Anion-exchange chromatography of normal and monoclonal serum immunoglobulins

Anion-exchange chromatography was used for separations of the three main immunoglobulin classes in human sera. The material included normal sera as well as sera with monoclonal immunoglobulin components. The Mono Q anion-exchange column was eluted with a stepwise gradient formed by 0.05 and 0.3 M phosphate buffers. No indications of negative effects on the sample components were observed and the protein recovery was high. A fast, easy and reproducible separation of immunoglobulins G, A and M was obtained.     

Enzyme-linked immunoglobulins and their clinical significance

Most of the enzymes used in routine biochemical tests have been detected in circulating enzyme-linked immunoglobulin (Ig) complexes (E-Ig). The complexes often cause unexplained hyperenzymemia and give anomalous patterns on isozyme electrophoresis. However, E-Ig does not appear to be associated with any pathological conditions. The present study was undertaken to determine the clinical characteristics of patients with E-Ig. More than 42,000 patients selected at random and 10,000 blood donors were screened for lactate dehydrogenase (LDH)-linked Ig complexes (LDH-Ig), amylase (Amy)-linked Ig complexes (Amy-Ig), alkaline phosphatase (AP)-linked Ig complexes (AP-Ig) and creatine kinase (CK)-linked Ig complexes (CK-Ig). LDH-Ig and Amy-Ig were screened by electrophoresis for routine isozyme analysis and identified by precipitin reactions. AP-Ig and CK-Ig were screened and identified by counter immunoelectrophoresis. The incidence of all E-Ig ranged from 0.18% to 0.61% in patients and from 0.03% to 0.23% in blood donors. The incidence curves of E-Ig were age-related, generally increasing with age. The curves of E-IgG's for different enzymes resembled each other in shape but those of E-IgA's did not. A positive correlation between E-IgG and autoimmune diseases was present. Consequently, it is concluded that both autoimmune diseases and age are the common clinical factors involved in E-IgG. No specific diseases were found for cases with E-IgA. However, it is suggested that some CK-IgA, but not all, is caused by elevated serum CK activity.     

Determination of nitrazepam and its main metabolites in urine by thin-layer chromatography and direct densitometry

A method for the direct quantitative densitometry of nitrazepam and its main metabolites (7-aminonitrazepam, 7-acetamidonitrazepam and 2-amino-5-nitrobenzophenone) in urine was developed. The unchanged drug and its metabolites were extracted with benzene-dichloromethane (4:1), subjected to thin-layer chromatography, and determined by direct ultraviolet densitometry. Recovery experiments showed that the method was quantitative. The limit of detection was 5 ng/ml for 2-amino-5-nitrobenzophenone and 10 ng/ml for other compounds. The method was applied to the determination of nitrazepam and its metabolites excreted in human urine after administration of 10 mg of the drug.     

Determination of netilmicin in serum by thin-layer densitometry and fluorescence polarization immunoassay

Summary A newly developed thin-layer chromatographic method (TLC) for the quantitative determination of netilmicin in serum is described and compared with fluorescence polarization immunoassay (FPIA). The TLC procedure involves solid-phase extraction of the aminoglycoside from serum and chromatography on reversed-phase thin layer. Post-chromatographic derivatization is carried out using 2,2-diphenyl-1-oxa-3-oxonia-2-boratanaphthalene (DOOB) as fluorescence reagent. The calibration curve for netilmicin in serum is linear in the range 1.0–5.0 g/ml and the detection limit is about 0.2 g/ml (correlation coefficient r=0.9963, mean coefficient of variation V_XO=±5.3 %). A total of 25 serum samples from patients treated with netilmicin was measured by TLC and the results were compared with those of FPIA (Abbott TDx). There is no statistically significant difference between the two procedures (y=–0.35+1.094·x, Passing/Bablok). Thus the TLC-procedure is an alternative for the determination of netilmicin in serum, that possesses the necessary sensitivity of other comparable methods.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthday.     

Electrophoretic analysis of enzyme-linked immunoglobulins and their clinical significance

The appearance of circulating enzyme-linked immunoglobulin complexes (E-Ig) is common for most enzymes used in clinical biochemical tests. The presence of E-Ig may result in altered enzyme activity in serum and interfere with the measurement of isoenzymes, and is thus of diagnostic importance. E-Ig can be identified by confirming that the binding protein is an immunoglobulin by its reaction with specific anti-human immunoglobulin antibodies. Currently, the presence of E-Ig in an individual is regarded as a benign phenomenon, not indicative of any particular disease process. However, it is becoming clear that E-IgG are closely associated with autoimmune states.     

ETAAS determination of nickel in serum and urine

Methods for the direct determination of Ni in human blood serum and urine by electrothermal atomic absorption spectrometry (ETAAS) are described. Hydrogen peroxide was proposed as matrix modifier, assisting thermal decomposition of proteins during the ashing step. A pyrolysis temperature of 1,200 degrees C was found to be optimal while 2,100 degrees C and 2,200 degrees C were found to be optimal atomizing temperatures for Ni in serum and urine respectively. Calibration was performed by using a calibration curve prepared with aqueous standard solutions of Ni (glycine must be used as modifier for Ni in aqueous solutions). The limits of detection, defined as the blank values plus 3 times the standard deviation of the blank values, were 0.2 microg/L for both serum and urine samples. Relative standard deviations for serum samples with concentrations of Ni in the range 0.5-2 microg/L were 10-15% and for urine samples with Ni concentrations in the range 0.5-2.5 microg/L were 8-10%.     

Quantitative determination of tolazoline in serum and urine

A high-performance liquid chromatographic procedure is described for the analysis of tolazoline in serum and urine. This assay procedure is suitable for the analysis of micro-samples (50 or 100 microliters serum and 100 microliters urine). Samples are extracted in a single step and injected into a reversed-phase high-performance liquid chromatography system for detection at 210 nm. The clinical applicability of this assay is demonstrated by the determination of tolazoline serum and urine concentrations in neonates. In addition, the presence of urine conjugates and the extent of serum protein binding were investigated. This assay procedure has the required sensitivity (0.1 microgram/ml), accuracy and precision for both routine monitoring and pharmacokinetic characterization of tolazoline in neonates and adults.     

Assay of naproxen in rat serum by high-performance thin-layer chromatography/densitometry

A simple, sensitive, and reproducible high-performance thin-layer chromatographic method using densitometry is presented for the determination of naproxen in rat serum. Only 0.1 mL serum was used for extraction. Separations were performed on 10 x 10 cm plates coated with silica gel, with toluene-ethyl acetate-acetic acid (82 + 15 + 3) as the mobile phase. Benzophenone was used as the internal standard. Quantification was performed by densitometry at 260 nm. The response response for naproxen was linear (r = 0.992) over the range 2-100 mg/L. Method validation demonstrated good recoveries (92-96%), sensitivity (limit of quantitation, 1 mg/L), repeatability of sample application (4%), repeatability of the method (8%), and intermediate precision (5%). The procedure was applied to the quantitation of naproxen in rat serum.     

GLC assay of khellin in human serum and urine

Summary A sensitive, specific and rapid GLC assay for khellin in serum and urine has been developed using flame ionization The method involves the addition of hyoscine as an internal standard, extraction with chloroform and the use of a 3% OV-17 on Gas Chromosorb column. The suitability of this procedure for serum sample analysis from rats after oral administration of the drug is reported.     

Extraction--spectrophotometric determination of oxalate in urine and blood serum

An extraction--spectrophotometric method is described for the determination of oxalate, based on the formation of a mixed ligand vanadium (V)--mandelohydroxamic acid--oxalate complex. The complex was extracted into a solution of trioctylmethylammonium chloride (Adogen 446) in toluene and the absorbance measured at 535 nm. The experimental variables and interferences in this determination were studied. The detection limit is 0.5 microgram ml-1 and the range of application is between 2 and 8 micrograms ml-1. The method was applied to the determination of oxalate in urine and blood serum.     

Electrophoresis of serum isoenzymes and proteins following acute myocardial infarction

The clinical significance of the serum enzymes creatine kinase (CK, EC 2.7.3.2), lactate dehydrogenase (LD, EC 1.1.1.27) and aspartate aminotransferase (EC 2.6.1.1), and the isoenzymes CK 1-3 and LD 1-5, in acute myocardial infarction (AMI) is reviewed. Particular attention is given to electrophoretic analysis of the isoenzymes (and the CK isoforms/subforms) following AMI and thrombolytic therapy. Other protein markers for the monitoring of AMI, including myoglobin and muscle contractile proteins, are also discussed and the potential for the detection of new marker proteins using high-resolution two-dimensional electrophoretic methods is demonstrated. Whilst emphasis is placed upon electrophoretic methods the value of complementary immunoassays is acknowledged in order to maintain a balanced perspective.     

On chromium direct ETAAS determination in serum and urine

A simple, accurate and reliable method for direct electrothermal atomic absorption spectrometric (ETAAS) determination of chromium in serum and urine samples without any preliminary sample pretreatment is described. Instrumental parameters are optimized in order to define: the most suitable atomizer, optimal temperature program and efficient modifier. An appropriate quantification method is proposed taking into account a matrix interference study. Pyrocoated graphite tubes and wall atomization, pretreatment temperature of 700 °C, atomization temperature of 2600 °C, hydrogen peroxide as modifier and standard addition calibration are recommended. The accuracy of the method proposed for Cr determination in serum and urine was confirmed by comparative analysis of parallel samples after wet or dry ashing as well as by the analysis of two certified reference materials: Serum, Clin Rep 1 and Lypochek Urine, level 1. The detection and determination limits achieved for both matrices are 0.08 μg/L and 0.15 μg/L respectively. The relative standard deviation varied between 15 and 18 % for the chromium content in the samples in the range 0.08–0.2 μg/L and between 4 and 7 % for the chromium content in the range 0.2–2.0 μg/L for both matrices.      

Profile analysis of chondroitin sulfates in human urine and serum

A highly sensitive high-performance liquid chromatographic method with fluorometric postcolumn labeling using 2-cyanoacetamide was developed for the profile analysis of chondroitin sulfates (ChS) in normal human urine and serum. Over-sulfated disaccharide units such as di- or trisulfated unsaturated disaccharides in urine were estimated and unsaturated 6-sulfated disaccharide (delta Di-6S) was found as a major component from ChS in urine, although only small amounts of delta Di-6S from ChS were present in serum.