Serum sphingomyelin species profile is altered in hematologic malignancies.

Sphingomyelin (SM) plays key roles in regulating cell membrane fluidity and in intracellular signal transduction. However, little is known as to whether alterations in SM concentration or SM species distribution are linked pathological conditions. The present study examined SM concentrations and species profiles in serum taken from patients with hematologic malignancies. Serum was collected from normal subjects and from patients with B-cell lymphoma, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and acute lymphatic leukemia/ lymphoblastic lymphoma (ALL/LBL). Serum SM species distribution was analyzed using electrospray ionization mass spectrometry/ mass spectrometry (ESI MS/MS). Serum lipids concentration were measured using enzymatic assays. Normal and hematologic malignancy sera were similar in terms of total serum SM and phosphatidylcholine (PC) concentrations and SM/PC ratio. However, all hematologic malignancy sera had lower levels of SM species containing saturated odd chain fatty acids (OCFAs) in the side chain compared to normal serum. In addition, the proportion of SM species with saturated (C20 and C22) and mono unsaturated fatty acids (C18, C20, C22) were lower in MDS patient serum compared to normal serum. The present study revealed that the serum SM species profile in patients with hematologic malignancies differed from that of normal subjects despite total serum SM and PC concentrations and SM/PC ratios being similar between the various cancer groups and the normal group.

Helicobacter pylori infection and its related factors in junior high school students in Nagano Prefecture, Japan.

BACKGROUND: There have been few reports on Helicobacter pylori (H. pylori) infection in asymptomatic Japanese children and adolescents. We hypothesized that the prevalence of H. pylori infection is very low among Japanese children and that clinical variables such as serum pepsinogen and iron levels are associated with H. pylori infection. MATERIALS AND METHODS: We conducted a cross-sectional analysis of a sample of 454 junior high school students aged 12-15 years in four areas in Nagano Prefecture. A commercial ELISA kit (E-plate Eiken H. pylori antibody) was used to measure IgG antibody against H. pylori. Serum pepsinogen and iron levels were also measured using standard methods. A urea breath test was performed for seropositive students. RESULTS: The overall prevalence of H. pylori was 3.1% (14/454). There were no significant differences in H. pylori prevalence among mountain, rural, and urban areas. The mean level of both serum pepsinogen (PG I) and PG II was significantly increased in the seropositive subjects compared with the seronegative subjects. When the cutoff values for adults (PG I: 70 ng/mL and PG I/II ratio: 3) were used, 4 of 14 subjects had PG I ≤70 ng/mL and PG I/II ratio ≤3. The results of a logistic regression analysis showed that low serum iron levels were significantly associated with H. pylori infection (P=.02). CONCLUSIONS: The prevalence of H. pylori infection is as low as 3% among junior high school students aged 12-15 years in Japan. The disappearance of H. pylori is accelerating in Japanese children.

Importance of cystatin C and uric acid levels in the association of cardiometabolic risk factors in Japanese junior high school students.

BACKGROUND: Serum cystatin C (CysC), a novel marker of renal function, is associated with the components of metabolic syndrome in adults. Little is known about the utility of CysC and its association with cardiometabolic risks in young subjects. METHODS AND RESULTS: In a cohort of 454 Japanese junior high school students, the distribution of serum CysC levels and associated variables were analyzed. CysC levels were significantly higher in boys than in girls (0.92±0.10mg/L vs. 0.77±0.08mg/L, p<0.001). CysC was significantly correlated with serum creatinine (r=0.473, p<0.001), and serum uric acid (SUA) (r=0.546, p<0.001). Multivariable regression analysis revealed significant associations between CysC and SUA in all subjects (ß=0.241, p<0.001), and in boys and girls separately (ß=0.264 and 0.240, respectively, both p<0.001). Importantly, subjects with elevation of both serum CysC and SUA levels had the highest ratio of triglyceride to high-density lipoprotein cholesterol. CONCLUSIONS: CysC had significant associations with both creatinine and SUA in Japanese junior high school students. The concomitant elevation of serum CysC and SUA levels was associated with subclinical lipid metabolism dysregulation, and suggested the presence of cardiometabolic risk accumulation.

Rapid quantitative analysis of human serum sphingomyelin species using MALDI-TOF mass spectrometry with lipid hydrolase treatment.

BACKGROUND: Sphingomyelin (SM) is a key component of extracellular membranes and lipoproteins, and plays roles in cell signaling and as a component of lipoproteins. SM species differ in terms of fatty acid (FA) composition. However, no simple, rapid, quantitative assay for identifying different SM species has yet been reported. In this study, lipid hydrolase treatment and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to identify serum SM species. METHODS: Sera were collected from healthy young individuals. To identify SM species, sera were treated with phospholipase A2 and lipoprotein lipase, and lipids were extracted using the standard chloroform/methanol (2/1 v/v) method. RESULTS: We detected 15 peaks from serum using MALDI-TOF MS, which were assigned to SM species bound with FA components ranging from C15:0 to C24:2. The most prominent serum SM species was SM [C16:0], which accounted for approximately 26% of serum SM. Some SM species contained an odd-carbon FA (C15, C21, and C23), and these accounted for approximately 4% of serum SM. The reproducibility of major SM species within and between application positions on MS-sample plate was CV=3.0%-7.9% and CV=3.1%-6.8%, respectively. The concentration and dilution ratio were linearly related. The SM species composition of 10 healthy young subjects showed a similar profile. CONCLUSIONS: We developed a rapid, and quantitative method for identifying serum SM species using lipid hydrolase treatment and MALDI-TOF MS. This method will be suitable for clinical laboratory studies to examine the associations between SM species and disease states.

[Rapid and Easy Measurement of Serum Fatty Acid Composition of Neonates, Infants and Young People Using the Gas Chromatography Mass Spectrometry].

A variation of the fatty acid composition is closely associated with the clinical state of inflammatory disorder and metabolic syndrome. The analysis of serum fatty acid composition of neonates and infants has been measured hardly at the laboratory, because a large quantity of serum was required for an analysis and the measurement procedure was cumbersome. We examined the rapid and easy analysis in a small amount of serum using the combination methods of the gas chromatography mass spectrometry (GC MS) and the quick transmethylation. The serum fatty acid composition of neonates and infants were compared with the young people. The serum fatty acids with the internal standard material were performed transmethylation using the microwave, and then the lipids were extracted. The fatty acid esters were analyzed by GC MS with capillary column, and the statistical procedure used nonparametric method. The repeatability of each fatty acid concentration was CV = 5-11.3% (n = 5) with serum 50 µl. The lowest quantity of sample was possible to measure with 13 µl of serum. The total serum fatty acid, saturated fatty acid and monounsaturated fatty acid levels did not show a significant difference at all age, but the polyunsaturated fatty acid (PUFA) level of neonates and infants was significantly lower than young people, p = 0.007. The four main PUFA exclusive of α-linolenic acid showed a significant difference. The fatty acid composition with small quantities serum was measured by the rapid and accurate method using the GC MS and the microwave. The serum PUFA concentrations have fluctuated according to growth, therefore it was necessary to evaluate serum fatty acid composition in each age category.

[Abnormal Serum Total Protein Measurement by Lipoprotein-X in an Infant with Biliary Atresia].

Lipoprotein-X (LP-X) in cholestatic jaundice causes abnormal reaction in assays for low-density lipoprotein-cholesterol, but the effects on other test items are unknown. Here, we report an infant with biliary atresia showing abnormal reaction in total serum protein assay using the biuret method, and lipoprotein-X (LP-X) was then detected. In this 11-month-old female infant, jaundice was observed at 2 months old, and a diagnosis of biliary atresia was made. On biochemical tests at 12 months old, the total serum protein concentrations detected by the biuret method were very high, and the response curve and linearity of dilution were abnormal. LP-X was detected by agar electrophoresis. In addition and recovery experiments with normal serum fractionation of the patient's LP-X-rich lipoprotein fraction prepared by ultracentrifugation, normal γ-globulin fractionation showed an abnormal reaction by the biuret method. In infants with biliary atresia, we showed that the total serum protein assay by the biuret method was influenced by LP-X-rich lipoprotein, which may be caused by abnormal reaction of LP-X and γ-globulin. [Case Report].

[Laboratory tests of serum lipid molecules using mass spectrometry].

Serum lipids have a variety of molecular frameworks and fatty acid side chains, and these molecules include various metabolites and derivatives. Lipid molecules influence the physicochemical properties of the plasma membrane and intracellular transport, and their metabolites are often bioactive substances. Fatty acid molecules are also precursors of lipid mediators, and are involved in the regulation of inflammatory reactions. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is used in various fields in the clinical laboratory. Serum phosphatidylcholine (PC), lysoPCGSphingomyelin, triglycerides, cholesterol ester, sulfatide, phosphatidylethanolamine, and phosphatidylglycerol can be analyzed by MALDI-TOF MS. We devised an analytical procedure for serum ceramide and glycolipids from partially purified serum lipids. Investigation of lipid molecular species is used for the characterization of disorders of lipid metabolism. MS methods for use in routine laboratory tests require both precise and accurate measurement, and sample preparation procedures should be both simple and quick. (Review).

Consumption of nonfat milk results in a less atherogenic lipoprotein profile: a pilot study.

BACKGROUND: An increase in plasma low-density lipoprotein (LDL) is a well-known risk factor in the development of atherosclerosis. Dairy consumption may lower the risk of atherosclerosis; however, studies on the effects of milk on cardiovascular risk factors are still scarce. We were interested in investigating whether the intake of milk improves the atherogenic lipoprotein profile. AIMS: We investigated the effects of consuming whole or nonfat milk on plasma lipoprotein composition in healthy Japanese subjects as a pilot study. METHODS: Normolipidemic subjects consumed 500 ml of whole milk (whole milk group; n=7) or nonfat milk (nonfat milk group; n=7) every day for 2 weeks. RESULTS: The consumption of nonfat milk resulted in a lowering of plasma triglyceride (TG) and phospholipid levels and TG level in high-density lipoprotein (HDL) and increased the plasma apolipoprotein (apo) C-III level. In addition, the TG/cholesterol ratios in HDL and LDL were significantly decreased, and LDL particles became larger. In contrast, the only changes observed following whole milk consumption were increases in the plasma levels of apoC-III and apoE. CONCLUSIONS: These findings suggest that consumption of nonfat milk, but not whole milk, may result in a less atherogenic lipoprotein profile, and that the constituents of nonfat milk may improve lipid metabolism.

Low-dose rosuvastatin improves arterial stiffness in high-risk Japanese patients with dyslipdemia in a primary prevention group.

BACKGROUND: The treatment effects of rosuvastatin on arterial stiffness were assessed and compared to those of fluvastatin in high-risk Japanese patients with dyslipidemia in a primary prevention group. METHODS AND RESULTS: Patients were randomly assigned to either 2.5-5 mg/day of rosuvastatin (Group A) or 20-40 mg/day of fluvastatin (Group B) and followed up for 12 months. In Group A (n=38), there was a progressive reduction in brachial-ankle pulse wave velocity (baPWV) along with a decrease in the low-density lipoprotein cholesterol/high-density lipoprotein cholesterol (L/H) ratio and high-sensitivity C-reactive protein (hsCRP), and the change in baPWV correlated significantly with that of the L/H ratio and that of hsCRP after rosuvastatin treatment. In Group B (n=37), although fluvastatin achieved a significant improvement in baPWV, L/H ratio, and hsCRP, baPWV was significantly greater than that in Group A and showed a significant correlation with that of hsCRP alone after fluvastatin treatment. In a subgroup of patients (n=26), switching from fluvastatin to rosuvastatin further improved baPWV and the L/H ratio without altering hsCRP after 12 months. CONCLUSIONS: Low-dose rosuvastatin would be more effective than fluvastatin in improving arterial stiffness in high-risk Japanese patients with dyslipidemia. The results suggest that improvement in arterial stiffness by rosuvastatin mainly depends on its strong lipid-lowering effects, whereas that by fluvastatin is strongly dependent on the pleiotropic effects, especially an anti-inflammatory action.

Association between serum uric acid levels and cardiometabolic risk factors among Japanese junior high school students.

BACKGROUND: The present study was designed to examine whether serum uric acid (SUA) levels were associated with cardiometabolic risk factors and to determine optimal cut-offs for SUA to identify multiple risk factors among Japanese junior high school students. METHODS AND RESULTS: A total of 958 students (518 boys and 440 girls, aged 12.1-15.0 years) who were enrolled between April 2005 and June 2008 were divided into 4 groups according to SUA quartiles. Compared with the lowest quartile of SUA, prevalence of abdominal obesity, hypertension, and dyslipidemia was significantly increased in the highest quartile in boys and that of abdominal obesity was increased in the highest quartile in girls. The adjusted odds ratios (95% confidence interval) of the highest quartile of SUA for 2 or more cardiometabolic risk factors were 2.59 (1.16-5.79) for boys and 1.54 (0.43-5.56) for girls. Receiver operating characteristic curve analysis demonstrated that the most appropriate cut-offs for SUA to identify multiple cardiometabolic risk factors were 6.4 mg/dl for boys and 4.9 mg/dl for girls. CONCLUSIONS: SUA was strongly associated with the prevalence of cardiometabolic risk factors among male Japanese junior high school students. The present study may provide insights into the role of SUA in the school screening system for the development of educational programs on prevention of lifestyle-related diseases among school children.

High-molecular-weight beta-amyloid oligomers are elevated in cerebrospinal fluid of Alzheimer patients.

There is accumulating evidence that soluble amyloid-beta (Abeta) oligomers, rather than amyloid fibrils, are the principal pathogenic species in Alzheimer disease (AD). Here, we have developed a novel enzyme-linked immunosorbent assay (ELISA) specific for high-molecular-weight (HMW) Abeta oligomers. Analysis of Abeta oligomers derived from synthetic Abeta 1-42, by size-exclusion chromatography (SEC), revealed that our ELISA specifically detected HMW Abeta oligomers of 40-200 kDa. Using this ELISA, we detected significantly higher (P<0.0001) signals in cerebrospinal fluid (CSF) samples from 25 patients with AD or mild cognitive impairment (MCI), compared to 25 age-matched controls. As a test for discriminating between the AD/MCI and control groups, the area under the curve in receiver operating characteristic analysis for the CSF HMW Abeta oligomers was greater than that for CSF Abeta x-42. Furthermore, the CSF levels of HMW Abeta oligomers showed a negative correlation with Mini-Mental State Examination scores in the AD/MCI group. We conclude that the CSF HMW Abeta oligomers detected by our ELISA could be useful as a diagnostic marker for AD, and also as a potential surrogate marker for disease severity. Our results support the idea that soluble HMW Abeta oligomers play a critical role in the pathogenesis and progression of AD.

Characterization of cysteine and homocysteine bound to human serum transthyretin.

BACKGROUND: Cysteine-bound transthyretin (TTR) in serum is believed to be associated with deposition of amyloid in familial amyloidotic polyneuropathy (FAP). We examined the binding of cysteine and homocysteine with TTR, and the effect of such binding on TTR structure. METHODS: The concentration of serum cysteine and homocysteine was measured using HPLC and fluorescence detection. TTR was analyzed using isoelectric focusing (IEF), SDS-polyacrylamide gel electrophoresis (PAGE) and mass spectrometry. RESULTS: Incubation of serum at 4 degrees C resulted in an approximate doubling of the amount of both cysteine and homocysteine bound to TTR. Incubation of serum TTR with cysteine-HCl at 37 degrees C markedly altered the IEF and SDS-PAGE profiles of TTR. The main ion peaks of TTR were observed at m/z 13,761 and 13,881, and assigned as free TTR and TTR+cysteine respectively. Incubation of purified TTR with dithiothreitol followed by cysteine-HCl resulted in loss of the latter peak and an increase in the m/z 13,761 peak, while incubation with cysteine-HCl alone did not cause such a change. CONCLUSION: Cysteine- and homocysteine-bound TTR was more easily denatured by cysteine-HCl than free TTR. Binding of cysteine and homocysteine may alter the structure and characteristics of serum TTR, and may facilitate TTR denaturation and deposition.

Specific, rapid, and sensitive enzymatic measurement of sphingomyelin, phosphatidylcholine and lysophosphatidylcholine in serum and lipid extracts.

OBJECTIVE: Human serum sphingomyelin (SM) and phosphatidylcholine (PC) play important roles in the development of atherosclerosis. However, there are no rapid and sensitive methods for SM and PC measurement. The present report describes a novel enzymatic method for measuring SM, PC and lysophosphatidylcholine (lyso-PC) levels in plasma and lipid extracts. DESIGN AND METHODS: The total choline-containing phospholipids (total PL), SM and PC were measured using a two-reagent system involving specific enzymes for choline-based phospholipids. The procedure was performed using either microplate or automatic analyzer technology. The concentration of lyso-PC was calculated by subtracting the concentration of SM plus PC from the total PL concentration. RESULTS: Assay results showed linear correlations between sample concentration and absorbance. The within-run and between-run coefficients of variation for PC, SM, and lyso-PC concentrations were 2.0-4.4% for the microplate analyzer and 0.9-2.9% for the automatic analyzer. Analysis of normal human serum showed that the total PL concentration strongly correlated with the SM plus PC concentration (r=0.9850). There were moderate correlations between serum PC and SM levels (r=0.6228) and between serum PC and lyso-PC levels (r=0.7806). SM, PC, and lyso-PC levels in normal human serum (n=50) were 0.54+/-0.07, 1.99+/-0.22 and 0.60+/-0.15 mmol/L, respectively. CONCLUSION: The present enzymatic method allowed for rapid, simple, and accurate measurement of SM, PC, and lyso-PC levels in lipid extracts and in serum. The method is suitable for both microplate and automatic analyzer assays.

Fluvastatin improves arterial stiffness in patients with coronary artery disease and hyperlipidemia: a 5-year follow-up study.

BACKGROUND: The present study was designed to test the hypothesis that fluvastatin might improve arterial stiffness, as assessed with pulse wave velocity (PWV), in patients with coronary artery disease (CAD) and hyperlipidemia over the long term. METHODS AND RESULTS: Ninety-three patients were randomly assigned to either fluvastatin (group A, n=50) or bezafibrate (group B, n=43) and followed for 5 years. There was no difference in the clinical findings between the 2 groups. In group A, there was a progressive reduction in the brachial-ankle PWV along with a decrease in serum low-density lipoprotein-cholesterol (LDL-C) and C-reactive protein (CRP) by 12 months after fluvastatin, and the improvement was maintained until 5 years after treatment. In group B, despite identical lowering of the serum lipid, PWV was progressively increased. In group A, the percentage change in PWV correlated significantly with that of the serum CRP (r=0.49, p<0.001), but not with that of the serum LDL-C after treatment. CONCLUSIONS: The beneficial vascular effects of fluvastatin persisted for a long period in patients with CAD and hyperlipidemia. Its anti-inflammatory action might contribute to the favorable effects on arterial stiffness.

Analysis of human serum lipoprotein lipid composition using MALDI-TOF mass spectrometry.

This study used matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify all lipid classes in human serum lipoproteins. After the major lipoproteins classes were isolated from serum by ultracentrifugation, the lipids were extracted and mixed with 2,5-dihydroxybenzoic acid (2,5-DHB) dissolved in Folch's solution (chloroform/methanol 2:1, v/v). MALDI-TOF MS analysis of the samples identified phospholipids (PLs), lysophospholipids (lysoPLs), sphingolipids (SLs), triglycerides (TGs), cholesteryl esters (CEs), and free cholesterol; it also showed the characteristics of individual fatty acid chains in serum lipids. MALDI-TOF MS allowed analysis of strongly hydrophobic and non-polar molecules such as CEs and TGs as well as hydrophilic molecules such as phospholipids. Direct analysis of fatty acids was not possible. The concentrations of lipids were not consistent with the ion peak intensities, since the extent of polarity affected the ionization characteristics of the molecules. However, lipid molecules with similar molecular structures but various fatty acid chains, such as phosphatidylcholine (PCs), were analyzed quantitatively by MALDI-TOF MS. Quantitative measurement of cholesterol was possible with the use of an internal standard. This study shows that MALDI-TOF MS can be used for direct investigation and quantitative analysis of the phospholipid composition of serum lipoproteins.

[Abnormal laboratory data due to interaction between immunoglobulins and other serum proteins].

Two cases with abnormal laboratory data due to interaction between immunoglobulins and other serum proteins are described. Case 1 was a patient with lactate dehydrogenase (LD) -IgG3 complex whose serum LD value was moderately elevated. Case 2 were nondiabetic patients with IgA type M-proteinemia who had significantly increased serum fructosamine (FRA). The IgG3 in Case 1 was found to be conjugated to LD by immunoprecipitation assay. The LD-IgG3 complex was easily dissociated by affinity chromatography on 5'-AMP or Cibacron Blue F3G-A. The relative molecular weights of the patient's gamma3 chains and light chains were 67,000 and 28,000, respectively, by Western blotting, which corresponded to the expected values. However, the patient's IgG3 did not react to the anti-kappa and anti-lambda light chain antibodies in Immunofixation electrophoresis. Serum FRA concentrations were higher in patients (Case 2) with IgA type M-proteinemia or polyclonal hyper-IgA than those with the IgG type or IgM type. The sera from the patients with IgG or IgM type M-proteinemia had FRA only at the position of albumin, but 11 of 13 sera with IgA type M-proteinemia stained for glycoprotein at the position of the M-protein band as well as the albumin band. The abnormal precipitin arcs of IgA-albumin complex were observed in 11 of 13 sera from patients with IgA type M-proteinemia that were glycosylated at the position of M-protein band.

Degradation of pre-beta-high density lipoproteins and their binding activity to human blood monocytes.

We have previously reported that high density lipoprotein3 (HDL3), apolipoprotein A-I (apoA-I) rich lipoprotein, binds specifically to the surface of human blood monocytes. Pre-beta-HDL with a pre-beta mobility on agarose gels is an apoA-I (MW 28 kDa)-rich and a lipid-poor lipoprotein. In the present study, we found that pre-beta-HDL purified by ion-exchange chromatography was susceptible to degradation if isolated in the absence of anti-proteases, resulting in the smaller lyso-pre-beta-HDL. The mass of lyso-pre-beta-HDL was confirmed using a delayed extraction matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (DE-MALDI-TOF MS), which showed a fragment of approximately 22,378.9 Da. We further investigated limited proteolysis of apo A-I purified from human plasma HDL with various proteases, and cleavage appeared to be limited to the C-terminal end of apo A-I (amino acids 188-223). The ability of pre-beta-HDL and lyso-pre-beta-HDL to compete for HDL binding to monocytes was determined using a flow cytometry-based assay. Pre-beta-HDL competed efficiently for binding whereas lyso-pre-beta-HDL was significantly less effective. The data may indicate that the binding sites on monocytes specifically recognize apoA-I. We suggest that limited proteolysis around amino acids 188-223 of apo A-I may affect lipid binding, which may in turn affect HDL structure and function.

Characterization of factor XII Tenri, a rare CRM-negative factor XII deficiency.

Factor XII Tenri (Y34C), a rare cross-reacting material (CRM)-negative factor XII deficiency, was identified in a 71-yr-old Japanese woman with angina pectoris. In the patient's plasma, factor XII activity and antigen levels were only 1.6% and 5.0%, respectively, of those seen in a normal subject. Immunoblot analysis showed that the secreted factor XII Tenri existed not only as a monomer (76 kDa), but also in complexes with apparent molecular weights of approximately 115, 140, 190, 215, and 225 kDa. After reduction with 2-mercaptoethanol, the factor XII Tenri contained in the complexes was completely converted to monomeric form on immunoblot patterns. It appeared that some of the secreted factor XII Tenri formed several types of disulfide-linked complexes, including a factor XII-alpha1-microglobulin complex, through a newly generated Cys residue. The monomeric form of factor XII Tenri, like normal factor XII, was degraded into 2 major fragments with molecular weights of approximately 45 kDa and 30 kDa following mixing with activated partial-thromboplastin-time measuring reagent (cephalin and ellagic acid), whereas the factor XII Tenri that formed the complexes was not. This indicates that the factor XII Tenri present in disulfide-linked complexes with other proteins (and itself) is not converted to active forms, suggesting that attached proteins obstruct or delay the activation of factor XII via an inhibition of its binding to a negatively charged surface in vitro.

Purification and measurement of HDL3-binding proteins in human peripheral blood mononuclear cells.

We previously identified a binding site for high density lipoprotein-3 (HDL3) on the surface of human peripheral blood monocytes. Here we describe the purification and measurement of HDL-binding proteins present on these cells. Purification of HDL-binding proteins was achieved by chromatography using DEAE ion-exchange, wheat germ lectin, and apoHDL3 affinity columns. Subsequent use of SDS-PAGE and ligand blotting lead to the identification of two major proteins with apparent molecular masses of 100 and 120 kDa, plus several smaller proteins that appeared to be degradation products. Analysis of purified HDL-binding proteins by reverse-phase HPLC showed that the two main proteins at 100 and 120 kDa were eluted in 65 and 70% acetonitrile, respectively, indicating the proteins are strongly hydrophobic. To measure the amount of HDL-binding protein present in CHAPS-solubilized human mononuclear cells, a 96-well plate assay was developed that was based on the same principles as the purification method. The present study of HDL-binding proteins in mononuclear cells advances our understanding of the physiological roles and fate of HDL.