[Genetic tests and items to be noted at the diagnostic laboratory].
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Confidentiality↗
Biomedical subjects
M Tozuka
Publications and source records attributed to M Tozuka.
The identification of specific high density lipoprotein3 binding sites on human blood monocytes using fluorescence-labeled ligand.
We previously reported the identity and purification of two HDL3-binding proteins in rat liver plasma membranes. As these proteins are candidate high density lipoprotein (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 identification of HDL3-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 human HDL3, 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-HDL3 and human monocytes was concentration-dependent. Although very low density (VLDL) and low density lipoprotein (LDL) were ineffective competitors and HDL2 only partially competed for binding, a 50-fold concentration of HDL3 did compete effectively for binding of FITC-HDL3 to human monocytes. Trypsin treatment reduced the FITC intensity of monocytes, showing that a portion of cell-associated FITC-HDL3 remained bound to the cell surface. Two major HDL-binding proteins were identified in CHAPS-solubilized human mononuclear cells by ligand blotting, using HDL3 as the ligand. Both showed similar binding parameters, specificity, and molecular weight identical to HB1 and HB2 from rat liver plasma membrane. We conclude that corresponding candidate HDL receptors or a similar receptor complex also exist on human blood monocytes.
Binding Sites↗
Fibrinogen Matsumoto III: a variant with gamma275 Arg-->Cys (CGC-->TGC)--comparison of fibrin polymerization properties with those of Matsumoto I (gamma364 Asp-->His) and Matsumoto II (gamma308 Asn-->Lys).
Fibrinogen Matsumoto III (M-III) is a dysfibrinogen identified in a 66-year-old woman with rectal cancer. The fibrinogen level determined by the thrombin-time method was markedly decreased in preoperative coagulation tests of her plasma. Three fibrinogen polypeptide-chain gene fragments from the proposita were amplified by the polymerase chain reaction method, then sequenced. The triplet CGC encoding the amino acid residue gamma275 was replaced by TGC, resulting in the substitution of Arg->Cys. There have been previous reports of nine families with the same alteration, nine families with an Arg->His variant and one family with an Arg->Ser variant in this residue, which has been shown to be one of the most important amino acids in the 'D:D' interaction site. In addition, there are three silent mutations in the Aalpha-chain gene and two mutations in the intron of the Bbeta-chain and the gamma-chain gene. However, none of these mutations is thought to be the cause of the dysfunctional fibrinogen. The thrombin-catalyzed fibrin polymerization in the presence of 1 mM Ca ions was markedly delayed in purified M-III. Its lag period was longer than those of Matsumoto II (M-II; gamma308Asn->Lys) and Matsumoto I (M-I; gamma364Asp-His). gamma364Asp is one of the most important residues in the polymerization pocket of the 'D:E' interaction site and gamma308Asn is located in the vicinity of a high affinity Ca2+ binding site in the D-domain, gamma311-336. The maximum slope of the polymerization curve for M-III was about 4-fold steeper than that for M-1 but less steep than that for M-II. These results may suggest that the tertiary structure of the polymerization pocket plays a more important role in the lateral aggregation of protofibrils than that of the 'D:D' interaction site.
Aged↗
Characterization of apolipoprotein E-containing lipoproteins in cerebrospinal fluid: effect of phenotype on the distribution of apolipoprotein E.
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 <1.006, 1.063-1.125, and 1.125-1.21 kg/L. Neither apoE nor apoAI was detected in the fraction with a density range of 1.006-1.063 kg/L. The diameters of the lipoprotein particles with densities of <1.006, 1.063-1.125, and 1.125-1.21 kg/L were 16.7 +/- 3.1, 14.0 +/- 3.2, and 11.6 +/- 2.8 nm (mean +/- SD, n = 200), respectively. All of these lipoproteins exhibited a spherical structure. The distribution profile of apoE-containing lipoproteins was affected by the apoE phenotype. A relatively large amount of apoE-containing lipoproteins was isolated from the fraction with a density >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 <1.006 kg/L obtained from CSF associated with apoE4. 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.
Apolipoproteins E↗
[Clinical evaluation of PCR method for detection of cytomegalovirus DNA].
Polymerase chain reaction (PCR) to detect Cytomegalovirus (CMV)-DNA from the clinical specimens is useful to diagnose CMV infection. Eighty-one specimens of 31 patients including peripheral blood, bronchioalveolar lavage fluid, biopsy tissues, feces, urine, sputum and etc. and normal peripheral blood from 59 volunteers were used in this study. After DNA extraction each samples was amplified by the seminested PCR using primers recognizing sequences in the Immediate-early gene of CMV. This PCR method specifically detected more than 10 virus copies even in the presence of the genomic DNA. CMV-DNA was detected in only one of 59 normal peripheral bloods (1.7%). Six of 31 patients were clinically diagnosed as CMV infection by anti-CMV therapy. These 6 patients were positive in the peripheral blood by PCR for CMV, and 5 of them were positive in other samples. However, 3, 5 and 1 of 25 patients, who were clinically diagnosed as not having CMV infection, were also positive in peripheral blood, in the other samples and in both, respectively. The PCR method was able to examine any clinical samples. To examine both the peripheral blood and the samples from infected organs is helpful for the diagnosis of CMV infection.
Adolescent↗
[Genetic examination in clinical laboratory].
Genetic technology is finding active application today in the field of clinical laboratory medicine. Genetic examinations are divided into following three main classes: 1) examination for infectious disease according to the detection of the gene derived from bacteria or viruses, 2) examination for inherited disease according to molecular analysis of the genetic variation, 3) examination for oncogene according to molecular analysis of genetic abnormalities. At present, the main genetic examination in a large number of laboratories is for infectious disease because of its relatively simplified technique and high demand. The division of genetics is not a new independent section of clinical laboratory, but rather an ultramodern and powerful tool for existing divisions, such as biochemistry, serology, hematology, microbiology, and pathology. Genetic technology quickly provides results with high sensitivity and reliability, and plays a role at the core of the clinical laboratory. We should remember that the genetic technology is a great present given to clinical laboratories, however, it will eventually change into only one of the routine examinations according to the method of used. Examinations utilized in the clinical laboratory must be well established and standardized. Genetic examinations are no exception to that rule. These tests require a remarkably high precision since the results have an extraordinarily important meaning. There are more than 8,000 inherited diseases for instance. It is difficult to cover all examinations for those 8,000 in one laboratory. We need a network of laboratories that possess a genetic division, so that the examinations for as many inherited disease as possible can be comprehensively offered.
Clinical Laboratory Techniques↗
Plasminogen Kanagawa-I, a novel missense mutation, is caused by the amino acid substitution G732R.
A new dysplasminogen, plasminogen Kanagawa-I, was identified in a healthy male with no previous thrombotic episodes. His plasma plasminogen (PLG) activity was 51.4% of that of normal pooled plasma (reference interval 70-130%) and the antigen level was 94.2% of that of normal pooled plasma (reference interval 80-150%). Nucleotide sequencing revealed a heterozygous G to A transition in exon 18, which resulted in an amino acid substitution of G732R. Both the proband's father and paternal grandfather were heterozygous for this mutation. Interestingly, the grandfather was found to be a compound heterozygote for plasminogen Kanagawa-I and Tochigi (A601T), so that his plasminogen activity and antigen level was 7.7% and 87.2% of that of normal pooled plasma, respectively. However, he has never been affected by significant thrombosis.
Adult↗
[Measurement of fibrinogen binding to platelets by flow cytometry: evaluation method for reflecting platelet activation].
Platelet aggregation, induced by agonist-mediated activation of membrane glycoprotein (GP) IIb/IIIa, and binding of fibrinogen to GPIIb/IIIa, is commonly analyzed using an aggregometer in the clinical laboratories. However, this method has a limitation to get precise results on the samples with small number of platelet (less than 100,000/1) or hyperlipidemia. Recently, flow cytometry has been used to evaluate platelet function due to the detection of fibrinogen binding to activated platelets using fluorescence labeled fibrinogen or anti-fibrinogen antibody. However, the appropriate rule for evaluation of the results has not been established yet. We converted a ratio of fibrinogen binding platelets to a velocity per unit concentration of ADP as follows: a difference of two ratios of fibrinogen binding platelets on neighboring two ADP concentrations was divided by a difference of ADP concentrations. It was considered to be a mean velocity between the two ADP concentrations. We adopted the range of ADP concentration, which gave the maximum velocity, as an index of platelet activation. If the peak of maximum velocity move toward lower or higher ADP concentration, it means hyper- or hypoactivation of the platelets, respectively. The objectivity of this method may make it a useful technique for clinical examination of platelet function.
Adenosine Diphosphate↗
[New approach from lipoprotein disorders to atherosclerosis].
To make diagnosis arteriosclerosis directly by biochemical markers is not easy, but to identify risk factors by biochemical markers is useful. Lipoprotein disorder is one such risk factor. Low density lipoproteins (LDL), remnants and small LDL were high risks of coronary disease in Japanese. Moreover, those incidences were significantly higher in diabetes mellitus, especially with nephropathy, and latter two lipoproteins frequently coexisted. Oxidizability of small LDL was the highest among LDLs, indicating that small LDL promotes atherosclerosis by forming oxidized lipids, which enhance complicated lesion of atherosclerosis. The mechanism by which the remnant is retained remains unknown. We measured LPL mass in preheparin serum. Preheparin LPL mass was negatively correlated with triglyceride, and positively correlated with high density lipoprotein cholesterol. Further more, preheparin LPL mass was lower in remnant-positive persons, indicating that preheparin LPL mass might be involved in remnant clearance. Understanding the role and catabolism of LPL itself requires further study.
Arteriosclerosis↗
[Determination of neutrophil function by measuring superoxide production with whole blood flow cytometry].
The function of neutrophil can be evaluated by measuring oxidative metabolism using chemiluminescence, tetrazolium dye reduction or the others. Those results are not always satisfactory which would be caused by subtle difference in each preparation of the reagents and the lack of reproducibility. Recently, flow cytometric procedures for semi-quantitating superoxide production in neutrophils have been developed to evaluate their function. This procedure, which requires only small amount of whole blood, can easily and rapidly yield reproducible and reliable data. In this study, we optimized analytical conditions and then determined reference interval to evaluate neutrophil function of patients with various disorders. Optimal concentrations and incubation times of DCFH-DA and PMA were 5 mumol/l for 15 minutes and 25 micrograms/ml for 20 minutes, respectively. Production of superoxide in neutrophil was represented by relative fluorescence intensity(RFI) with assay coefficient of variance(CV) of 4.0-11.1%. Neutrophils had to be examined within 2 hours after venipuncture to obtain reliable data. Reference interval was determined as 170.4 +/- 58.7(mean +/- SD) RFI. Neutrophil function of patients with neutropenia, paroxysmal nocturnal hemoglobinuria(PNH), renal failure, systemic lupus erythematosus(SLE), myeloperoxidase deficiency, myelodysplastic syndrome(MDS), and diabetes mellitus were within the reference interval as evaluated by this method. Only neutrophils of chronic granulomatous disease, which is known to give clearly low superoxide production, showed actually decreased value. These results indicate that this procedure would be clinically useful for diagnosis of patient with impaired neutrophil function.
Adult↗
[Distribution of the apolipoprotein-E and -E-complexes in lipoproteins of human serum with the apolipoprotein-E4/E3 phenotype].
Distribution of apolipoprotein(apo) E4 and E3 in lipoproteins of serum with apoE4/E3 phenotype was analyzed. ApoE was eluted in two major peaks by gel chromatography; peak 1 and 2 corresponding to very- and intermediate-low density lipoprotein (VLDL + IDL) and high density lipoprotein2 (HDL2), respectively. ApoE in peak 1 (VLDL + IDL) consisted of monomers of 34 kDa, complexes with a high molecular weight (apoEs) of 100 kDa and with a small amount of apoE-AII complexes weighing 43 kDa. In contrast, apoE in peak 2 (HDL2) was composed mainly of apoE-AII complexes and apoEs complexes, and a small amount of monomers. Both apoE3 and E4 isoforms were detected in these peaks; E4 was more predominant in peak 1 while E3 was more predominant in peak 2. These findings suggest that different distributions of apoE3 and E4 in lipoprotein particles.
Apolipoprotein A-II↗
Influence of apolipoprotein E phenotypes on total serum cholesterol in Japanese children.
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Adolescent↗
A case of immunoglobulin A-lambda conjugated with lactate dehydrogenase-5 isoenzyme, causing an extremely high enzyme activity in serum.
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Antibody Specificity↗
[A study of low density lipoprotein in a patient with hyper-high density lipoprotein cholesterolemia with normal cholesterolester transfer protein].
We examined a patient with hyper-cholesterolemia with a high level of HDL2 and LDL-cholesterol in serum. The metabolism of lipoproteins in this case was different from that in well-known hyper-high density lipoproteinemia or hyper-low density lipoproteinemia, because the patient had normal levels of cholesterolester transfer protein, lipoprotein lipase and hepatic triglyceride lipase activity. This study describes the characterization of LDL obtained from the patient's serum. LDL from the patient was separated by ultracentrifuge, and analyzed by gradient PAGE. The molecular weight of two LDL from the patient have been estimated to be approximately 1250 and 1450kDa by polyacrylamide gel electrophoresis, and were larger than those of normal individuals and patients with typical hypercholesterolemia (approximately 1150kDa in molecular weight). The LDL from the patient was separated into three fractions by HPLC, and their lipid composition was not significantly different from that of normal LDL. The high level and large size of LDL from the patient may be caused by a reduction in the transfer of cholesterol from LDL to HDL2, or an equilibrium of cholesterol with the increased HDL2.
Aged↗
Possible link between a low prevalence of cardiovascular disease and mild dyslipidaemia: a study in Japanese patients with type 2 diabetes.
In 98 Japanese patients with Type 2 diabetes mellitus, serum total cholesterol, triglyceride, high density lipoprotein cholesterol (HDL-C), free fatty acid (FFA), and apolipoproteins (apo) A-I, A-II, B, C-II, C-III, and E were determined. The data were compared with those in 47 normolipidaemic normal controls. The total cholesterol value of the diabetic patients was also compared to that of a general population (n = 2227). The diabetic patients were separated into those with cardiovascular disease (n = 20) and without it (n = 78) and a comparison of clinical characteristics and dyslipidaemia was also performed. The diabetic patients had slightly but significantly higher FFA, LDL-C, apo B, C-II, C-III, E, and B/A-I, and lower apo A-I and A-II compared to the normal controls. The total cholesterol level of the diabetic patients (5.17 +/- 0.96 mmol-1) was not significantly higher than that of the general population (5.12 +/- 0.91 mmol-1). By multivariate stepwise discriminant analyses, only total cholesterol significantly discriminated the patients with and without cardiovascular disease. In Japanese patients with Type 2 diabetes, a diabetic population with a very low prevalence of cardiovascular disease, high total cholesterol is a risk factor for developing cardiovascular disease. Nevertheless, a markedly low prevalence of cardiovascular disease in Japanese with Type 2 diabetes compared to Caucasian counterparts may partly be due to the mildness of dyslipidaemia.
Aged↗
Identification and characterization of apolipoprotein(AII-E2-AII) complex in human plasma lipoprotein.
A new apolipoprotein complex designated as the apo(AII-E2-AII) complex was identified in the lipoprotein fractions of human plasma with apoE phenotypes containing apoE2 (E4/E2, E3/E2, and E2/E2). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by an immunoblotting assay using anti-apoE or anti-apoAII antibodies, established that the apo(AII-E2-AII) complex, with a molecular weight of 58,000, was identical to the complex consisting of apoE and apoAII, and that it also dissociated following reduction with beta-mercaptoethanol. This new complex was also demonstrated to be distinct from the apo(E-AII) complex and apoE monomer by isoelectric focusing, in the samples that were not treated with beta-mercaptoethanol. In apoE phenotype E3/E2, the apo(AII-E2-AII) complex was primarily included in the high-density lipoprotein (HDL, 1.063 < d < 1.21 g/ml) fraction, but was also observed in a small quantity in the very-low-density lipoprotein (VLDL, d < 1.006 g/ml) fraction. For further characterization, the apo(AII-E2-AII) complex was isolated by preparative SDS-PAGE, and no contamination of apo(E-AII) complex and apoE monomer was detected by immunoblotting assay using an anti-apoE antibody. It was confirmed by an enzyme-linked immunosorbent assay (ELISA) system that a molecular ratio between apoAII monomer and apoE in the isolated apo(AII-E2-AII) complex was approx. 2, when the apo(E-AII) complex was used as a standard with the ratio of 1:1. It indicates that the apo(AII-E2-AII) complex is formed from two molecules of apoAII monomer and one molecule of apoE.(ABSTRACT TRUNCATED AT 250 WORDS)
Apolipoprotein A-II↗
Determination of the structural domain of ApoAI recognized by high density lipoprotein receptors.
There is good evidence that high density lipoprotein (HDL) interacts with high affinity sites present on hepatocytes. The precise nature of the ligand recognized by putative HDL receptors remains controversial, although there is a consensus that apolipoprotein AI (apoAI) is involved. This suggestion would be strengthened if a biologically active site demonstrating a high affinity for the receptor could be isolated. Cyanogen bromide fragments (CF) of apoAI (CF1-CF4) were complexed with phospholipid, and their ability to associate with the receptor was compared in various binding studies. Careful analysis of the concentration-dependent association of 125I-labeled dimyristoyl phosphatidylcholine (DMPC) recombinants to rat liver plasma membranes revealed high and low affinity binding components. As all DMPC recombinants displayed the low affinity binding component, it was postulated that this interaction was independent of the protein present in the particle and may well represent a lipid-lipid or lipid-protein association with the membranes. Only 125I-labeled CF4.DMPC displayed a high affinity binding component with similar Kd and Bmax (8 x 10(-9) M, 1.6 x 10(-12) mol/mg plasma membrane protein) to that of 125I-labeled AI.DMPC (7 x 10(-9), 1.4 x 10(-12) mol/mg plasma membrane protein). Similarly, egg yolk phosphatidylcholine complexes containing CF4 (CF4.egg PC) showed higher affinity binding than CF1-egg yolk phosphatidylcholine complexes confirming the results obtained with DMPC complexes. Furthermore, ligand blotting studies showed that only 125I-labeled CF4.DMPC associated specifically with HB1 and HB2, two HDL binding proteins recently identified in rat liver plasma membranes. We conclude that a region within the carboxyl-terminus of apoAI is responsible for the interaction with putative HDL receptors present in rat liver plasma membranes.
Amino Acids↗
Development of an enzyme-linked immunosorbent assay of apolipoprotein E-AII complex in plasma.
Measurement of apolipoprotein (apo) E-AII complex in human plasma is important in determining the role of apoE in lipoprotein metabolism. In this paper, we demonstrate a new and simple method to determine apoE-All complex by using an enzyme-linked immunosorbent assay. Anti-apoE IgG (goat) was used as a capture antibody, and captured apoE-All complexes were detected by an anti-apoAll (rabbit) horseradish peroxidase-conjugated anti-rabbit IgG (goat) system. With this method, apoE-All complex was specifically determined without the interference of apoAll and was not affected by apoE monomer less than 250 mg/L. The content of the complex in reference serum, a normolipidemic serum pooled from five subjects with phenotype E3/E3, was arbitrarily defined as 100%. The coefficients of variation were 3.5%-6.3% within assay and 8.8%-11.6% between assays.
Apolipoproteins E↗