Relationship between lipoprotein- and oxidation-related variables and atheroma lipid composition in subjects undergoing coronary artery bypass graft surgery.

The relationship between atheroma lipid composition and serum lipoprotein and oxidation measurements has not been fully explored. To address this question, we studied serum, plasma, and aortic wall specimens from 66 subjects undergoing coronary artery bypass graft surgery. The lipid composition of aortic specimens was characterized in terms of cholesterol ester and cholesterol crystal plus phospholipid by using hot-stage polarizing light microscopy; tissue oxidation status was assessed by measuring conjugated dienes. Serum lipoprotein-related measurements included total cholesterol, triglyceride, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, apolipoproteins B and AI, and lipoprotein(a). Oxidation status was assessed by measuring LDL mobility, thiobarbituric acid-reactive substances, LDL conjugated dienes, and IgG and IgM autoantibodies against oxidized LDL. Fasting blood glucose was also determined. Lesion cholesterol crystal plus phospholipid content was associated inversely with serum HDL cholesterol levels (r=-0.279, P=0.029) and positively with fasting blood glucose (r=0.359, P=0.016), LDL mobility (0.276, P<0.05), and IgM autoantibodies against oxidized LDL (r=0.272, P=0.037). There was also a significant relationship between the level of aortic tissue conjugated dienes and plasma LDL mobility (r=0.332, P=0.007). In multivariate analysis, IgM autoantibodies against oxidized LDL, fasting blood glucose, and LDL mobility, in descending order of significance, together accounted for 35% of the variability in aortic lesion cholesterol crystal plus phospholipid content. These data support direct and independent roles for oxidation and hyperglycemia in the pathophysiology of atherosclerosis.

Effect of low-density lipoprotein (LDL) antigen source on an enzyme-linked immunosorbent assay for autoantibodies against oxidized LDL.

We examined the effect of antigen source on an enzyme-linked immunosorbent assay (ELISA) for autoantibodies against oxidized low-density lipoprotein (LDL). Serum samples from 20 subjects with systemic lupus erythematosus (SLE) and from 20 controls were assayed for immunoglobulin G (IgG) and immunoglobulin M (IgM) autoantibodies against oxidized LDL, using either a pooled or individual (n = 3) LDL preparation as antigen. For IgG autoantibodies against oxidized LDL there was a relationship (r approximately 0.5, P < 0.01) between data obtained using individual versus pooled antigen preparations. Bias plots demonstrated consistent inverse, concentration-dependent relationships (r approximately -0.6, P < 0.001). The difference in IgG autoantibodies against oxidized LDL levels between SLE patients and controls was underestimated (39-58%) when assays used individual rather than pooled LDL antigen. For IgM autoantibodies against oxidized LDL the direct relationships were stronger (r approximately 0.8, P < 0.001) and the concentration-dependent relationships weaker (r approximately -0.3, significance variable) than for IgG autoantibodies against oxidized LDL. Variations between LDL preparations suggested that a pooled antigen would give a more stable assay. Thus, LDL antigen source is important in assays for both IgG and IgM autoantibodies against oxidized LDL.

Analytical evaluation of particle-enhanced immunonephelometric assays for C-reactive protein, serum amyloid A and mannose-binding protein in human serum.

Against a background of growing interest in more sensitive assays for quantifying various acute phase proteins, we evaluated the performance of recently developed tests for C-reactive protein (CRP), serum amyloid A (SAA) and mannose-binding protein (MBP) on the Behring nephelometer II (BNII). Sample results outside the calibration ranges of 3.5 to 220 mg/L for CRP, 3.3 to 215 mg/L for SAA and 0.09 to 5.6 mg/L for MBP were automatically re-measured at another dilution. The lower limits of detection were 0.01, 0.7 and 0.01 mg/L for CRP, SAA and MBP, respectively. The coefficients of variation (CV) for intra- (n > or = 20) and inter- (n > or = 15) assay precision were < 5.2% and < 8.5%, respectively, for the three proteins at concentrations representing low, normal and high. Linearity for each method was within 5% of the expected values throughout the calibration range. We observed no significant interference from bilirubin (up to 300 mg/L) or haemoglobin (up to 10 g/ L) for the three tests. Method comparison studies performed for CRP and SAA yielded the following results: y (CRP on BNII) = 0.75x (ELISA, Hemagen) -0.25 mg/L (r = 0.981, Sy/x = 2.1 mg/L; y (SAA on BNII) = 1.44x (ELISA, Hemagen) -9.9 mg/L (r = 0.972, Sy/x = 6.9 mg/L), where ELISA is enzyme-linked immunosorbent assay. Reference intervals established in 261 adult blood donors (aged 36.2 +/- 9.0 years) were found to be log-normal with 2.5th, 50th, and 97.5th centiles of < 0.17, 1.00 and 10.1 mg/L for CRP, < 0.84, 2.10 and 9.70 mg/L for SAA; and 0.30, 1.28 and 4.10 mg/L for MBP. We observed no relationship with CRP concentration and age; however, SAA levels increased with age while MBP levels decreased. The BNII provides a simple, rapid and sensitive system for measuring CRP, SAA and MBP in human serum.

The relationship between apolipoprotein E and serum oxidation-related variables is apolipoprotein E phenotype dependent.

To examine the relationship between apolipoprotein E and serum oxidation status, we assayed apolipoprotein E level, apolipoprotein E phenotype, and levels of lipid peroxides and transition metal ions and their binding proteins in sera from apparently healthy individuals. The study group included 129 women aged 22-63 years and 53 men aged 22-56 years. Among subjects with apolipoprotein E 4/3 phenotype, lipid peroxide levels were higher compared with E 3/2 phenotype (786 +/- 182 nmol/l vs. 659 +/- 174 nmol/l, P = 0.015), and ceruloplasmin levels were slightly higher compared with apolipoprotein E 3/3 phenotype (0.28 +/- 0.08 mg/l vs. 0.26 +/- 0.06 mg/l, P = 0.035). In the study group as a whole, there were significant associations between serum apolipoprotein E level, and serum levels of ceruloplasmin (r = 0.266, P < 0.001) and ferritin (r = 0.2, P < 0.007). Among subjects with apolipoprotein E 4/3 phenotype, there was a significant association between serum apolipoprotein E and lipid peroxide levels (r = 0.470, P < 0.01), which was not apparent among subjects with E 3/3 or E 3/2 phenotypes. In multivariate analysis, apolipoprotein E phenotype was a small but significant independent contributor to variation in serum lipid peroxide levels. These data suggest that there may be heterogeneity among apolipoprotein E phenotypes in their relationships with serum lipid oxidation status.

Anti-oxidized LDL antibodies and antiphospholipid antibodies in healthy subjects: relationship with lipoprotein- and oxidation-related analytes.

IgG autoantibodies against malondialdehyde-modified LDL (alpha oxLDL), antiphospholipid antibodies (APA) and oxidation- and lipoprotein-related analytes were assayed in sera from healthy subjects (51 males, 115 females, aged 22-63 years). alpha OxLDL levels were associated (P < 0.03) with IgG alpha cardiolipin (r = 0.18), IgM alpha cardiolipin (r = 0.17) and IgM alpha phosphatidyl-serine (r = 0.16) but not with age, cholesterol, triglyceride, apolipoproteins B and AI, lipoprotein(a), lipid peroxides, ceruloplasmin, copper, ferritin, transferrin or iron. APA levels were inversely associated with levels of both oxidation- and lipoprotein-related analytes. Ferritin (3.5%) and alpha oxLDL (1.4%) contributed independently to variation in IgG alpha cardiolipin levels, and apo B (2%) to variation in IgM alpha cardiolipin levels. These associations are small, indicating that there are no major biological associations between the measured variables. The lack of association between alpha oxLDL and lipoprotein- or oxidation-related analytes suggests that the relevant antigen is not in serum.

Oxidation-related analytes and lipid and lipoprotein concentrations in healthy subjects.

The relations between oxidation-related analytes and lipoprotein risk factors for coronary heart disease are poorly understood. To address this issue, ceruloplasmin, copper, iron, ferritin, cotinine, lipid peroxides, cholesterol, triglyceride, apoB, apoA-I, and lipoprotein(a) levels were measured in sera from apparently healthy subjects (51 men and 115 women). Pairwise comparisons revealed strong positive associations (P < .001) of copper and ceruloplasmin with lipid peroxides, total cholesterol, triglycerides and apoB, of transferrin with apoA-I and cholesterol, and of ferritin with triglycerides. Serum levels of oxidation-related analytes did not differ between smokers and nonsmokers. In multivariate analysis, serum copper was the major independent determinant of serum lipid peroxide level, accounting for 15% of the variability in concentration (ferritin accounted for 1.6%). Copper and ceruloplasmin accounted for 20.5% of the variation in triglyceride levels; triglycerides and apoB accounted for 12% of the variability in ferritin levels; apoB and apoA-I accounted for 9% of the variability in transferrin levels. The data suggest that serum copper contributes to lipid peroxidation in vivo. There are significant associations between lipoprotein and transition metal-related analytes, and further work is needed to elucidate the physiological basis for these relations.

ELISA of IgG antibody to oxidized low-density lipoprotein: effects of blocking buffer and method of data expression.

We describe an ELISA for serum IgG antibodies against malondialdehyde-modified low-density lipoprotein (mLDL). Optimal antigen concentration, serum dilution, and dilution of enzyme-conjugated second antibody were 25 mg/L, 1:250, and 1:5000, respectively, when 5 g/L human serum albumin was used for blocking. When data were expressed as mLDL/LDL (the ratio of IgG binding to mLDL vs LDL), within-run and between-run CVs were 7.0% and 8.9%, respectively. Antibody concentrations expressed as mLDL/LDL or as mLDL-LDL (the difference between IgG binding to mLDL and to LDL) were higher in women with systemic lupus erythematosus (n = 20) than in controls (n = 20) (P < 0.001). With bovine serum albumin or Superblock blocking buffers, only the mLDL-LDL data were significant. Thus, the choice of blocking agent and the method of data expression should be carefully considered when assaying IgG antibodies against mLDL.

Apolipoprotein (a): a comparison of isoforms identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by sodium dodecyl sulfate-agarose gel electrophoresis.

Lipoprotein(a) resembles low density lipoprotein in structure, except that a unique apolipoprotein (apo), apo(a), is linked to apo B-100. Variations in the number of sequence repeats in the apo(a) gene give rise to a range of isoforms. Depending on the method used, 6-30 apo(a) isoforms have been observed; however, the correspondence of these different isoforms has not been reported, making between-study comparisons difficult. In the present study we address this question by characterizing the apo(a) phenotypes of 48 sera using two previously reported separation methods, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 3-12% gels) and SDS-agarose gel electrophoresis. In addition, the molecular weight of each isoform was estimated using haptoglobin 2-2 polymers as molecular weight standards. Among the 48 sera, 15 distinct apo(a) isoforms were separated by SDS-PAGE and 28 by SDS-agarose gel electrophoresis. There was excellent correlation between the two nomenclature systems (r = -0.97, p < 0.001, by rank correlation), and the ranges were totally overlapping, with the same two isoforms being identified as the largest and smallest by either method. The apparent molecular mass range for the isoforms was 294-624 kDa, which is in close agreement with the theoretical molecular mass range of 238-643 kDa, calculated from the sequence and carbohydrate content of recombinant apo(a). The disparity in number of isoforms between methods was expected, due to the poorer separation of apo(a) by SDS-PAGE; 3.1 +/- 1.7 (median, 2.0) SDS-agarose isoforms were combined for each SDS-PAGE isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

Background staining in immunoblot assays. Reduction of signal caused by cross-reactivity with blocking agents.

Certain serum samples produce high background in Western and direct immunoblot assays that detect human serum IgG against specific antigens. We determined that this was due to a reaction between endogenous IgG and the membrane blocking agent (we refer to this as blocking-specific background). Using milk as blocking agent, we screened 107 sera by Western immunoblot or checkerboard immunoblot assays, and found that 6.5% of sera had background intensities sufficient to interfere with the interpretation of final results. Blocking-specific background was also observed using bovine serum albumin and other animal protein-based blocking agents. As the primary antibody in these immunoblot assays was human IgG, we investigated human serum albumin as a blocking agent; this approach eliminated the problem of blocking-specific background.

The molecular weights of twelve apolipoprotein(a) variants, determined using haptoglobin 2-2 polymer molecular weight standards.

Apolipoprotein(a) [apo(a)] variants were characterized in 398 sera by immunoblotting: (a) by molecular weight, using a haptoglobin 2-2 polymeric series as standards, and (b) by nomenclature, using serum pools containing previously characterized apo(a) variants as standards. The haptoglobin 2-2 standard curve (172-859 kDa) alleviates the necessity of obtaining molecular weights by extrapolation. Among the 398 sera, 40.2% had double apo(a) bands (54 phenotypes), 58.0% had a single apo(a) band and 1.8% were null (no bands observed). An inverse, though non-monotonic, relationship was observed between apolipoprotein(a) molecular weight and serum lipoprotein(a) [Lp(a)] concentration. Due to the large size of apo(a) and the relatively small increment between variants (15-16 kDa), molecular weight could not be used alone to characterize variants. Even with a CV of 3-4%, there was an overlap between variant molecular weight estimates. However, in combination with the identification of variants by comparison with standards, the haptoglobin 2-2 standard curve could be used to obtain mean molecular weight estimates for each variant. 12 distinct variants were identified among the sera, with apparent mean molecular weights of 314, 388, 410, 433, 454, 466, 503, 519, 528, 543, 553 and 572 kDa, respectively. These molecular weight estimates are consistent with the theoretical molecular weight range for apo(a) variants, calculated from sequence and carbohydrate analysis, of 238-643 kDa.

Application of checkerboard immunoblotting (CBIB) to the detection of anti-viral IgG in human serum.

In the present study, we have begun to investigate the possibility of using checkerboard immunoblotting (CBIB) as a semi-quantitative screening tool for detecting human serum IgG against specific viral antigens. The viral antigens studied were Epstein-Barr, herpes simplex I and II, cytomegalovirus, varicella zoster, rubella, rubeola, and mumps. Western immunoblotting experiments using these partially purified preparations demonstrated that there were apparently no interactions between IgG from non-immune sera and the respective viral antigen preparations. The CBIB assay was evaluated using sera of known positive or negative immune status for the viral antigens. There was excellent agreement between the results of CBIB and the results of alternative methods for evaluating immune status: all discrepancies (1/18 sera for mumps, 3/18 sera for rubeola, and 1/28 sera for rubella) involved sera with borderline results, either by CBIB or by the alternative method. Therefore, although further work is required to define the method in terms of sensitivity and clinical specificity, and to refine positive/negative cutpoint criteria for certain antigen components, our preliminary experience suggests that CBIB has considerable potential in the efficient and inexpensive screening of sera for the presence of IgG against a panel of viral antigens, so as to identify subjects at risk for infection.

Effect of sample storage on the assay of lipoprotein(a) by commercially available radial immunodiffusion and enzyme-linked immunosorbent assay kits.

Lipoprotein(a) [Lp(a)] was measured by both a radial immunodiffusion (RID) kit from Immuno AG (Zurich, Switzerland) and a Tint Elize enzyme-linked immunosorbent assay (ELISA) kit from CytRx Biopool Ltd. (Umeå, Sweden) in serum samples that had been stored at -20 and -70 degrees C for six months. Storage temperature had no significant effect on the Lp(a) concentrations obtained by either method. After six months, mean Lp(a) degradation was 46% (95% confidence interval, 34-58%) with the RID kit; the ELISA data could not be compared between time points. In fresh sera, Lp(a) concentrations obtained by RID were 41% higher than by ELISA (because of differences in assay calibration materials), but in paired measurements of a set of 215 samples stored at -40 degrees C for an average of 10 years, Lp(a) concentrations were 62% lower by RID. This suggests that RID is more sensitive to the effects of long-term storage than is ELISA.