 and 10. From the results of this sample, patients in the main trial were divided into three groups with different degrees of trauma: group 1 (n = 22) with low surgical trauma; group 2 (n = 20) with extensive abdominal operations; group 3 (n = 17) with total endoprosthesis of the hip. A 25-40% perioperative decrease in cholesterol, triglycerides, lipoprotein classes (alpha-, beta- and pre-beta-lipoproteins) and apolipoprotein A1 and B was found during the first 24 h after surgical trauma. Thereafter, the above parameters showed a tendency toward more or less complete normalization by day 10. In contrast, C-reactive protein (CRP), initially increased by a factor of 8-10, returned to the normal concentration range by postoperative day 10. The amount of cholesterol loss was low in group 1 (-16%), but considerable in group 2 (-38%) and group 3 (-35%) when compared with preoperative levels. This cholesterol loss was mainly due to a decrease in beta-lipoproteins (LDL), but also in alpha-lipoproteins (HDL). It can be concluded from these results that a sudden decrease in cholesterol containing serum lipoproteins occurs in relation to the size of a surgical trauma.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatty acid modulation of HepG2 cell cholesterol biosynthesis and esterification.

We investigated the effects of medium supplementation with increasing amounts of different fatty acids on HepG2 cell cholesterol biosynthesis and esterification. Up to 200 microM 16:0 led to an increase in cholesterol secretion/synthesis of 60%/40%; 18:1, 18:2, or 18:3 decreased secretion/synthesis by 35%/25%, 65%/65%, and 60%/60%; 80 microM 20:5 caused a reduction of 75%/50%. Compared to 200 microM 16:0, 18:2 led to a 50% reduced cellular cholesterol ester biosynthesis; the effect of 30 microM 20:5 was comparable to that of 18:2 while the addition of 200 microM 18:1 raised esterification. Supplementation of 18:2 reduced the cellular cholesterol ester content by 50%; 16:0 led to an increase of 80%. The effects of saturated and unsaturated fatty acids seem to be related to their number of double bonds and could be due to changes in membrane phospholipid fatty acid composition.

Liquid chromatographic analysis of phenobarbital, phenytoin, and theophylline.

Sera from the routine of therapeutic drug monitoring were assayed for phenobarbital, phenytoin, and theophylline with three different methods: fluorescence polarization immunoassay as the standard procedure, the new CEDIA assays within a multicenter evaluation and HPLC which is known to yield results with a high specificity. CVs for between-day imprecision ranged from 2.6-8.6%, depending on the concentration of the drugs. There was a tendency to lower CVs for the HPLC procedure. Accuracy was verified with commercial control materials and spiked sera and proved to be satisfactory for all three methods and parameters. The linear range was approx. twice as wide for the HPLC compared with the other methods. The method comparisons were quite favorable. Deviations occurred mainly in the subtherapeutic concentration range.

Results of the multicenter evaluation of the CEDIA Theophylline assay.

We report on the results of the multicenter evaluation of the CEDIA Theophylline assay on Boehringer Mannheim/Hitachi analyzers in 15 clinical laboratories in Europe and U.S.A. Main items of investigation were imprecision, recovery of control sera, interlaboratory survey and method comparisons using patient samples. Imprecision was found to be comparable to other routine methods. An advantage of the CEDIA assay can be seen in the good interlaboratory transferability of results. The new test has been shown to measure very accurately particularly by comparison with HPLC procedures revealing highly correspondent results. The reagent can be used up to one month using multiple recalibration. Due to its high practicability and reliability the CEDIA Theophylline assay can be recommended as a very suitable routine method for therapeutic drug monitoring on random access analyzers like Boehringer Mannheim/Hitachi analysis systems.

Results of the multicenter evaluation of the CEDIA Phenytoin assay.

Thirteen clinical evaluation sites in Europe and U.S.A. investigated the CEDIA Phenytoin assay on Boehringer Mannheim/Hitachi analyzers with respect to imprecision, recovery of control sera, interlaboratory survey, linearity and method comparisons using patient samples. The linear dose-response relationship up to 40 micrograms/mL was confirmed by all participants. Imprecision at therapeutic analyte concentrations equalled that of other routine methods. Recovery of controls was found in a +/- 6% range for target values assigned by the CEDIA assay. The good interlaboratory transferability of the CEDIA assay was confirmed with control material and human samples. The reconstituted reagent can be used up to one month using weekly recalibration. In method comparison studies good correlations to other routine methods were obtained. Results in analyte-free human sera did not deviate systematically from the zero-point. Thus, the accuracy in patient sera has been shown for the CEDIA Phenytoin assay.

Results of the multicenter evaluation of the CEDIA Phenobarbital assay.

The CEDIA Phenobarbital assay has been evaluated in twelve clinical laboratories in Europe and U.S.A. on Boehringer Mannheim/Hitachi analysis systems. The evaluation focused on the analysis of imprecision and accuracy. Within-run and between-day coefficients of variations of the new assay were comparable to those of established routine methods. As demonstrated in an interlaboratory survey study with controls and human sera, results obtained in different laboratories showed a good agreement. The CEDIA Phenobarbital assay measured very accurately, as particularly confirmed by comparison with HPLC. It can be recommended as a reliable and practicable test for monitoring of phenobarbital on Boehringer Mannheim/Hitachi analyzers used in routine clinical chemistry.

Results of the multicenter evaluation of the CEDIA cortisol assay.

The present paper describes the multicenter evaluation of the CEDIA Cortisol test for total cortisol. The observed linearity of the test was between 1.2 and 50 micrograms/dL cortisol. The limit of detection was calculated as 1.2/dL. Imprecision studies covering the diagnostically relevant range (5-20 micrograms/dL cortisol) yielded coefficients of variation between 1.7-8.9% (within-run) and 2.7-10.5% (between-day). An interlaboratory survey using 41 human samples and three control sera demonstrated that the new CEDIA Cortisol assay has a good interlaboratory transferability. Method comparison studies between the CEDIA Cortisol test and EIA, FIA, FPIA, and various RIAs yielded an acceptable level of agreement and concordant results in most cases. Low cross-reactivity of the antibody used in the new cortisol assay was observed with precursors or metabolites of cortisol. Especially, dexamethasone did not cross-react. However, prednisolone, 6-methylprednisone, and corticosterone showed cross-reactivities. No limitation by endogenous interferences was observed. The CEDIA Cortisol assay permits the precise, fast and sufficiently specific determination of cortisol. Furthermore, it offers the advantages of a non-radioactive assay and can be performed conveniently on Boehringer Mannheim/Hitachi analyzers in combination with routine clinical chemistry.

Improved method for enzymic determination of cholesterol in lipoproteins separated by electrophoresis on thin layer agarose gels.

The cholesterol of lipoproteins, separated electrophoretically on thin layer agarose films, is visualised and quantitated by incubating the gels in an enzymic reagent containing cholesterol esterase and cholesterol dehydrogenase. The individual fractions are quantitated by scanning densitometry. No sample pretreatment is necessary. All major fractions are detected readily. The accuracy of the determination is similar to that of ultracentrifugation. On average, imprecision is 3.1% for beta-, 7.0% for pre beta-, and 4.8% for alpha-lipoprotein cholesterol. Concentration and colour development are linear up to 8 mmol/l cholesterol in a given lipoprotein fraction. The results from the direct enzymic procedure for beta-, pre beta- and alpha-lipoprotein cholesterol are compared with those from quantitative lipoprotein electrophoresis after precipitation with phosphotungstic acid and bivalent cations and with those from different precipitation methods using dextran sulphate and polyethylene glycol. The new method has several advantages: high specificity; lack of dependence on the actual composition of the lipoproteins; lack of interference from coprecipitated proteins in the gel, e.g. fibrinogen or paraproteins; and insensitivity to lipolysis and high free fatty acid concentrations caused by heparin application or ageing of the specimen (at least for alpha-lipoprotein cholesterol quantitation). In its convenience and simplicity of operation, and the simple calculation of results, the method is similar to standard protein electrophoresis. The proposed method is therefore suggested as a standard method for elucidating lipoprotein disorders.

Enzymatic determination of lipids in human bile without bilirubin interference: reliable assessment of the cholesterol saturation index (CSI).

We describe a simple and rapid, but nevertheless precise and accurate method for the enzymatic determination of the main lipid constituents in human bile. Interfering bile pigments, especially bilirubin are eliminated by the use of aminopropyl bonded phase columns ("Bond-Elut") prior to the enzymatic measurement of cholesterol and lecithin. Intra-assay imprecision was between 3.1 and 4.9% CV, while the inter-assay figures were rather higher at 4.6 to 7.5% CV. Recoveries of bile salts, lecithin and cholesterol were between 94 and 103%. In contrast, the direct enzymatic determination in native bile produces falsely low results: lecithin from 5 to 20%, cholesterol from 25 to 40% of the true value. The results of both enzymatic methods correlated well with commonly accepted procedures for phospholipid and cholesterol determination. When compared with methods of bile lipid analysis involving solvent extraction, the column separation followed by enzymatic determination has the advantage of being simpler and less time consuming, without need of high-cost equipment, e.g. gas chromatography.