RESUMO
Clinical laboratory studies utilizing residual specimens after laboratory testing have markedly contributed to the development of medical science. However, according to recent rules regarding ethical aspects, the ethical committee of the Japanese Society of Laboratory Medicine (JSLM) developed ethical guidelines for treating residual specimens. In this symposium, several ethical problems concerning the application of such specimens to laboratory investigation were raised and discussed. Concerning whether or not each informed consent should be ob- tained before the secondary use of the residual specimens, this matter itself revealed serious disagreements among institutes. Therefore, the ethical committee of JSLM has to propose uniform and widely acceptable guidelines for the effective utilization of laboratory specimens, aiming at the advancement of laboratory medi- cine. [Review].
Assuntos
Manejo de Espécimes/ética , Guias como AssuntoRESUMO
The immature platelet fraction (IPF%) is a parameter for the automatic quantification of reticulated plate- lets and is considered to reflect platelet production. We evaluated IPF% measurements using an automated analyzer, the Sysmex XN. We measured the platelet counts and the IPF% values in 35 healthy subjects and 275 patients with various diseases using both the XN analyzer and a conventional analyzer, the Sysmex XE. Significant correlations in the platelet count and the IPF% were observed between the XN results and the XE results. In the same samples, significant inverse correlations were observed between the platelet count and the IPF% in both the XN and XE results. The coefficient of variation values for the platelet count and the IPF% measurements obtained using the XN analyzer were lower than those obtained using the XE ana- lyzer. In patients who had undergone hematopoietic stem cell transplantation, the IPF% measurements obtained using the XN analyzer increased several days before platelet recovery. IPF% measurements performed using the XN analyzer are adequate for clinical use. This parameter may be a useful marker for the prediction of platelet recovery. [Original].
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Plaquetas/citologia , Contagem de Plaquetas/métodos , Automação Laboratorial , Humanos , Reprodutibilidade dos TestesRESUMO
Paraproteinemia is a condition induced by an increase in paraprotein. Paraprotein is a monoclonal immu- noglobulin produced by plasma cells as a result of aging or malignancy. Paraprotein often induces a variety of laboratory test abnormalities by interfering with laboratory test reagents. The haptoglobin (Hp) measurement in a 55-year-old woman with IgM-lambda type paraproteinemia associated with lymphoplasmacytic lymphoma was found to be falsely low because of white-turbidity caused by an abnormal reaction. An in- creased absorbance was observed at 596 nm after the addition of the buffer reagent and was reduced by dilution with saline. This result indicates the existence of interfering substances in the patient's sample. To identify the inhibitors, we obtained the white-turbidity pellet by centrifugation of a mixture of the patient's serum and the Hp buffer reagent. A high IgM concentration was observed in the white-turbidity pellet. Moreover, a correlation was observed in a time series between the IgM concentration in sera and the extent of the turbidity during the Hp measurement. These results indicated that IgM was the main component of the white-turbidity. Next, we showed that the addition of the white-turbidity pellet to normal serum caused an increase in absorbance and a false low Hp value. A correlation was also observed in a time series be- tween the IgM concentration in sera and the rate of the Hp reduction. These results suggest that the false low Hp measurements were due to IgM present in the white-turbidity. In conclusion, false low values of Hp may occur in patients with IgM-lambda type paraproteinemia. Therefore, the presence or absence of an increase in absorbance after the addition of the buffer reagent in a time-course reaction may be required.
Assuntos
Haptoglobinas/análise , Imunoglobulina M/sangue , Cadeias lambda de Imunoglobulina/sangue , Paraproteinemias/diagnóstico , Reações Falso-Negativas , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
The measured concentration of thyroid stimulating hormone (TSH) differs depending on the reagents used. Harmonization of TSH is crucial because the decision limits are described in current clinical practice guide- lines as absolute values, e.g. 2.5 mIU/L in early pregnancy. In this study, we tried to harmonize the report- ed concentrations of TSH using the all-procedure trimmed mean. TSH was measured in 146 serum samples, with values ranging from 0.01 to 18.8 mIU/L, using 4 immunoassays. The concentration of TSH was highest with E test TOSOH and lowest with LUMIPULSE. The concentrations with each reagent were recalculated with the following formulas: E test TOSOH 0.855x-0.014; ECLusys 0.993x+0.079; ARCHITECT 1.041x- 0.010; and LUMIPULSE 1.096x-0.015. Recalculation eliminated the between-assay discrepancy. These formulas may be used until harmonization of TSH is achieved by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC).
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Tireotropina/sangue , Humanos , Inquéritos e QuestionáriosRESUMO
BACKGROUND: The migration of T cell to atherosclerotic lesions is proposed to be involved in the pathogenesis of the atherosclerosis. Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid released from activated platelets, exerts a variety of responses such as cell migration and proliferation, and reportedly induces T cell migration. Accordingly, platelet-T cell interactions may exist based on T cell responses triggered by platelet-derived S1P. METHODS: S1P was measured using two-step lipid extraction followed by high-performance liquid chromatography (HPLC) separation while other phospholipids were determined by an enzymatic assay. The expression of S1P and lysophosphatidic acid receptors on Jurkat T cells was examined by RT-PCR and flow cytometry. Jurkat cell migration by S1P and the supernatant of activated platelets (SAP) was evaluated by a modified Boyden's chamber assay. RESULTS: S1P1 receptor was confirmed to be expressed on Jurkat T cell by RT-PCR and flow cytometry. S1P at 10-100 nM induced strong Jurkat cell migration, which was inhibited by the S1P1 (and S1P3) antagonist VPC23019 and the Gi inactivator pertussis toxin (PTX). We found that the supernatant (releasate) of human platelets activated by collagen stimulation, which contains S1P abundantly, induced Jurkat cell migration and that the migration was inhibited by VPC23019 and PTX. In addition, human serum, into which platelet contents (including S1P) are fully released, induced the Jurkat cell migration, which was also inhibited by VPC23019. CONCLUSIONS: Our findings suggest that platelet-derived S1P induces Jurkat T cell migration possibly via S1P1. S1P may be a key molecule involved in the responses triggered by platelet-T cell interactions, including atherosclerosis.
Assuntos
Plaquetas/metabolismo , Movimento Celular , Lisofosfolipídeos/fisiologia , Esfingosina/análogos & derivados , Comunicação Celular , Humanos , Células Jurkat , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/fisiologiaRESUMO
BACKGROUND: The measurement of antinuclear antibodies (ANA) is used for screening of connective tissue diseases (CTD) in the laboratory. ANA detection is performed by indirect immunofluorescence (IF) assay on HEp-2 cells from human larynx carcinoma. However, it lacks specificity for the identification of specific diseases and antigen reactivity. The aim of the present study was to evaluate the EliA CTD Screen (EliA), a new enzyme fluoroimmunoassay (Phadia AB, Uppsala, Sweden) for detection of ANA in human serum. PATIENTS AND METHODS: The study involved a total of 732 serum samples, 200 from healthy donors, 297 from patients with CTD and 235 from patients with rheumatoid arthritis, vasculitis syndrome and relative disease of CTD. For all sera, ANA was measured by IF, commercial assay (MESACUP) and EliA. RESULT: The sensitivity and specificity of EliA were 73.7% and 78.7%, respectively, whereas those of MESACUP were 80.8% and 64.7%, respectively. Area under the receiver operating curves for EliA, MESACUP and IF were 0.821, 0.786 and 0.730, respectively. The concordance rate between EliA and MESACUP was 84.2%. These discrepancies between those 2 assays were found in 84 sera. Further investigation were done by each ANA antigen tests for the discrepant results of EliA in 83 sera. The discrepancies might be occurred by antigen difference or non-specific response. CONCLUSION: AUC results showed that the diagnostic performance of EliA was superior to MESACUP and IF. EliA had a good performance as method for screening of CTD.
Assuntos
Anticorpos Antinucleares/sangue , Fluorimunoensaio/métodos , Técnicas Imunoenzimáticas/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antinucleares/imunologia , Doenças do Tecido Conjuntivo/sangue , Doenças do Tecido Conjuntivo/diagnóstico , Doenças do Tecido Conjuntivo/imunologia , Feminino , Humanos , Masculino , Programas de Rastreamento/métodos , Pessoa de Meia-IdadeRESUMO
The erythrocyte sedimentation rate (ESR) has been used as an index for inflammatory conditions, such as infectious diseases, autoimmune diseases, and malignancies. The ESR values of a 37-year-old male with marked leukocytosis due to chronic myeloid leukemia showed remarkable differences between two devices of the same model (Ves-Matic 30, DIESSE Diagnostica Senese). From the appearance of the tested tube after the ESR measurement, the values obtained using one device might have been falsely low, whereas the values obtained using the other device were likely to have been accurate. The difference of the ESR values between the two devices might have occurred by the false detection of transmitted light during the transition from the erythrocyte layer to the leukocyte layer. These findings suggest that in cases with marked leukocytosis the accuracy of ESR should be confirmed with the appearance of the test tube.
Assuntos
Sedimentação Sanguínea , Leucócitos/patologia , Leucocitose/sangue , Leucocitose/diagnóstico , Adulto , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/complicações , Masculino , Ciência de Laboratório MédicoRESUMO
Sphingolipids have been recently elucidated to be not only mere components of the plasma membrane but also bioactive mediators which can induce various biological responses. Among these lipids, sphingomyelin(SM) and sphingosine 1-phosphate (Sph-1-P) are proposed to be involved in the pathogenesis of atherosclerosis: SM is abundant in atherosclerotic lesions and Sph-1-P is bound to HDL and attributes to the anti-atherosclerotic properties of HDL at least partly. Therefore, Sph-1-P and SM can be useful biomarkers for atherosclerotic disorders. However, at present, the measurement of Sph-1-P and SM levels has not been brought into clinical practice, yet. The main obstacle is the difficulty in measuring these sphingolipids precisely, rapidly, and conveniently. Recently, we have developed new methods for measuring Sph-1-P (HPLC method) and SM (enzymatic method). These methods are easy to be introduced into clinical laboratory testing because they do not need any special techniques and equipment. With this method for SM, we have demonstrated that the SM concentration was significantly higher in subjects with acute coronary syndrome. In this paper, we reviewed the possibility of sphingolipids as biomarkers for atherosclerotic disorders.
Assuntos
Aterosclerose/diagnóstico , Esfingolipídeos/metabolismo , Síndrome Coronariana Aguda/diagnóstico , Síndrome Coronariana Aguda/metabolismo , Aterosclerose/metabolismo , Biomarcadores/metabolismo , Humanos , Lisofosfolipídeos/sangue , Caracteres Sexuais , Esfingolipídeos/química , Esfingosina/análogos & derivados , Esfingosina/sangueRESUMO
BACKGROUND & AIMS: Mitochondrial isoenzyme of creatine kinase (MtCK) is reportedly highly expressed in hepatocellular carcinoma (HCC). Clinical relevance of serum MtCK activity in patients with HCC was assessed using a novel immuno-inhibition method. METHODS: Among patients with cirrhosis caused by hepatitis B or C virus, 147 patients with HCC (12 with the first occurrence and 135 with recurrence) and 92 patients without HCC were enrolled. RESULTS: Serum MtCK activity was higher in cirrhotic patients with HCC than in those without HCC or healthy subjects. Elevated serum MtCK activity in HCC patients decreased after radiofrequency ablation. In case of prediction of HCC, MtCK had a sensitivity of 62.6% and a specificity of 70.7% at a cut-off point of 8.0 U/L, with an area under the receiver operating curve of 0.722 vs. 0.713 for alpha-fetoprotein (AFP) and 0.764 for des-gamma-carboxy prothrombin (DCP). Among the HCC patients, serum MtCK activity was elevated in 52.9% individuals with serum AFP level < 20 ng/ml and 63.2% individuals with serum DCP level < 40 mAu/ml. Even in patients with a single HCC ≤ 2 cm, the sensitivity of serum MtCK activity for the prediction of HCC was 64.4%, which was comparable to the overall sensitivity. This increased activity was due to an increase in ubiquitous MtCK, not sarcomeric MtCK, and the enhanced mRNA expression of ubiquitous MtCK was observed in cell lines originating from HCCs in contrast to healthy liver tissues. CONCLUSIONS: Serum MtCK activity merits consideration as a novel marker for HCC to be further tested as for its diagnostic and prognostic power.
Assuntos
Carcinoma Hepatocelular/enzimologia , Creatina Quinase Mitocondrial/sangue , Isoenzimas/sangue , Neoplasias Hepáticas/enzimologia , Recidiva Local de Neoplasia/enzimologia , Idoso , Biomarcadores/sangue , Creatina Quinase Mitocondrial/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Precursores de Proteínas/sangue , Protrombina , RNA Mensageiro/análise , alfa-Fetoproteínas/análiseRESUMO
The performance of a chemical luminescence test reagent "Immulyze IL-2R II" with an automated immune chemiluminescent system "IMMULITE 2000XPi" for the measurement of serum soluble IL-2 receptor in clinical samples was investigated. The satisfactory results were obtained for the reproducibility, precision, linearity, and sensitivity, and no interference with hemolysis, bilirubin, chyle or intrafat was observed. A significant correlation was found between the values of sIL-2R measured by the Cell-free N IL-2R and those obtained by the IMMULYZE IL-2R II. The measurements were stable regardless of the methods of sample preservation, or repeated freeze-thawing procedures. Elevated concentrations of sIL-2R over 1,000 U/mL were found in multiple types of collagen diseases or severe cases of allergic diseases, indicative that sIL-2R levels might correlate with the severity of autoimmune diseases. In patients with lymphoma, sIL-2R levels correlated with the lactate dehydrogenase (LD) activity. Among the lymphoma cases with sIL-2R levels over 1,000 U/mL, the majority (84%) had significantly higher levels of LD, and among them, 81% were at the clinical stage IV. We observed that sIL-2R levels increased from the early stages of lymphoma, while LD activities increased at the advanced stages. Our present findings suggest that sIL-2R is a promising marker for the diagnosis of autoimmune and allergic diseases, and also for the diagnosis and staging of lymphomas.
Assuntos
Doenças Autoimunes/sangue , Automação Laboratorial/métodos , Biomarcadores Tumorais/sangue , Imunoensaio/métodos , Medições Luminescentes/métodos , Linfoma não Hodgkin/sangue , Receptores de Interleucina-2/sangue , Doenças Autoimunes/imunologia , Humanos , Linfoma não Hodgkin/imunologia , Receptores de Interleucina-2/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , SolubilidadeRESUMO
The performance of a latex agglutination test (Mediace TPLA) in the detection of anti-treponemal antibody was evaluated in comparison with chemical luminescence tests (LumipulsII-N and Architect TPAb) in 346 cases. Anti-treponemal antibody was further determined by immunochromatography and immunoblotting tests and additionally evaluated by a serological test for syphilis with lipoidal antigens. The total concordance rate between the latex agglutination test and chemical luminescence tests ranged from 96% to 97%: the positive concordance rate ranged from 96% to 97%, and the negative concordance rate, from 97% to 98%. The latex agglutination test showed two false positive cases, and each chemical luminescence test showed two false positive cases, respectively. In eight cases, only the latex agglutination test showed negative results; all specimens contained anti-treponemal antibodies. However, none of these was considered to be a false positive and each was treated as syphilis based on the results of confirmatory analysis with immunochromatography and immunoblotting tests and a serological test for syphilis. The discordant results in the latex agglutination test and chemical luminescence tests may be caused by the different antigenisity of each test. With detailed analysis of those sera treated as syphilis, each specimen was found to contain various antibodies against syphilitic antigens, suggesting that there was a different specificity of native and recombinant antigens. Based on the present results for the comparison between the latex agglutination test and chemical luminescence tests, it was considered that further investigation is necessary to clarify the anti-treponemal antibody profile of syphilis at the disease stage.
Assuntos
Anticorpos Antibacterianos/análise , Testes de Fixação do Látex , Treponema/imunologia , Reações Falso-Positivas , Humanos , Medições Luminescentes , Sorodiagnóstico da Sífilis/métodosRESUMO
Measurements of autoantibodies are served for diagnosis of autoimmune diseases in routine. Results of UniCAP EliA (Phadia), based on fluorescence-enzyme immunoassay, were compared to those of the current ELISA method, MESACUP DNA-II test [ds] and MESACUP-2 test (MBL) with total of 404 sera. The full automated instrument of UniCAP 250 was used for measurement of UniCAP EliA. The CVs of within day reproducibility (n=10) were 3.0-9.6% by UniCAP EliA, meanwhile 1.7-11.7% by MESACUP. The CVs of between day reproducibility(5 days) were 1.0-11.8% by UniCAP EliA, meanwhile 1.0-19.9% by MESACUP. The concordance of U1RNP, SS-A/Ro, SS-B/La, Scl-70 and Jo-1 between UniCAP EliA and MESACUP were 89.5-100%, but the positive concordance in dsDNA, Sm andJo-1 showed lower concordance percentage (40.0-62.9%). UniCAP EliA had better reproducibility than MESACUP. Some sera showed discrepant results between UniCAP EliA and MESACUP. These discrepancies might be occurred by the purification of antigens or different measurement principle of kits. The antigens' purification of UniCAP EliA seemed enough for the routine tests, and the results from UniCAP EliA would give high clinical importance.
Assuntos
Autoanticorpos/sangue , Técnicas Imunoenzimáticas/instrumentação , Ensaio de Imunoadsorção Enzimática , Humanos , Reprodutibilidade dos TestesRESUMO
Because of a growing need for good quality and safety in medical care, it is necessary for clinical laboratories to improve quality and competence, and for these improvements to be disclosed to clinicians and patients. To this end, each clinical laboratory should be evaluated and accredited by an international third-party organization. We, the Department of Clinical Laboratory, the University of Tokyo Hospital, achieved ISO15189 accreditation in January 2007. Thereafter, the Department of Blood Transfusion, the Department of Infection Control, and our system for specified health checkup also obtained ISO15189 accreditation in March 2008. Based on this ISO15189 accreditation, quality-certified laboratory test results are now reported and, therefore, clinicians can perform safe medical care for their patients. Furthermore, ISO15189 is useful for clinical trials and international collaboration in medical research. We hope our ISO15189 accreditation will be helpful in its dissemination in Japan, thereby playing a role in clinical laboratory standardization, benefiting both clinicians and patients.
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Acreditação/normas , Acreditação/tendências , Técnicas de Laboratório Clínico/normas , Técnicas de Laboratório Clínico/tendências , Laboratórios Hospitalares/normas , Laboratórios Hospitalares/tendências , Humanos , Cooperação InternacionalRESUMO
Panic values are laboratory test levels suggesting life-threatening conditions of patients. When these values are obtained, clinical doctors must start treatment promptly. However, the definition of panic values varies between laboratories. Herein, the management of panic values (in the field of clinical chemistry) at the University of Tokyo Hospital is described.
Assuntos
Química Clínica/normas , Emergências , Humanos , Valores de ReferênciaRESUMO
BACKGROUND: Serum amyloid A (SAA), which is one of the acute phase proteins, alters the structure of HDL by associating with it during circulation. We focused on whether SAA influences the values of HDL-cholesterol (HDL-C) measurements when using a homogeneous assay. METHODS: HDLs were isolated by ultracentrifugation from serum samples of 248 patients that were stratified into three groups based on their serum SAA concentrations (low: SAAâ¯≤â¯8⯵g/mL; middle: 8â¯<â¯SAAâ¯≤â¯100⯵g/mL; and high: SAAâ¯>â¯100⯵g/mL). HDL-C concentrations of the serum samples measured by the homogeneous assay were compared with the total cholesterol concentrations of HDL fractions isolated by ultracentrifugation. RESULTS: HDLs obtained from patients with low SAA concentrations were separated into their general particle sizes and classified as HDL2 and HDL3 by native-gel electrophoresis. On the other hand, HDLs obtained from patients with high SAA concentrations occasionally showed distributions different from the typical sizes of HDL2 and HDL3, such as extremely small or large particles. Nevertheless, HDL-C concentrations measured using the homogeneous assay were strongly correlated with those measured using the ultracentrifugation method, regardless of the SAA concentrations. However, the ratios of HDL-C concentrations obtained by the homogeneous assay to those obtained by the ultracentrifugation method for patients with high SAA concentrations were significantly lower than those of patients with low SAA concentrations. CONCLUSIONS: A large amount of SAA attached to HDL altered the HDL particle size but did not essentially affect HDL-C measurement by homogeneous assay.
Assuntos
HDL-Colesterol , Proteína Amiloide A Sérica , HDL-Colesterol/sangue , HDL-Colesterol/química , HDL-Colesterol/isolamento & purificação , Feminino , Humanos , Masculino , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/isolamento & purificação , Proteína Amiloide A Sérica/metabolismoRESUMO
BACKGROUND: Autotaxin (ATX), a tumor cell motility-stimulating factor, regulates the blood concentrations of lysophosphatidic acid (LPA), an important and multi-functional bioactive lipid, through its lysophospholipase D activity (lysoPLD). The introduction of ATX measurements into clinical laboratory testing is urgently needed. METHODS: Anti-human ATX monoclonal antibodies were produced by immunization of recombinant human ATX expressed in a baculovirus system. An immunoassay for the quantitative determination of ATX was established, and human serum samples were assayed. RESULTS: The within-run and between-run precision, interference, detection limit, and linearity studies were satisfactory. The central 95 percentile reference interval for the serum ATX antigen concentration in healthy subjects was 0.468-1.134 mg/l (n=120) and was strongly correlated with the serum lysoPLD activity. The ATX concentration was significantly (p<0.001) higher in women (0.625-1.323 mg/l) than in men (0.438-0.914 mg/l). The serum ATX concentrations were increased in patients with chronic liver diseases and decreased in postoperative prostate cancer patients but were not altered in nephrosis patients. Thus, serum ATX antigen concentrations could be used to discriminate these hypoalbuminemia conditions. CONCLUSIONS: The present ATX antigen assay may be useful for clinical laboratory testing.
Assuntos
Hipoalbuminemia/sangue , Hipoalbuminemia/diagnóstico , Técnicas Imunoenzimáticas/métodos , Complexos Multienzimáticos/sangue , Fosfodiesterase I/sangue , Pirofosfatases/sangue , Antígenos/sangue , Doença Crônica , Feminino , Saúde , Humanos , Hepatopatias/metabolismo , Masculino , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Nefrose/sangue , Fosfodiesterase I/genética , Fosfodiesterase I/isolamento & purificação , Fosfolipase D/metabolismo , Diester Fosfórico Hidrolases , Neoplasias da Próstata/sangue , Neoplasias da Próstata/cirurgia , Pirofosfatases/genética , Pirofosfatases/isolamento & purificaçãoRESUMO
BACKGROUND: Since sphingosine-1-phosphate (Sph-1-P) plays an important role as an extracellular mediator through interaction with specific cell surface receptors, especially in the area of vascular biology and immunology/haematology, determination of its plasma concentration may become important from the clinical viewpoint. Thus, we attempted to develop a method of measuring the plasma Sph-1-P concentration for use in the clinical laboratory setting. METHODS: After two-step lipid extraction, Sph-1-P was coupled with o-phthaldialdehyde, and the resultant fluorescent derivative was separated by high-performance liquid chromatography. C17-Sph-1-P was used as the internal standard, instead of dihydrosphingosine-1-phosphate, which had been used previously for the same purpose but was actually detected in plasma. RESULTS: Our procedures for preparing the plasma samples and assay Sph-1-P were found to be satisfactory for clinical laboratory testing. The plasma Sph-1-P concentrations were significantly higher in men (413.1 +/- 52.0 nmol/L; mean +/- SD) than in women (352.4 +/- 39.7 nmol/L). Unexpectedly, strong positive correlations were found between the plasma Sph-1-P concentration and red blood cell (RBC)-related parameters, rather than platelet-related parameters. CONCLUSIONS: Our present study confirmed the possibility of the clinical introduction of plasma Sph-1-P measurement, and in addition, suggested that RBCs may be involved in the regulation of plasma Sph-1-P concentrations.
Assuntos
Análise Química do Sangue/métodos , Eritrócitos/metabolismo , Saúde , Lisofosfolipídeos/sangue , Esfingosina/análogos & derivados , Humanos , Esfingosina/sangueRESUMO
BACKGROUND: Lysophosphatidic acid (LPA) plays important roles in a variety of biological responses, especially in the area of vascular biology, and the determination of its plasma concentration is believed to be important. Several mechanisms are known to be involved in the metabolism of LPA. METHODS: To identify factors that may determine the plasma concentrations of this important bioactive lipid, we examined its concentrations using an enzymatic cycling assay and related parameters in 146 healthy subjects. RESULTS: The LPA concentration was significantly higher in women (mean +/- SD, 0.103 +/- 0.032 micromol/L; n = 47) than in men (0.077 +/- 0.026 micromol/L; n = 99). A multiple regression analysis showed a strong positive correlation between the plasma LPA concentration and serum lysophospholipase D (lysoPLD) activity, while the LPA concentration was correlated with the plasma lysophosphatidylcholine (LPC) concentration only in men. Other lipid-related parameters were only slightly correlated or were not correlated with the LPA concentration. CONCLUSIONS: Our findings suggested that conversion from LPC by lysoPLD might be the major route for LPA production in plasma.
Assuntos
Saúde , Lisofosfolipídeos/sangue , Diester Fosfórico Hidrolases/metabolismo , Feminino , Humanos , Modelos Lineares , Masculino , Padrões de ReferênciaRESUMO
Background Human mercaptalbumin and human non-mercaptalbumin have been reported as markers for various pathological conditions, such as kidney and liver diseases. These markers play important roles in redox regulations throughout the body. Despite the recognition of these markers in various pathophysiologic conditions, the measurements of human mercaptalbumin and non-mercaptalbumin have not been popular because of the technical complexity and long measurement time of conventional methods. Methods Based on previous reports, we explored the optimal analytical conditions for a high-performance liquid chromatography method using an anion-exchange column packed with a hydrophilic polyvinyl alcohol gel. The method was then validated using performance tests as well as measurements of various patients' serum samples. Results We successfully established a reliable high-performance liquid chromatography method with an analytical time of only 12 min per test. The repeatability (within-day variability) and reproducibility (day-to-day variability) were 0.30% and 0.27% (CV), respectively. A very good correlation was obtained with the results of the conventional method. Conclusions A practical method for the clinical measurement of human mercaptalbumin and non-mercaptalbumin was established. This high-performance liquid chromatography method is expected to be a powerful tool enabling the expansion of clinical usefulness and ensuring the elucidation of the roles of albumin in redox reactions throughout the human body.
Assuntos
Biomarcadores/análise , Cromatografia Líquida de Alta Pressão/métodos , Albumina Sérica/análise , HumanosRESUMO
Estimation of the function as well as the amount of high-density lipoprotein (HDL) is required to predict the risk of cardiovascular disease development. Cholesterol efflux capacity (CEC) is the key metric for determining the antiatherosclerotic function of HDL. However, the assay methods currently used to calculate CEC are not ideal for clinical use as they require the culture of cells. In the present study, we developed a novel CEC assay using immobilized liposome-bound gel beads (ILGs), containing fluorescently labeled cholesterol, as a substitute for cultured cells. When apolipoprotein B-100 depleted serum, obtained by polyethylene glycol precipitation, was used as the cholesterol acceptors, the basic properties of this method, such as the available range of HDL-cholesterol, efflux temperature and time, and normalization parameters, indicate that this method is sufficient to estimate CEC. Furthermore, the CEC values obtained with this ILG method were also correlated with those obtained with a conventional method using THP-1 macrophages derived foam cells and 3H-cholesterol as a tracer (r = 0.932). Overall, this novel cholesterol efflux assay method is a realistic and effective alternative to current methods in the field while also being easier to use in clinical laboratories as neither cell culture, radioisotope nor ultracentrifugation is required.