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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.

BackgroundSulfatides are found in a variety of tissues and serum lipoproteins. Sulfatide is a molecular species composed of various sphingoid bases, fatty acids and sugar chains; therefore, rapid analysis of the qualitative structure is important in clinical assessment.MethodsIn this study, sulfatide-rich fractions were isolated from serum lipids, and the sulfatide species were analysed by negative ion mode using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).ResultsSulfatide species identified in human serum included two different sugar chains, eight sphingoid molecules and various fatty acid side chains including hydroxy fatty acids. In total, 64 galactosyl sulfatides (SM4s) and 49 lactosyl sulfatides (SM3) were identified. Quantitatively, the amount of SM3 was less than 1% of the amount of SM4s. The fatty acids of SM4s of healthy serum ( n = 8) were predominantly C16:0 and a hydroxylation C16:0 (C16:0h), followed by very long chain fatty acids (VLCFAs) predominant species, and SM3 was a major component of VLCFAs.ConclusionThis present study described a simple method of human serum sulfatide analysis using MALDI-TOF MS. This method is suitable for clinical laboratories and is likely to increase the understanding of the roles of sulfatide species in both physiological and disease states.

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.

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].

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 2.5-5mg/day of rosuvastatin (Group A) or 20-40mg/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. (Circ J 2011; 75: 2660-2667)

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. (Circ J 2010; 74: 1570 - 1577)

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 °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 °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.

Article, CLINICAL BIOCHEMISTRY. 41(14-15); 1211-1217 (2008)

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

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.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, p0.001). CysC was significantly correlated with serum creatinine (r=0.473, p0.001), and serum uric acid (SUA) (r=0.546, p0.001). Multivariable regression analysis revealed significant associations between CysC and SUA in all subjects (β=0.241, p0.001), and in boys and girls separately (β=0.264 and 0.240, respectively, both p0.001). Importantly, subjects with elevation of both serum CysC and SUA levels had the highest ratio of triglyceride to high-density lipoprotein cholesterol.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.

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.

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.

By making comparisons with the usual manual method, we evaluated an automatic fluorescent image analyzer (Image Titer, Tripath Imaging, Burlington NC), the software for which was developed to simplify measuring indirect immunofluorescent antinuclear antibodies (FANAs). In this new system, images of the stained sample are displayed, and it measures the FANA titer and staining pattern using only 1 slide per subject and does not required the staining of a series of diluted samples as does the manual method. This system showed good reproducibility and linearity for 4 types of control serum samples (with homogeneous, speckled, discrete speckled, and nucleolar staining patterns). In 132 serum samples, consistency between the methods was 100% for the FANA staining pattern and 93.9% for the FANA titer. The Image Titer system detected each pattern in samples with 2 mixed patterns. This system should partly reduce labor and lead to results with minimum differences among individuals, including newly trained persons. A variety of antinuclear antibodies (ANAs) are found in serum samples obtained from patients with autoimmune diseases. Identification of the corresponding antigens and measurement of the antibody titer are useful for diagnosis and to inform about the probable progress and prognosis of the disease. 1-3 At present, with HEp-2 cultured cells being used as a substrate, the indirect immunofluorescent antinuclear antibody (FANA) test is in use in many facilities for the measurement of ANAs. The FANA test permits determination of the corresponding antibodies by classification of staining types and has the advantage of permitting detection of a great variety of ANAs. 4,5 Therefore, it is considered a useful primary screening test for the identification of ANAs. On the other hand, the FANA test is not convenient for the treatment of a large number of samples since the assay of antibody titer by this test requires a complex dilution procedure. 6 Enzyme-linked immunosorbent assay (ELISA) FANA tests have been developed as routine screening tests. The method is convenient for large number of samples; however, a classification of staining types is not obtained. 7-11

Apolipoprotein (apo) E and its polymorphism are linked to the pathogenesis of late-onset and sporadic Alzheimer's disease (AD). ApoE facilitates the deposition and fibrillogenesis of beta-amyloid (Abeta), and may participate in Abeta clearance. We recently found that apo(E-AII) complex binds to Abeta much more strongly than does monomeric apoE. Here, we investigated the effect of apoAII on the interaction between apoE and Abeta. Addition of apoAII to apoE monomers increased the binding of apoE2 and apoE3 to Abeta(1-42), presumably following the formation of apo(E3-AII), apo(E2-AII), and apo(AII-E2-AII) complexes. This increased binding was not seen in the case of apoE4. When neuroblastoma cells were cultured in media containing Abeta(1-42) and a mixture of apoE3 and apoAII, intracellular Abeta was significantly reduced and cell viability was maintained at a higher level than in cells cultured without apoAII. ApoE2 itself seemed to act as an inhibitor of the endocytosis of Abeta, and we did not observe a significant effect of apoAII on the movement of Abeta in apoE2-containing medium. However, cell viability could be maintained at a higher level (as with apoE3) by adding apoAII to apoE2, despite the reduced viability of cells incubated without apoAII. In medium containing apoE4, both the amount of Abeta accumulated into cells and the cell viability were unchanged by the presence of apoAII in the medium. In addition, apoE4 itself was toxic, as previously suggested. These findings demonstrate that the type of apo(E-AII) complex present could underlie the isoform-specific role of apoE in the pathogenesis of AD.

Apolipoprotein E (apoE) is believed to be closely involved in the pathogenesis of Alzheimer's disease (AD) because of its ability to bind to β-amyloid (Aβ), the primary component of senile plaques. The presence of cystein residues in apoE2 and apoE3 allows these isoforms to form disulfide-linked complexes, such as apo(E–AII) complex and apo(AII–E–AII) complex. A 50-kDa complex [which corresponded to apo(E–AII)–Aβ, because it reacted with any of the three antibodies, anti-apoE, anti-apoAII, or anti-Aβ] was detected by immunoblot analysis in native cerebrospinal fluid (CSF) obtained from nondementia patients with the apoE phenotype E3/E3. However, a band considered to represent apoE–Aβ was not observed. The dissociation constant (Kd) values obtained for the specific binding of recombinant apoE2, apoE3, and apoE4 to Aβ1–42 were 48.1 ± 2.2 nM, 63.7 ± 2.1 nM, and 75.9 ± 1.8 nM, respectively. In contrast, the binding affinity of the partially purified apo(E3–AII) complex to Aβ1–42 was very high, the Kd being 5.5 ± 0.5 nM. No basic difference was observed between lipidated and nonlipidated apoE in terms of the characteristics of the binding of apoE isoforms to Aβ1–42; however, lipidation reduced the binding capacity of each isoform in a dose-dependent manner. These findings seem consistent with the generally accepted idea that apoE4 is a risk factor for AD, insofar as only apoE4 is unable to form a complex with apoAII owing to its lack of a cystein residue. In addition, it is possible that apoE3 monomer (and possibly apoE2 monomer), like apoE4 but unlike apo(E–AII) complex, can act as a risk factor in the pathogenesis of AD. J. Neurosci. Res. 58:301–307, 1999. © 1999 Wiley-Liss, Inc.

We investigated the exact prevalence of dementia of Alzheimer type (DAT) and apolipoprotein E (ApoE) phenotypes in 106 Japanese Down's syndrome (DS) patients. Among these patients 16 were diagnosed as having DAT. The prevalence of DAT was 0% in the 30- to 39-year-old group, 16% in the 40- to 49-year-old group, and 38% in those over 50 years old. The frequency of the epsilon4 allele in DS patients with DAT was 18.8%, which was considerably higher than that of nondemented DS patients (4.5%) and Japanese nondemented controls (6.7%). Especially, the frequency of the epsilon4 allele in DS patients who developed DAT under 50 years was significantly higher (28.6%). DS patients certainly develop DAT at earlier ages but the prevalence of DAT in each group of patients was lower than previously recognized. It is very likely that the ApoE epsilon4 is a risk factor for DAT even in DS patients with a genetic predisposition to Alzheimer's disease.

This study was designed to delineate the nature of beta-cell dysfunction in a model of genetically determined nonobese diabetes, the Goto-Kakizaki (GK) rat. Pancreatic beta-cell function was analyzed immediately after weaning and 5 weeks thereafter, comparing animals with or without insulin treatment during the interval. In 3.5-week-old GK rats, fasting plasma glucose was mildly elevated with normoinsulinemia, and the islet insulin content was reduced by 33%. When incubated with 3-30 mM glucose in vitro, the GK rat islets showed reduced glucose sensitivity, i.e. the EC50 values were 19.5 and 15.9 mM, and the Hill constants for the positive cooperativity 2.1 and 4.2 in the islets of GK and the control rats, respectively. On the other hand, the maximum response to glucose was not attenuated when reduced islet insulin content was considered. In 8.5-week-old GK rats hyperglycemia worsened and glucose-stimulated insulin release by the islets more severely impaired. A daily insulin injection from the 3.5-8.5 weeks of age significantly lowered plasma glucose in the GK rat, accompanied by a marked suppression of both basal (with 3 mM glucose) and glucose (6-30 mM)-stimulated insulin release by the islets. In the GK rat, beta-cell dysfunction develops by the age of 3.5 weeks, and insulin treatment during the subsequent 5 weeks accelerates its progression.

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.

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.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.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.

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

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.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, p0.001), but not with that of the serum LDL-C after treatment.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.

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.

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.

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.

Hypertriglyceridemia is an independent risk factor for atherosclerosis. This risk is most likely due to accumulation of circulating triglyceride rich lipoproteins with heterogeneous particles. The identification and characterization of these triglyceride rich lipoproteins is important to detect abnormality of triglyceride metabolism. In the present study, we developed a new method that combines ultracentrifugation and agarose gel electrophoresis with triglyceride- and cholesterol-staining. We investigated 40 subjects with hypertriglyceridemia. Triglyceride rich lipoproteins with very light density were recovered in the aqueous fraction after ultracentrifugation (17,000 x g, 15 min). The lipoproteins recovered in the aqueous fraction contained chylomicrons, if present, their remnants, and light-VLDL (d1.000 g/ml) containing apoB-100, but not normal VLDL (d1.006 g/ml) and IDL. Triglyceride rich lipoproteins in the aqueous fraction were characterized by electrophoresis patterns of triglyceride- and cholesterol-staining. Forty patients with hypertriglyceridemia were separated into 8 groups according to their electrophoretic patterns. In lipoproteins recovered in the aqueous fraction from each group, the triglyceride level was correlated with the respective cholesterol level. In summary, a system using ultracentrifugation and agarose gel electrophoresis with triglyceride- and cholesterol-staining is useful for characterization of triglyceride rich lipoproteins and their remnants.

Apolipoprotein (apo) J, abundant in cerebrospinal fluid (CSF), is known to play a role in the pathogenesis of Alzheimer's disease (AD); however, the mechanism remains obscure. To characterize the apoJ-containing lipoproteins in CSF, we compared the distribution of apoJ in CSF lipoprotein partides with those of apoE and apoAI. CSF lipoproteins (fractionated by ultracentrifugation, gel-filtration chromatography, and agarose-gel electrophoresis) were characterized by immunoblot analysis using anti-apoJ, anti-apoE, and anti-apoAI antibodies. Immunoprecipitation and immunoabsorption were used to clarify the combinations in which these apolipoproteins exist. All of the apoJ in CSF was in the fraction with density ofor = 1.250 g/ml after ultracentrifugation; relatively little apoE and apoAI was in that fraction. In gel-filtration chromatography, the main peak of apoJ-containing lipoprotein particles was clearly distinguishable from those of apoE- and apoAI-containing lipoproteins. Immunoabsorption and agarose-gel electrophoresis indicated that the dominant apoJ-containing lipoprotein partides did not contain apoE. These findings indicate that a significant fraction of the apoJ present in CSF does not co-exist with apoE or apoAI within the same particles. Immunoprecipitation revealed two types of particles: one that contains no apoAl but apoE and another that contains no apoE but apoAI. These results show that several subfractions of lipoprotein particles exist in CSF, differing from each other in their combinations of apoE, apoJ, and apoAI. We concluded that there are at least 9 forms or combinations (including free apolipoproteins) of apoJ, apoE, and apoAl in the CSF.

Functional heterogeneity of pancreatic islets was systematically analyzed for the first time using freshly isolated single rat pancreatic islets. First, 60 islets were sequentially exposed to 3, 9.4, 15.6, and 24.1 mM glucose for 30 min each in incubation experiments: 36 (60%) responded in a concentration-dependent and 19 (32%) in an all-or-none manner, and 5 (8%) islets did not respond to high glucose. As a group, the larger the islet, the higher the beta cell glucose sensitivity. However, glucose-stimulated elevation of [Ca2+]i in the beta cell. insulin/glucagon ratio in the islet, and expression of glucose transporter 2, glucokinase, and pancreatic duodenal homeobox factor-1 in the beta cell were not significantly related to islet size. Second, 50 islets were stimulated with 16.7 mM glucose in perifusion. A biphasic insulin release was found in 39 (78%), and no or little first phase response in 11 (22%) islets, irrespective of the islet size. Nevertheless, when the response was plotted as a group, it was clearly biphasic. Islet size, insulin content and the amount of insulin release were positively correlated with each other. In conclusion, there are size-related and size-unrelated functional diversity among pancreatic islets. The reason for such heterogeneity remained to be determined.

BACKGROUND AND AIMS—Helicobacter pylori locate not only on the apical surface of surface mucous cells but also in the mucous gel layer covering the gastric mucosa. The present study was undertaken to observe the mucous gel layer itself and any H pylori in this layer at the electron microscopic level, and to determine whether H pylori proliferate in this layer. METHODS—We examined resected human stomachs (five cases, fixed in Carnoy's solution, paraffin embedded) under the light microscope, and gastric biopsy specimens (10 cases, fixed in glutaraldehyde with or without osmium, epoxy embedded) under the electron microscope. We performed histochemical staining for gastric mucins and immunostaining for H pylori, gastric gland mucous type mucins, and intestinal mucins. RESULTS—Under the electron microscope, surface mucous cell type mucins and gland mucous cell type mucins in the mucous gel layer covering gastric mucosa without intestinal metaplasia showed reticular and band like structures, respectively. H pylori were frequently found as small aggregates within the mucous gel layer of surface mucous cell type mucins, and H pylori within these aggregates were seen dividing. H pylori were frequently found in the mucous gel layer of the surface mucous cell type mucins along the border with the layer of gland mucous cell mucins. Occasionally, H pylori were trapped by frayed thin threads of the gland mucous cell type mucins. CONCLUSIONS—The two types of gastric mucins in the mucous gel layer differ in ultrastructure. H pylori preferentially colonise and form microcolonies within the mucous gel layer of surface mucous cell type mucins. Mucins from gland mucous cells may disturb the movement of H pylori within the mucous gel layer. Keywords: gastric mucin; Helicobacter pylori; immunohistochemistry; surface mucous gel layer

The specificity of monoclonal antibodies against gastric mucins (designated as HIK1083, PGM 36, and PGM 37) was studied immunohistochemically in normal, metaplastic, and neoplastic human tissues. These antibodies labeled class III mucin-producing cells identified by paradoxical concanavalin A staining in normal stomach, duodenum (Brunner gland), biliary tract, and main pancreatic duct; in mucinous metaplasia of pancreas and gallbladder; and in adenocarcinomas of stomach (90%), bile duct (80%), gallbladder (100%), pancreas (80%), lung (100% of goblet cell type adenocarcinomas), ovary (67% of mucinous carcinomas), and uterine cervix (100% of adenoma malignum tumors). Normal and neoplastic cells of esophagus, colon, salivary gland, kidney, endometrium, breast, prostate, and liver, as well as normal small intestine, lung, and uterine cervix, were all negative. The antibodies used should be valuable for the detection of class III mucin and class III mucin-producing cells in normal, metaplastic, and neoplastic tissues.

To analyze both hemoglobin (Hb) and globin chain variants, we modified a commonly used method, capillary isoelectric focusing (CIEF), with detection at 280 nm. The samples were hemolysates prepared from red blood cells, and globin chains obtained from the hemolysates by treatment with cold acidified acetone. When the migration time for the internal reference, carbonic anhydrase I (isoelectric point, pI 6.60), was taken as 1.0, the migration ratio for Hb A0 in normal human blood was 0.877 +/- 0.004 (mean +/- SD, n = 9), and those of the alpha- and beta-globin chains were 0.673 +/- 0.004 and 0.847 +/- 0.005 (mean +/- SD, n = 4), respectively. The ratio of peak heights between the beta- and alpha-globin chains (beta/alpha) in the normal Hbs obtained from four subjects was almost constant at 2.5 +/- 0.1 (mean +/- SD). This ratio indicates which of the globin chains includes a mutation (if one exists). When an Hb variant, Hb Hoshida (in which Gln is substituted for Glu at residue 43 in the beta-globin chain), was analyzed by this method, two main peaks were observed (migration ratios 0.836 and 0.877, corresponding to an abnormal and the normal Hb, respectively). An additional peak with an abnormal migration ratio of 0.788 was also detected in the globin chain profiles. The ratio of peak heights between normal beta- and alpha-globin chains was 1.57, indicating that a mutation exists in the beta-globin chain. We thus established a convenient system using CIEF that provides a rapid and reproducible method for the random analysis of both Hb and globin chain variants.

Class III mucin, identified by paradoxical concanavalin A staining, is confined to gastric gland mucous cells and is an essential component of the gastric surface mucous gel layer. The pretreatment required has hampered the application of this method to electron microscopic studies. Antibody HIK1083 reacts selectively with class III mucins. The present study was undertaken to explore, electron microscopically, the immunoreactivity of the human stomach to HIK1083. We examined normal mucosa from resected human stomachs (five cases; formalin-fixed, paraffin-embedded) and gastric biopsy specimens from patients with early gastric cancer [nine cases; glutaraldehyde- and osmium-fixed, epoxy-embedded (seven cases) and half-strength Karnovsky's solution-fixed, Lowicryl K4M-embedded (two cases)]. Immunostaining with HIK1083 and anti-lysozyme antibody was examined under light and electron microscopes. Gland mucous cells were labeled with HIK1083, and lysozyme was detected in some gland mucous cells and surface mucous cells. Electron microscopically, the secretory granules of gland mucous cells contained a single electron-dense core. HIK1083-positive mucins and lysozyme coexisted in the secretory granules of gastric gland mucous cells. HIK1083-reactive mucins and lysozyme were distributed in the matrix and in the dense core of these secretory granules, respectively. HIK1083 can be used for electron immunohistochemistry.

Background: Apolipoprotein (apo) E, one of the main apolipoproteins in the central nervous system, may play an important role in lipid metabolism; however, the details of its function are poorly understood. In this study, we characterized apoE-containing lipoproteins in cerebrospinal fluid (CSF) and examined the effect of apoE phenotype on the distribution of apoE among the lipoprotein fractions.Methods: CSF lipoproteins were fractionated by gel filtration and ultracentrifugation, and then characterized by electrophoresis, immunoblot, electron microscopy, and analysis of apoE, total cholesterol, and phospholipid concentrations.Results: The ratio of sialylated to nonsialylated apoE was higher in CSF than in serum. However, the fundamental forms containing apoE homodimers or heterodimers [such as apo(E-AII) and apo(AII-E2-AII) complexes] were similar in CSF and serum. apoE-containing lipoproteins were fractionated at densities of 1.125 kg/L obtained from CSF associated with apoE2 or apoE3. This tendency was more obvious in CSF associated with apoE2 than in CSF without apoE2. apoE-containing lipoproteins were predominantly observed in the fraction with a density of Conclusions: The lipoproteins in CSF have a unique composition that is different from that of the lipoproteins in plasma. However, the differences in diameter between the CSF fractions were not as large as for the serum fractions. Our data suggest that the apoE phenotype may affect the distribution profile of apoE-containing lipoproteins in the CSF. This would mean that the metabolism of apoE-containing lipoproteins depends on the apoE isoform present.

We previously reported the identity and purifica- tion of two HDL 3 -binding proteins in rat liver plasma mem- branes. As these proteins are candidate high density lipo- protein (HDL) receptors and probably multifunctional, including a role in HDL metabolism, we have considerable interest in identifying corresponding proteins that are present in human tissue. This report describes the identifi- cation of HDL 3 -binding sites on human monocytes with the use of fluorescence microscopy and flow cytometry assay. After the incubation of mononuclear cells from human blood with fluorescein isothiocyanate (FITC)-labeled hu- man HDL 3 , fluorescence micrographs showed dense signals of fluorescent grains on monocytes, but not lymphocytes. A significant increase in FITC intensity on monocytes, but not lymphocytes, was observed by flow cytometry analysis, and the interaction between FITC-HDL 3 and human monocytes was concentration-dependent. Although very low density (VLDL) and low density lipoprotein (LDL) were ineffective competitors and HDL 2 only partially competed for binding, a 50-fold concentration of HDL 3 did compete effectively for binding of FITC-HDL 3 to human monocytes. Trypsin treatment reduced the FITC intensity of monocytes, show- ing that a portion of cell-associated FITC-HDL 3 remained bound to the cell surface. Two major HDL-binding proteins were identified in CHAPS-solubilized human mononuclear cells by ligand blotting, using HDL 3 as the ligand. Both showed similar binding parameters, specificity, and molecu- lar weight identical to HB 1 and HB 2 from rat liver plasma membrane. We conclude that corresponding candidate HDL receptors or a similar receptor complex also exist on hu- man blood monocytes. —Hidaka, H., E. Hidaka, M. Tozuka, J. Nakayama, T. Katsuyama, and N. Fidge. Identification of specific high density lipoprotein 3 binding sites on human blood monocytes using fluorescence-labeled ligand. J. Lipid Res. 1999. 40: 1131-1139.

Background: Apolipoprotein (apo) E may be related to the development of Alzheimer disease, but data on apoE in cerebrospinal fluid (CSF) are limited. The aim of the present study was to measure apoE in CSF and relate its concentrations to apoE phenotype and CSF lipids.Methods: We adapted an assay for CSF apoE sensitivity using an ELISA. It allowed us to measure CSF apoE with sufficient reproducibility and precision.Results: The within- and between-run CVs were 5 year group (n = 34; 8.82 ± 3.31 mg/L). The mean concentrations of total cholesterol (TC) and phospholipid (PL) in CSF were 2.68 ± 2.16 and 6.50 ± 2.84 mg/L (n = 52), respectively. Although no significant differences in TC or PL in the CSF were found with respect to sex or age, the concentrations in subjects with the apoE phenotype E4/E3 were significantly lower than in those with E3/E3 and E3/E2. The concentrations of apoE, TC, and PL in CSF did not correlate with those in plasma. The time-related fluctuations in CSF apoE were independent of those in total protein and IgG. CSF apoE was significantly correlated with TC and PL concentrations in the CSF, but not with the number of cells in the CSF.Conclusions: These findings support the idea that apoE and lipids are unable to cross the blood-brain barrier and that their concentrations in CSF may reflect production in central nervous tissue.

This is the first systematic study on the pancreatic beta cell function in the heterozygous mouse with targeted disruption of the beta cell glucokinase gene. The heterozygotes' beta cell displayed the following characteristics: (1) impaired glucose sensitivity with normal glucose responsiveness, (2) poor discrimination of alpha and beta glucose anomers, and (3) normal response to glucose in the presence of 25 mM K+ and 150 microM diazoxide. Both the first and the second phases of glucose-stimulated insulin release were depressed. Although the heterozygotes were mildly hyperglycemic, insulin treatment further suppressed beta cell function, implying the beta cell glucose toxicity is not the cause of impaired glucose sensitivity. The data are compatible with the glucokinase glucose sensor concept inasmuch as glucose sensitivity is reduced in the heterozygotes' beta cell. The anomeric malaise and preservation of the ATP-sensitive K+ channel-independent glucose action were considered due to chronic hyperglycemia.

It has been shown that the Apolipoprotein E (ApoE) epsilon4 allele increases the risk of developing Alzheimer's disease (AD) and lowers the age of its onset. ApoE has also been suggested to be a common facilitating factor in the different types of amyloidoses. However, the association of ApoE epsilon4 with the onset of disease in various types of amyloidoses has not been extensively investigated. Type I familial amyloid polyneuropathy (FAP) is one form of systemic amyloidosis in which ApoE co-localizes with amyloid deposits. We examined 54 patients with type I FAP and found that there was no significant effect of either ApoE epsilon2 or epsilon4 allele on the age at onset. Our results suggest that ApoE4 is not a facilitating factor in the development of FAP, transthyretin amyloidosis.

P-243 Hyperuricemia is Associated with the Presence of Lifestyle-Related Diseases Among Male Japanese Junior High School Students Minoru Hongo1, Hiroya Hidaka2, Shigeko Sakaguchi3, Uichi Ikeda4, Yoshikazu Yazaki5, Goro Tsuruta6, Kenichi Koike6. 1Department of Cardiovascular Medicine, Shinshu University, Japan, 2Department of Biomedical Laboratory Medicine, Shinshu University School of Health Sciences, Japan, 3Department of Nursing, Shinshu University School of Health Sciences, Japan, 4Department of Cardiovascular Medicine, Shinshu University School of Medicine, Japan, 5Department of Cardiovascular Medicine, National Matsumoto Hospital, Japan, 6Department of Pediatrics, Shinshu University School of Medicine, Japan