Fibrinogen is an antioxidant that protects beta-lipoproteins at physiological concentrations in a cell free system.

Oxidation of beta-lipoproteins has been linked to the development of arteriosclerosis. Using a copper mediated cell free system to oxidize beta-lipoproteins, we found that beta -lipoproteins isolated from plasma were less susceptible to oxidation than lipoproteins from serum and that this was probably due to inhibition by fibrinogen, because removal of fibrinogen from plasma enhanced oxidation, while addition of fibrinogen restored inhibition. Fibrinogen inhibited conjugated diene formation and peroxide formation assayed by the xylenol orange assay (absorbance+/-confidence interval: 0.155+/-0.007 with fibrinogen vs 0.255+/-0.014 without) and retarded copper mediated oxidation of apolipoproteins in low density lipoproteins, reducing the distance of electrophoretic migration by 5 mm. The effect of fibrinogen was not due to chelation of copper, since it provided protection when hydrogen peroxide was substituted for copper as an oxidizing agent. At normal physiological concentration equivalents, fibrinogen showed superior antioxidant properties compared to albumin, melatonin, vitamin C and vitamin E and was superior to the vitamins when compared on an equimolar basis. Other studies have shown the fibrinogen to be more oxidizable than other major plasma proteins and to inhibit peroxide production. Because of its high mass concentration, we postulate fibrinogen is an important antioxidant protecting beta-lipoproteins in plasma and that it may be important in protecting lipoproteins in tissue spaces.

Understanding and identifying monoclonal gammopathies.

Monoclonal gammopathies reflect conditions in which abnormal amounts of immunoglobulins are produced by a clone that developed from a single pro-B germ cell. The condition may reflect a disease process or be benign. The primary purpose of this review is to emphasize routine clinical laboratory techniques that currently are recommended for use in identifying monoclonal gammopathies from serum and urine. Selection of the preferred technique and correct interpretation often is dependent on an understanding of the immunological basis and clinical sequelae associated with these conditions. For this reason, we first briefly discuss the structure, production, and nature of immunoglobulins, and then describe important features of the associated diseases. Finally, we discuss strengths and weaknesses of the techniques and make reference to current recommendations to facilitate optimal testing. We discuss in detail high-resolution electrophoresis, methods for quantifying immunoglobulins, immunofixation electrophoresis, problems associated with analysis of urine immunoglobulins, and identification of cryoglobulins and immune complexes.

Urine protein electrophoresis and immunofixation electrophoresis supplement one another in characterizing proteinuria.

Urine protein electrophoresis (UPE) is often considered to have limited usefulness in evaluating proteinuria that is not associated with gammopathies. Unusual protein bands that are detected by UPE are commonly characterized by immunofixation electrophoresis (IFE). In this paper, electrophoretic gel patterns are shown to illustrate the greater sensitivity of IFE, compared to UPE. However, UPE remains useful for three applications: (1) UPE provides distinctive patterns that can indicate the source of proteinuria and is useful in assessing renal diseases that are independent of gammopathy; (2) combined use of UPE and IFE can avoid misinterpretations and repeated analyses of urine proteins, and (3) UPE can be used in conjunction with IFE to improve the quantitation of Bence-Jones proteinuria (BJP).

Simpler technique for measuring oxidative susceptibility by heparin affinity column isolation of lipoproteins.

Variations in the in vitro oxidative susceptibility or resistance of lipoproteins have been used to test the effect of ingested antioxidants and may prove to be a marker for coronary artery disease. Here we describe a simple technique for isolating and oxidizing beta-lipoproteins that may have utility in the clinical laboratory. Electrophoretic profiles showed that beta-lipoproteins were separated from alpha-lipoproteins and essentially from most other serum proteins using heparin affinity columns. Lipoproteins were normalized in the reaction mixture by measuring apo B in the beta-lipoprotein eluate using an automated apo B method, which gave good recoveries of 106-112%. Copper mediated oxidation was monitored by measurement of conjugated dienes formation at 234 nm for 300 min. When the reaction mixture included beta-lipoprotein eluate containing 0.03 g/L of apoB and 5 micromol/L copper sulfate, conditions were effective for obtaining complete oxidation while allowing reproducible measurements, with between day coefficients of variation of 2.35%, 14.6%, and 10.5% for lag, propagation and plateau phases, respectively. Beta-lipoproteins isolated from serum were more susceptible to oxidation than beta-lipoproteins from plasma apparently due to inhibition of oxidation by fibrinogen which co-eluted with beta-lipoprotein from plasma. For this reason, we recommend using serum preserved with EDTA for this assay.

When is a heterophile antibody not a heterophile antibody? When it is an antibody against a specific immunogen.

Heterophile antibodies are antibodies produced against poorly defined antigens. These are generally weak antibodies with multispecific activities. Human anti-animal antibodies that develop as a result of treatments with animal immunoglobulins are antibodies with strong avidities, produced against well-defined antigens. Although heterophile antibodies and human anti-animal antibodies interfere with immunological assays by similar mechanisms, modes for identifying the sources of the antibodies and for circumventing or retarding the interference may differ. Unfortunately, there has not been a well-organized attempt to encourage correct definition of these antibodies. This problem of inexact definition is highlighted by recent articles in this Journal. In the present discussion, we examine the history leading to this problem and discuss the origins and the reasons that the nature of the antibody is important for rectifying the problem. We propose a simple nomenclature for general usage that should appropriately characterize these antibodies in most cases.

Mechanized lipoprotein(a) assay as a marker for coronary artery disease illustrates the usefulness of high lipoprotein(a) levels.

Only a few simple lipoprotein(a) [Lp(a)] assays are available in kit form for use in clinical laboratories. The present study compares the analytical and clinical performance of a mechanized immunonephelometric method to enzyme-linked immunosorbent assay. Clinical performance was evaluated by measuring lipoprotein markers in 191 patients, with the extent of stenosis defined by angiography. Analytically, both methods showed little or no correlation with cholesterol, high density lipoprotein cholesterol, elevated triglycerides, apo A-I and apo B, while they showed good agreement with one another (r = 0.88). The methods showed comparable well known differences between black and white persons. Logistic regression indicated that Lp(a) was a weak but independent marker for coronary artery disease (CAD). Receiver operator characteristic curve analysis showed an association with CAD only at higher Lp(a) concentrations. We conclude that Lp(a) at higher concentrations may be a contributory marker for CAD and that mechanized nephelometric assays for it can be used in the clinical laboratory.

Optimized automated apolipoprotein A-I assays as markers for coronary artery disease.

BACKGROUND: Studies are divided as to whether or not apolipoprotein A-I (apo A-I) is a better marker for coronary artery disease (CAD) than high-density lipoprotein cholesterol. We hypothesized that the detergent Tween 20, which is thought to expose antigenic sites in apo A-I, would improve automated kit apo A-I assays as a diagnostic marker for CAD. METHODS: Apolipoprotein A-I was assayed by two standard automated methods and by the same methods after serum samples and reagents had been treated with Tween 20. Serum samples were obtained from 226 consecutive male patients, age 40-70 years, presenting for angiography, except for defined exclusion characteristics. Patients were categorized into two groups on the basis of stenosis: (1) normal, all vessels <20% stenosis, n = 79, and (2) CAD, at least one vessel >70% stenosis, n = 147. Diagnostic accuracy was assessed by receiver operator characteristic stenosis curves and forward stepwise logistic regression, where adjustment was made for significant possible confounding characteristics and drugs. RESULTS: The optimal concentration of Tween 20 was found to be 0.5%. Receiver operator characteristic curves showed a greater area for apo A-I with Tween (area = 0.63 to 0.64) as compared to apo A-I without Tween (area = 0.60 to 0.62). Logistic regression indicated that apo A-I with Tween was a significantly better marker than high-density lipoprotein cholesterol. Receiver operator characteristic curves indicated that the ratio of modified apo A-I to apo B gave a significant improvement in area over the ratio of high-density to low-density lipoprotein cholesterol. CONCLUSIONS: Addition of Tween 20 to apo A-I assays improved diagnostic discrimination for CAD. The modified apo A-I assays were better markers than high-density lipoprotein cholesterol, and the ratio of apolipoproteins was significantly better markers than lipoprotein lipids. These findings may explain the discrepancies between studies comparing high-density lipoprotein cholesterol and apo A-I as markers for CAD. Our data suggest that a multicenter effort toward optimizing and clinically validating apo A-I test reagents may be worthwhile.

An algorithmic approach using kappa/lambda ratios to improve the diagnostic accuracy of urine protein electrophoresis and to reduce the volume required for immunoelectrophoresis.

The most sensitive routine method for identifying urinary monoclonal immunoglobulin kappa and lambda light chains, called Bence Jones proteins (BJPs), in clinical laboratories is immunofixation electrophoresis (IFE), but this procedure is time-consuming and expensive. As a result, many laboratories screen for paraproteins with urine protein electrophoresis (UPE), which is insensitive when low concentrations of BJP are present and is difficult to interpret with severe proteinuria. The purpose of this study was to determine whether kappa/lambda ratios can be used in conjunction with UPE to improve diagnostic reliability in identifying paraproteins, and decrease the need for IFE on all samples. Urine specimens from 243 patients were examined by UPE and kappa/lambda ratios, and compared with IFE. Due to poor analytical sensitivity, the urinary kappa or lambda concentrations could not be determined in many cases. As a result, many specimens showed kappa/lambda ratios that were indeterminate. Nevertheless, when both urinary kappa and lambda concentrations were undetectable, a BJP could be ruled out. A urinary kappa/lambda ratio between 0.75-3 also ruled out a BJP. The use of kappa/lambda ratios, in conjunction with UPE, resulted in a 52% decrease in the volume of IFE during the course of this study, with 100% sensitivity for detecting BJP.

Relationship between bilirubin, apolipoprotein B, and coronary artery disease.

Lipoprotein lipids and apo B from 254 male patients were compared with bilirubin as a risk factor for coronary artery disease (CAD). Patients were classified as: 1, Normal, all vessels < 20% stenosis, n = 83; or 2, CAD, at least one vessel > 70%, n = 171. Diagnostic accuracy was assessed by receiver operator characteristic curves (ROC). Corrections were made for possible confounding variables in the multivariate analysis. Upon ROC analysis, bilirubin showed an inverse relationship with risk of CAD, with areas under the ROC curve comparable to lipoprotein: however, bilirubin showed no discrimination below false positive frequencies of approximately 0.3. Logistic regression indicated that bilirubin was a weaker global marker than the lipoproteins and interacted with apo B. A highly significant correlation was found between bilirubin and apo B (p = 0.0025), but not with cholesterol, triglycerides, or high density lipoprotein cholesterol. Compared to lipoprotein markers, bilirubin provides little practical power of discrimination for CAD. Further studies of the affect of bilirubin on CAD must take its interaction with apo B into consideration.

Hemoglobin interference with urinary Bence Jones protein analysis on electrophoresis.

Two cases are described which show that on urine protein electrophoresis a paraprotein owing to hemoglobin cannot be distinguished from a Bence Jones protein. A simple method is described for confirming the presence of hemoglobin: a densitometric scan of the electrophoretogram at 415 nm, in which hemoglobin absorbs in a band coincident with the restriction. Furthermore, it is shown that other serum proteins, elevated levels of bilirubin, and Bence Jones protein do not interfere with this detection system.

Omeprazole therapy does not affect pharmacokinetics of orally administered ethanol in healthy male subjects.

The purpose of this study was to study the effect of high-dose omeprazole therapy (20 mg twice daily) on kinetics of moderate amounts of orally administered ethanol. Eight healthy men participated in the study. After an overnight fast, they drank 0.5 g/kg body weight ethanol over 20 min. Blood samples were drawn before and then every 20 min after ethanol ingestion for the next 3 h. Subjects then ingested omeprazole 20 mg twice daily for 6 days. On the seventh day, the same dose of oral ethanol was administered as before and blood samples drawn. Blood ethanol concentrations were determined. We fit a one-compartment model with first-order absorption and zero order elimination to the blood ethanol data with PCNONLIN (SCI Software, Lexington, KY, U.S.A.) separately for each subject before as well as after omeprazole therapy. Area under the curve was calculated using the trapezoidal rule. There were no differences in the peak concentration, time to peak concentration, area under the curve, or elimination rate constant for ethanol before and after omeprazole treatment. Omeprazole treatment (20 mg twice daily) does not affect the pharmacokinetics of orally ingested ethanol in healthy male subjects. Our results do not rule out a possible effect on psychomotor function as a result of a pharmacodynamic interaction.

Relationships between triglycerides, lipoproteins, glucose and coronary artery disease.

We examined the relationship of apolipoprotein B (apo B), glucose, triglycerides and other lipoprotein lipids to coronary artery disease (CAD). Using receiver-operating characteristic curves (ROC), we noticed that the triglyceride ROC curve crossed above other lipoprotein curves at a triglycerides level of approximately 1.4 g/l. We examined subgroups of < 1.4 g/l and > 1.4 g/l. ANOVA (F = 18.9, P < 0.0001) and stepwise logistic regression (P = 0.0002) indicated that triglycerides were the best predictor in the < 1.4 g/l group. The best markers in the > 1.4 g/l group were low density lipoprotein cholesterol and apo B. Glucose did not appear to significantly alter the predictive power of triglycerides. These data suggest that triglyceride appears to be an overall significant univariate marker for CAD because of its effect at lower concentrations. The strong relationship between CAD and triglycerides at low triglyceride levels may reflect increased levels of very low density lipoprotein metabolites in some individuals. We conclude that some triglyceride-rich particles are independently atherogenic, that glucose did not alter this relationship and that when the samples were split into those with high and low levels of triglycerides, triglycerides and apo B but not HDLC was a significant predictor of CAD.

Free light chains of immunoglobulins: clinical laboratory analysis.

The increased sensitivity of immunofixation electrophoresis (IFE) over prior electrophoretic methods has led to renewed interest in the study of free light chains. Here, we discuss problems associated with the identification of monoclonal free light chains (Bence Jones proteins) in urine. Besides reviewing the nature of the sample specimens and the assays themselves, we discuss the physiology, biochemistry, genetics, and immunological properties of these molecules. Direct measurement of kappa/lambda ratios may ultimately be useful, but all commercial methods available now lack sufficient sensitivity. IFE is the preferred method because of its sensitivity and ease of interpretation. There are, however, difficulties associated with the interpretation of urinary IFE patterns, because the technique does not include an intrinsic mechanism for antibody-antigen titration and because of its great sensitivity in the absence of quantification. Problems of interpretation are discussed.

Usefulness of various lactate dehydrogenase isoenzyme 1 profiles after myocardial infarction.

Isoenzyme profiles of lactate dehydrogenase (percent LD-1 of total LD, LD-1/LD-2 ratio, and absolute LD-1) have all been studied as late markers for myocardial infarction. It is known, however, that elevations of LD-5 frequently occur in this period as a result of liver congestion. Elevations of LD-5 may also occur as a result of complicating conditions. Such elevations could result in a reduced percent LD-1 of total LD, giving rise to false negatives. Receiver operating characteristic (ROC) curves were constructed for LD-1, LD-1/LD-2 and percent LD-1 of total LD from 285 specimens (124 patients) with suspected myocardial infarction. There was little difference in overall diagnostic power among the three assays. Using cutoffs determined from the ROC curves, 6 patients (18 specimens) were evaluated who appeared to be in the late period or who exhibited complicating conditions which could increase LD-5. In 14/18 specimens, increases in LD-5 resulted in false negatives by percent LD-1 of total LD. Only 5/18 specimens were false negatives by LD-1 or LD-1/LD-2. It is concluded that the percent LD-1 of total LD was affected by an increase in LD-5, and caution is recommended when using it.

The relative usefulness of automated apolipoprotein A-I and high-density lipoprotein cholesterol assays as markers for coronary artery disease.

Research studies have shown that apolipoprotein A-I (apo A-I) is a better marker for coronary artery disease (CAD) than high-density lipoprotein cholesterol (HDLC). Yet, it is unclear whether assays for apolipoprotein A-I, which is part of macromolecular lipoprotein complex, can perform as well when measured by routine automated clinical laboratory techniques. The purpose of this study was to compare automated apolipoprotein A-I assays with HDLC as a marker for CAD. The authors studied two groups of angiographically documented men, aged 44-70 years, 42 with CAD and 123 without CAD in an unmatched, controlled study. Standard clinical laboratory techniques for assaying HDLC, and automated kit rate immunonephelometric, end point immunonephelometric, and immunoturbidimetric assays for apolipoprotein A-I were used. High-density lipoprotein cholesterol was a better marker than apolipoprotein A-I, according to the Mann Whitney test; HDLC also showed better diagnostic sensitivity, specificity, and predictive value. Using a precipitation method, HDL3 was a better marker than HDL2, but not as good as total HDLC. The authors concluded that HDLC remains the best routine single CAD marker commonly available for evaluation of HDL status in a high-risk population.

Immunonephelometric/turbidimetric apolipoprotein B assays for the clinical laboratory.

Because apolipoproteins are a part of complex macromolecular particles, modifications to the assay system may substantially alter results of immunological measurement. Accuracy as analytical recovery cannot be effectively determined by adding exogenous apolipoproteins because antibody access differs from access to endogenous apolipoproteins. Clinical studies are essential for determining accuracy in terms of clinical effectiveness. Since different kit methods use different reagent systems, the purpose of the present study was to compare total cholesterol and LDL cholesterol as markers for coronary artery disease with apo B by automated rate nephelometric, end-point nephelometric and turbidimetric kit methods. The subjects were age matched, male patients with and without angiographically documented coronary artery disease. High correlation coefficients (0.95-0.96) between the assays for both the normal and disease groups indicate that the methods are providing similar information: apo B was a better marker for coronary artery disease (CAD) than total or LDL cholesterol on the basis of univariate, multivariate and Bayesian statistics and correlated best with non-HDL cholesterol. Apo B along with HDLC could explain the variability between the CAD and normal groups without LDLC, total C, or triglycerides.

Interference of polyclonal free light chains with identification of Bence Jones proteins.

We present a case in which kappa free light chains caused difficulty in interpreting classical urinary immunoelectrophoresis, but immunofixation electrophoresis (IFE) demonstrated the presence of a lambda-Bence Jones protein. Analysis of the urine by Ouchterlony double diffusion and IFE after gel-filtration chromatography showed that the difficulty was caused by the presence of large amounts of polyclonal free light chains. The workup also demonstrated that although IFE is the more sensitive and specific technique, IFE performed on concentrated urinary samples is especially subject to misinterpretation unless densely staining patterns are diluted and reassayed. This process of sample dilution provides a means for titrating antigen and antibody concentrations such that condition-specific patterns become visible on the gel. This workup also shows that, at some dilutions, polyclonal free light chains may migrate in the same manner as an oligoclonal band in a so-called ladder configuration. These bands were observed from both monomeric and dimeric fractions isolated by gel chromatography, consistent with reports that this pattern is largely linked to the isoelectric points of the molecules. We speculate that, in rare instances, the distinction between polyclonal and monoclonal kappa free light chains migrating as a ladder-banding pattern may be equivocal.

Human immunodeficiency virus infection and anion gap.

Although a normal or increased anion gap (AG) is commonly used to help assess acid-base balance, decreased AG has aided in the diagnosis of halogen ingestion and myeloma. Substantially increased levels of IgG cause a decrease in the AG. Patients with polyclonal increases in immunoglobulins, especially hepatic cirrhosis, also exhibit decreased anion gaps. Patients with human immunodeficiency virus (HIV) infection commonly show polyclonal increases in immunoglobulins. A case is reported of a patient with HIV infection who exhibited a decreased AG associated with increased polyclonal IgG (63 g per L). Unlike the electrophoretic profile of patients with hepatic cirrhosis, which commonly shows a beta-gamma-globulin bridge, reflecting a decreased immunoglobulin degradation, the profile of the patient with HIV infection was consistent with an increased immunoglobulin synthesis. Examination of sera from 18 additional HIV positive patients indicated that, in general, the AG of HIV infected patients with normal renal function is significantly higher than in normal persons. The significance of this finding is as yet unclear. Nevertheless, decreased AG was associated with increased IgG. This may complicate the use of the AG in evaluating HIV infected patients because of frequent elevations in IgG. These relationships are now in the process of further investigation. Nevertheless, it is suggested that, with appropriate history and physical, identification of a decreased anion gap in conjunction with a polyclonal increase in gamma-globulin may be reason to consider a work up for infection by HIV.