Publications by authors named "Seiichi Hashida"

23 Publications

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Urinary adiponectin excretion is an early predictive marker of the decline of the renal function in patients with diabetes mellitus.

J Diabetes Complications 2021 Apr 7;35(4):107848. Epub 2021 Jan 7.

Diabetes Therapeutics and Research Center, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan. Electronic address:

Aims: Since diabetes-associated kidney complication changes from diabetic nephropathy to diabetic kidney disease (DKD), more suitable biomarkers than urinary albumin are required. It has been hypothesized that urinary adiponectin (u-ADPN) is associated with the progression of DKD. We therefore evaluated the effectiveness of u-ADPN in predicting the decline of the renal function in patients with diabetes prior to end-stage renal disease.

Methods: An ultrasensitive immune complex transfer enzyme immunoassay (ICT-EIA) was used to measure total and high molecular weight (HMW) adiponectin separately. We evaluated the relationships between the creatinine-adjusted urinary total-ADPN and HMW-ADPN, albumin (UACR) and liver-type fatty acid binding protein (L-FABP) at baseline and the 2-year change of the estimated glomerular filtration rate (ΔeGFR).

Results: This 2-year prospective observational study included 201 patients with diabetes. These patients were divided into three groups according to their ΔeGFR: ≤-10 mL/min/1.73m, >-10 and ≤0 mL/min/1.73m, and >0 mL/min/1.73m. Jonckheere-Terpstra test showed that lower ΔeGFR was associated with higher u-HMW-ADPN (p = 0.045). In logistic regression analysis, u-HMW-ADPN was associated with ΔeGFR after adjusted age, sex, and basal eGFR.

Conclusion: Urinary HMW-ADPN could predict a declining renal function in patients with diabetes.
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http://dx.doi.org/10.1016/j.jdiacomp.2021.107848DOI Listing
April 2021

Development of fully automated and ultrasensitive assays for urinary adiponectin and their application as novel biomarkers for diabetic kidney disease.

Sci Rep 2020 09 28;10(1):15869. Epub 2020 Sep 28.

Department of Diabetes and Molecular Genetics, Ehime University Graduate School of Medicine, Ehime, Japan.

Glomerular filtration rate (GFR) and urinary albumin excretion rate (UAER) are used to diagnose and classify the severity of chronic kidney disease. Total adiponectin (T-AN) and high molecular weight adiponectin (H-AN) assays were developed using the fully automated immunoassay system, HI-1000 and their significance over conventional biomarkers were investigated. The T-AN and H-AN assays had high reproducibility, good linearity, and sufficient sensitivity to detect trace amounts of adiponectin in the urine. Urine samples after gel filtration were analyzed for the presence of different molecular isoforms. Low molecular weight (LMW) forms and monomers were the major components (93%) of adiponectin in the urine from a diabetic patient with normoalbuminuria. Urine from a microalbuminuria patient contained both high molecular weight (HMW) (11%) and middle molecular weight (MMW) (28%) adiponectin, although the LMW level was still high (52%). The amount of HMW (32%) and MMW (42%) were more abundant than that of LMW (24%) in a diabetic patient with macroalbuminuria. T-AN (r = - 0.43) and H-AN (r = - 0.38) levels showed higher correlation with estimated GFR (eGFR) than UAER (r = - 0.23). Urinary levels of both T-AN and H-AN negatively correlated with renal function in diabetic patients and they may serve as new biomarkers for diabetic kidney disease.
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http://dx.doi.org/10.1038/s41598-020-72494-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522970PMC
September 2020

Circulating osteocalcin as a bone-derived hormone is inversely correlated with body fat in patients with type 1 diabetes.

PLoS One 2019 3;14(5):e0216416. Epub 2019 May 3.

Diabetes Therapeutics and Research Center, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan.

The objective of the present study was to investigate the correlations between serum undercarboxylated osteocalcin (ucOC) or osteocalcin (OC) concentrations and %body fat, serum adiponectin and free-testosterone concentration, muscle strength and dose of exogenous insulin in patients with type 1 diabetes. We recruited 73 Japanese young adult patients with childhood-onset type 1 diabetes. All participants were receiving insulin replacement therapy. The correlations between logarithmic serum ucOC or OC concentrations and each parameter were examined. Serum ucOC and OC concentrations were inversely correlated with %body fat (r = -0.319, P = 0.007; r = -0.321, P = 0.006, respectively). Furthermore, multiple linear regression analyses were performed to determine whether or not serum ucOC or OC concentrations were factors associated with %body fat. Serum ucOC and OC concentrations remained significant factors even after adjusting for gender, HbA1c, body weight-adjusted total daily dose of insulin and duration of diabetes (β = -0.260, P = 0.027; β = -0.254, P = 0.031, respectively). However, serum ucOC and OC concentrations were not correlated with serum adiponectin or free-testosterone concentrations, muscle strength or dose of exogenous insulin. In conclusion, our study demonstrates the inverse correlation between serum ucOC or OC concentrations and body fat in patients with type 1 diabetes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0216416PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499427PMC
January 2020

The immune complex transfer enzyme immunoassay: Mechanism of improved sensitivity compared with conventional sandwich enzyme immunoassay.

J Immunol Methods 2018 08 5;459:76-80. Epub 2018 Jun 5.

Life Style Diseases, Institute of Health Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.

Immune complex transfer enzyme immunoassay (ICT-EIA) is one of the technologies which enables ultrasensitive measurements of protein biomarkers. The ICT-EIA uses two types of beads and sandwich-shaped immune complexes are transferred from the 1st bead to the 2nd bead in the assay. The purpose of the study is to reveal the reason why the ICT-EIA achieves ultrasensitive measurements by making a detailed comparison between conventional sandwich enzyme immunoassay (Sand-EIA) and ICT-EIA. ICT-EIAs for cytokines were developed and the sensitivities were compared with the sandwich EIAs. ICT-EIAs had about 100 times higher sensitivities because of markedly decreased non-specific signals derived from non-specific binding of detection antibody conjugates onto the polystyrene bead. The results have enabled us to show the importance of reducing non-specific signals in EIAs to obtain higher sensitivities. This methodology should be more valuable if combined with a different label detection system such as digital counting or immuno-PCR, which may enable the detection of single target protein molecules in the near future.
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http://dx.doi.org/10.1016/j.jim.2018.05.010DOI Listing
August 2018

A study of high-, middle- and low-molecular weight adiponectin in urine as a surrogate marker for early diabetic nephropathy using ultrasensitive immune complex transfer enzyme immunoassay.

Ann Clin Biochem 2018 Sep 30;55(5):525-534. Epub 2018 Jan 30.

2 Life Style Diseases, Institute for Health Sciences, Tokushima Bunri University, Tokushima, Japan.

Background For the early identification of patients at risk of developing diabetic nephropathy, we have developed an ultrasensitive immune complex transfer enzyme immunoassay to measure adiponectin in urine. Methods We developed immune complex transfer enzyme immunoassay for adiponectin and measured urinary adiponectin from 70 healthy subjects, 35 obese non-diabetic subjects and 20 patients with diabetes. Results The urinary adiponectin concentrations in patients with diabetes (3.3 ± 10.7 ng/mg creatinine) were significantly higher than those in obese subjects (0.54 ± 0.44; P < 0.01) and healthy subjects (0.46 ± 0.42; P < 0.001). The gel filtration elution profile of urine from healthy subjects showed traces of four immunoreactive peaks (high-, medium-, low-molecular weight and monomer molecules), despite the majority of blood adiponectin being high-molecular weight. However, urinary adiponectin molecules were more frequent in low-molecular weight as the estimate glomerular filtration rate decreased. Furthermore, as blood glucose concentrations rose, middle-molecular weight and high-molecular weight increased in urine. Further, urinary adiponectin concentrations correlated with estimate glomerular filtration rate ( r = -0.61, P < 0.001), but not urinary albumin. In addition, our analysis showed a significantly ( P < 0.001) higher value for urinary adiponectin in the G2 stage of chronic kidney disease classification where urinary albumin is not elevated. Conclusion Adiponectin increases in urine as renal function decreases, and urinary adiponectin may be useful as a surrogate marker for diabetic nephropathy risk.
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http://dx.doi.org/10.1177/0004563217748681DOI Listing
September 2018

Sequential cleavage of insulin receptor by calpain 2 and γ-secretase impairs insulin signalling.

Diabetologia 2016 12 30;59(12):2711-2721. Epub 2016 Sep 30.

Human Life Science, Tokushima Bunri University, Tokushima, Japan.

Aims/hypothesis: Soluble insulin receptor (sIR), the ectodomain of the insulin receptor (IR), has been detected in human plasma and its concentration paralleled that of blood glucose. We have previously developed an in vitro model using HepG2 liver-derived cells, which mimics changes in sIR levels in plasma from diabetic patients and shows that calcium-dependent proteases cleave IR extracellularly (a process known as shedding). The present study aimed to reveal the mechanisms of IR cleavage.

Methods: Using the in vitro model, we investigated the molecular mechanisms of IR cleavage, which is accelerated by high-glucose treatment. We also analysed the relationship between IR cleavage and cellular insulin resistance, and the correlation between plasma sIR levels and insulin sensitivity, which was assessed by the euglycaemic-hyperinsulinaemic clamp technique.

Results: Here, we determined that calpain 2, which is secreted into the extracellular space associated with exosomes, directly cleaved the ectodomain of the IRβ subunit (IRβ), which in turn promoted intramembrane cleavage of IRβ by γ-secretase. IR cleavage impaired insulin signalling and the inhibition of IR cleavage (by knockdown of calpain 2 and γ-secretase), restored IR substrate-1 and Akt, independent of IR. Furthermore, the glucose-lowering drug, metformin, prevented IR cleavage accompanied by inhibition of calpain 2 release in exosomes, and re-established insulin signalling. In patients with type 2 diabetes, plasma sIR levels inversely correlated with insulin sensitivity.

Conclusions/interpretation: Sequential cleavage of IR by calpain 2 and γ-secretase may contribute to insulin signalling in cells and its inhibition may be partly responsible for the glucose-lowering effects of metformin. Thus, IR cleavage may offer a new mechanism for the aetiology of insulin resistance.
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http://dx.doi.org/10.1007/s00125-016-4102-5DOI Listing
December 2016

Endoplasmic reticulum stress induced by tunicamycin increases resistin messenger ribonucleic acid through the pancreatic endoplasmic reticulum eukaryotic initiation factor 2α kinase-activating transcription factor 4-CAAT/enhancer binding protein-α homologous protein pathway in THP-1 human monocytes.

J Diabetes Investig 2016 May 18;7(3):312-23. Epub 2015 Nov 18.

Department of Diabetes and Molecular Genetics Ehime University Graduate School of Medicine Ehime Japan.

Aims/introduction: Resistin, secreted from adipocytes, causes insulin resistance in mice. In humans, the resistin gene is mainly expressed in monocytes and macrophages. Tunicamycin is known to induce endoplasmic reticulum (ER) stress, and reduce resistin gene expression in 3T3-L1 mouse adipocytes. The aim of the present study was to examine whether ER stress affects resistin gene expression in human monocytes.

Materials And Methods: The relationship between resistin messenger ribonucleic acid (mRNA) and ER stress markers mRNA was analyzed by reverse transcription polymerase chain reaction in isolated monocytes of 30 healthy volunteers. The effect of endotoxin/lipopolysaccharides or tunicamycin on resistin gene expression was analyzed in THP-1 human monocytes. Signaling pathways leading to resistin mRNA were assessed by the knockdown using small interfering RNA or overexpression of key molecules involved in unfolded protein response.

Results: Resistin mRNA was positively associated with immunoglobulin heavy chain-binding protein (BiP) or CAAT/enhancer binding protein-α homologous protein (CHOP) mRNA in human isolated monocytes. In THP-1 cells, lipopolysaccharides increased mRNA of BiP, pancreatic endoplasmic reticulum eukaryotic initiation factor 2α kinase (PERK) and CHOP, as well as resistin. Tunicamycin also increased resistin mRNA. This induction appeared to be dose- and time-dependent. Tunicamycin-induced resistin mRNA was inhibited by chemical chaperone, 4-phenylbutyric acid. The knockdown of either PERK, activating transcription factor 4 (ATF4) or CHOP reduced tunicamycin-induced resistin mRNA. Conversely, overexpression of ATF4 or CHOP increased resistin mRNA.

Conclusions: Endoplasmic reticulum stress induced by tunicamycin increased resistin mRNA through the PERK-ATF4-CHOP pathway in THP-1 human monocytes. ER stress could lead to insulin resistance through enhanced resistin gene expression in human monocytes.
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http://dx.doi.org/10.1111/jdi.12434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4847884PMC
May 2016

Immunoreactive insulin in diabetes mellitus patient sera detected by ultrasensitive ELISA with thio-NAD cycling.

Biotechniques 2015 Dec 1;59(6):359, 361-7. Epub 2015 Dec 1.

Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.

To minimize patient suffering, the smallest possible volume of blood should be collected for diagnosis and disease monitoring. When estimating insulin secretion capacity and resistance to insulin in diabetes mellitus (DM), increasing insulin assay immunosensitivity would reduce the blood sample volume required for testing. Here we present an ultrasensitive ELISA coupled with thio-NAD cycling to measure immunoreactive insulin in blood serum. Only 5 μL of serum was required for testing, with a limit of detection (LOD) for the assay of 10(-16) moles/assay. Additional recovery tests confirmed this method can detect insulin in sera. Comparisons between a commercially available immunoreactive insulin kit and our ultrasensitive ELISA using the same commercially available reference demonstrated good data correlation, providing further evidence of assay accuracy. Together, these results demonstrate our ultrasensitive ELISA could be a powerful tool in the diagnosis and treatment of not only DM but also many other diseases in the future.
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http://dx.doi.org/10.2144/000114355DOI Listing
December 2015

Development of a novel ultrasensitive enzyme immunoassay for human glutamic acid decarboxylase 65 antibody.

Ann Clin Biochem 2016 Jul 17;53(Pt 4):495-503. Epub 2015 Sep 17.

Institute for Health Sciences, Tokushima Bunri University, Tokushima, Japan

Background: We developed a novel, ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for determination of glutamic acid decarboxylase autoantibody concentrations in serum samples from patients with type 2 diabetes.

Methods: We developed an immune complex transfer enzyme immunoassay for glutamic acid decarboxylase autoantibody and measured glutamic acid decarboxylase autoantibody from 22 patients with type 1 diabetes, 29 patients with type 2 diabetes, and 32 healthy controls.

Results: A conventional ELISA kit identified 10 patients with type 1 diabetes and one patient with type 2 diabetes as glutamic acid decarboxylase autoantibody positive, whereas 15 patients with type 1 diabetes and six patients with type 2 diabetes were identified as glutamic acid decarboxylase autoantibody positive using immune complex transfer enzyme immunoassay.

Conclusions: Immune complex transfer enzyme immunoassay is a highly sensitive and specific assay for glutamic acid decarboxylase autoantibody and might be clinically useful for diabetic onset prediction and early diagnosis.
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http://dx.doi.org/10.1177/0004563215609639DOI Listing
July 2016

Subattomole detection of adiponectin in urine by ultrasensitive ELISA coupled with thio-NAD cycling.

Biophys Physicobiol 2015 12;12:79-86. Epub 2015 Nov 12.

Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Kagawa 769-2193, Japan.

Adiponectin is a hormone secreted from adipocytes, and it demonstrates antidiabetic, anti-atherosclerotic, antiobesity and anti-inflammatory effects. However, the patterns of change in urinary adiponectin levels in various diseases remain unknown, because only trace amounts of the hormone are present in urine. In the present study, we applied an ultrasensitive ELISA coupled with thio-NAD cycling to measure urinary adiponectin levels. Spikeand-recovery tests using urine confirmed the reliability of our ultrasensitive ELISA. The limit of detection for adiponectin in urine was 2.3×10(-19) moles/assay (1.4 pg/mL). The urinary adiponectin concentration ranged between 0.04 and 5.82 ng/mL in healthy subjects. The pilot study showed that the urinary adiponectin levels, which were corrected by the creatinine concentration, were 0.73±0.50 (ng/mg creatinine, N=6) for healthy subjects, versus 12.02±3.85 (ng/mg creatinine, N=3) for patients with diabetes mellitus (DM). That is, the urinary adiponectin levels were higher (P<0.05) in DM patients than in healthy subjects. Further, these urinary adiponectin levels tended to increase with the progression of DM accompanied with nephropathy. Our method is thus expected to provide a simple, rapid and reasonably priced test for noninvasive monitoring of the progression of DM without the requirement of special tools.
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http://dx.doi.org/10.2142/biophysico.12.0_79DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4736831PMC
August 2016

Development of in vitro model of insulin receptor cleavage induced by high glucose in HepG2 cells.

Biochem Biophys Res Commun 2014 Feb 6;445(1):236-43. Epub 2014 Feb 6.

Division of Molecular Genetics, Institute for Enzyme Research, Tokushima University, 3-18-15 Kuramotocho, Tokushima 770-8503, Japan. Electronic address:

Soluble insulin receptor (sIR), the ectodomain of IR, has been detected in human plasma, and its concentration parallels that of blood glucose in patients with diabetes. IR has a pivotal role in glucose homeostasis and diabetes development; therefore, cleavage of IR promoted by hyperglycemia is involved in insulin resistance and glucose toxicity. To elucidate the physiology of sIR, we developed an in vitro model mimicking the changes in sIR levels in plasma from patients with diabetes. Among four human cell lines that expressed IR, spontaneous cleavage of IR occurred only in HepG2 cells. The molecular characteristics of sIR derived from HepG2 cells were similar to those of sIR detected in human plasma. The concentration of sIR in the medium did not differ between basal and high-glucose conditions in the initial 24-h period, but increasing the duration of pre-stimulation (>48 h) led to a significant increase in sIR levels in cells exposed to high glucose. Additionally, glucose-dependent increment of sIR was reversible in this model. These results are consistent with the observation of plasma sIR in patients with diabetes. Using this model, O-linked N-acetylglucosamine modification was determined to be involved in high-glucose-induced IR cleavage. A calcium-dependent protease was shown to cleave IR extracellularly. These findings show that this in vitro model could be useful for determining the molecular mechanism underlying IR cleavage.
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http://dx.doi.org/10.1016/j.bbrc.2014.01.187DOI Listing
February 2014

Development of an ultra-sensitive enzyme immunoassay for human insulin autoantibodies.

Clin Biochem 2012 Sep 2;45(13-14):1086-91. Epub 2012 Jun 2.

Department of Health Science, University of Kochi, Kochi 781-8515, Japan.

Objectives: We developed an ultrasensitive enzyme immunoassay (ICT-EIA) for insulin autoantibody (IAA) measurements to better understand the pathophysiology of diabetes.

Design And Methods: We developed ICT-EIA for IAA and measured IAA in 24 patients with type 1 diabetes, 30 patients with type 2 diabetes, 30 patients with methimazole-treated Graves' disease, 20 patients with Hashimoto's disease, 9 patients with hyperinsulinemia, and 73 healthy control subjects.

Results: The conventional ELISA identified 3 patients with type 1 diabetes and 2 patients with type 2 diabetes as IAA positive, whereas 15 patients with type 1 diabetes, 7 patients with type 2 diabetes, and 4 patients with methimazole-treated Graves' disease were identified as IAA positive using ICT-EIA.

Conclusions: The ICT-EIA is an ultrasensitive and specific assay for IAA, and its use may provide a better understanding of the role of IAA in diabetes onset and progression.
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http://dx.doi.org/10.1016/j.clinbiochem.2012.05.027DOI Listing
September 2012

A novel ultra-sensitive enzyme immunoassay for soluble human insulin receptor ectodomain and its measurement in urine from healthy subjects and patients with diabetes mellitus.

Clin Biochem 2009 Sep 25;42(13-14):1468-75. Epub 2009 Jun 25.

Human Life Science, Tokushima Bunri University, Tokushima 770-8514, Japan.

Objective: For the early identification of patients at risk of developing diabetes mellitus, and to prevent the onset of diabetes by performing dietary counseling and exercise guidance, we have developed an ultra-sensitive immune complex transfer enzyme immunoassay (ICT-EIA) to measure soluble human insulin receptor ectodomain (sIRalpha) in urine which is collected non-invasively.

Design And Methods: We developed ICT-EIA for sIRalpha and measured urinary sIRalpha from 106 healthy volunteers, 35 obese volunteers and 42 patients with diabetes.

Results: The detection limit of ICT-EIA (0.04 pg/mL), using a urine sample of as little as 100 microL, was a few hundred-fold higher than that of conventional ELISA. Using ICT-EIA, the urinary sIRalpha level in patients with diabetes (9.7+/-20.1 pg/mg creatinine) was significantly higher than those in healthy volunteers (1.4+/-0.9; P<0.001).

Conclusion: ICT-EIA for sIRalpha may be useful as a good marker for evaluating diabetes risk.
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http://dx.doi.org/10.1016/j.clinbiochem.2009.06.014DOI Listing
September 2009

Adrenomedullin release in the rat mesenteric resistance artery.

Peptides 2005 Nov 2;26(11):2222-30. Epub 2005 Jun 2.

Department of Clinical Pharmaceutical Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Japan.

Adrenomedullin (AM) is a potent vasodilator peptide whose major source is the vascular wall. In the present study, the mechanism of release of AM was investigated in the rat mesenteric resistance artery. The isolated mesenteric vascular bed was perfused with Krebs solution at a constant flow rate (5 ml/min) and AM in the perfusate was measured by a highly sensitive enzyme immunoassay (Immunoenzymometric assay; IEMA) method. In preparations without endothelium, spontaneous release of AM was detected in the perfusate (68.7+/-5.8 fmol/ml, n=45). Periarterial nerve stimulation (PNS, 4 and 8 Hz) caused 11.4+/-3.9% (4 Hz) and 9.1+/-3.5% (8 Hz) decreases in the spontaneous release of AM. Removal of Ca2+ from the medium did not affect the spontaneous AM release, but abolished the PNS-induced inhibition of spontaneous AM release. Perfusion of 10nM calcitonin gene-related peptide (CGRP) or 0.1 microM capsaicin (inducer of CGRP release) inhibited significantly the spontaneous AM release. PNS (8 Hz)-induced inhibition of spontaneous AM release was antagonized by CGRP(8-37) (CGRP receptor antagonist). These results suggest that AM is mainly released from vascular smooth muscle cells of the rat mesenteric artery and endogenous or exogenous CGRP inhibits AM release.
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http://dx.doi.org/10.1016/j.peptides.2005.04.014DOI Listing
November 2005

Study of blood metabolism and urinary excretion of chymopapain following intradiscal injection using a high-sensitivity enzyme immunoassay.

J Orthop Sci 2005 ;10(2):206-13

Matsubashikawano Orthopaedic Hospital, 2-2-13 Matsubashi, Miyazaki, 880-0013, Japan.

To develop chymopapain-induced chemonucleolysis as an established treatment, it is necessary to determine the kinetics of chymopapain in blood and urine following intradiscal injection. To investigate the rate of blood metabolism and urinary excretion of chymopapain following intradiscal injection, we developed a high-sensitivity enzyme immunoassay for chymopapain. The sensitivity for this assay was 1 pg/tube (40 amol). After injecting chymopapain into the nucleus pulposus of humans, levels of blood chymopapain were measured by enzyme immunoassay. The level of chymopapain in blood decreased gradually, with a half-life of 2-3 days. The half-life for urinary excretion was a little longer, at 3 days. It was also found that chymopapain in blood was not present as a free molecule but formed a complex that had a molecular weight of about 120 kDa. These findings suggest that most chymopapain would not have activity in blood.
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http://dx.doi.org/10.1007/s00776-004-0872-6DOI Listing
January 2006

Disassociated increases of adrenomedullin in the rat cerebrospinal fluid and plasma after salt loading and systemic administration of lipopolysaccharide.

Peptides 2004 Apr;25(4):609-14

Department of Physiology, University of Occupational and Environmental Health, Iseigaoka 1-1, Yahatanishi-ku, Kitakyushu 807-8555, Japan.

To determine the role of adrenomedullin (AM) in the fluid electrolyte homeostasis and endotoxin shock, cerebral spinal fluid (CSF) and plasma were sampled from rats after respective challenges. The AM levels were measured by a highly sensitive immunoassay. The AM levels in the CSF of the rats anesthetized with ether (10.7 +/- 0.60 fmol/ml) were significantly higher than those with isoflurane 5.17 +/- 0.70 fmol/ml, P < 0.01), while the plasma level did not differ significantly. The CSF levels of the rats received 2% saline drinking increased to 3 and 4 folds at day 5 and day 7, respectively, while the plasma levels did not differ from controls at both time points. The AM levels in CSF or plasma increased to 1.5 and 3 folds at 1.5 h after intraperitoneal (i.p.) administration of lipopolysaccharide (LPS, 5 mg/kg), reached 6.5 and 30 folds at 6 h, respectively, while no change was observed in the controls. The present findings suggest that AM in the CSF is regulated independently from that in the plasma, the centrally synthesized AM plays and important role in the regulation of the fluid electrolyte homeostasis. Furthermore, the circulatory AM plays an important role in the endotoxin shock.
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http://dx.doi.org/10.1016/j.peptides.2004.02.001DOI Listing
April 2004

Plasma adrenomedullin is closely correlated with pulse wave velocity in middle-aged and elderly patients.

Hypertens Res 2003 Nov;26(11):887-93

First Department of Internal Medicine, Miyazaki Medical College, University of Miyazaki, Miyazaki, Japan.

Arterial stiffness as measured by pulse wave velocity (PWV) is a major predictor of cardiovascular disease. Adrenomedullin (AM), a hypotensive peptide, works as a compensatory factor for arterial sclerosis. The aim of this study was to investigate the relationship between PWV and the plasma concentration of AM in risk-loading patients. One hundred and twenty-six inpatients aged 30 to 75 years with or without varying degrees of atherosclerosis were investigated. Patients with heart and/or renal failure were excluded. The PWV was measured using an automatic waveform analyzer, and the plasma AM level was measured using a newly developed, hypersensitive immunoenzymometric assay system. The PWV increased with the increasing number of cardiovascular risk factors and organ damage in the patients. A positive correlation between the PWV and AM level was observed (r=0.375, p<0.0001, n=126). Seventy-four patients were receiving antihypertensive medications; medication did not affect the correlation. Multivariate regression analysis revealed that the PWV was significantly and independently associated with age, systolic blood pressure, and AM level. These results indicate that the plasma AM concentration could serve as a marker of advanced arterial sclerosis as estimated by increased PWV.
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http://dx.doi.org/10.1291/hypres.26.887DOI Listing
November 2003

Development of an ultrasensitive enzyme immunoassay for human proadrenomedullin N-terminal 20 peptide and direct measurement of two molecular forms of PAMP in plasma from healthy subjects and patients with cardiovascular disease.

Clin Biochem 2004 Jan;37(1):14-21

Department of Biochemistry, Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan.

Objective: Proadrenomedullin N-terminal 20 peptide (PAMP) processed from an adrenomedullin precursor is a potent hypotensive peptide. It was anticipated that a mature form of PAMP (m-PAMP) and an intermediate PAMP-gly existed together in the blood. To measure concentrations of PAMPs in human plasma directly, we have developed a highly sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay, ICT-EIA).

Design And Methods: PAMP was reacted simultaneously with 2,4-dinitrophenyl (DNP)-biotinyl-bovine serum albumin (BSA)-anti-PAMP Fab' conjugate and anti-PAMP Fab'-beta-D-galactosidase conjugate. The immune complex that was formed was initially trapped onto a polystyrene bead coated with anti-DNP IgG, and then transferred onto a second polystyrene bead coated with streptavidin. The resulting three-component complex was then assayed fluorometrically.

Results: The detection limits of ICT-EIA for both m-PAMP and PAMP-gly were 0.1 pmol/l with as little as 10 microl of plasma, and were a hundred times higher than with conventional radioimmunoassay (RIA). Using ICT-EIA, we determined that the plasma concentrations of m-PAMP and PAMP-gly in 51 healthy volunteers were 0.51 +/- 0.19 and 1.15 +/- 0.38 pmol/l (mean +/- SD), respectively. Both plasma m-PAMP and PAMP-gly concentrations in patients with a variety of diseases, including hypertension, heart failure, chronic renal failure, and hemodialysis, were significantly higher than those in healthy subjects. In addition, both plasma m-PAMP and PAMP-gly concentrations in patients with New York Heart Association (NYHA) class I-IV heart failure were increased in proportion to clinical severity.

Conclusions: These sensitive and specific ICT-EIAs may be used as a powerful tool for investigating the cardiovascular system in patients with heart failure.
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http://dx.doi.org/10.1016/j.clinbiochem.2003.09.007DOI Listing
January 2004

Glycine-extended adrenomedullin exerts vasodilator effect through amidation in the rat aorta.

Regul Pept 2003 May;113(1-3):109-14

First Department of Internal Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.

Human adrenomedullin (hAM) is an endogenous peptide that has potent vasodilator activity. Mature AM is biosynthesized from its intermediate form, glycine-extended AM (AM-gly), by carboxy-terminal amidation. AM-gly is generally considered to be biologically inactive but is a major molecular form in human and rat plasma. The present study demonstrated that recombinant human AM-gly (hAM-gly) elicits potent vasodilator effect on isolated rat aorta. In aortic rings, hAM-gly produced dose-dependent (0.1-100 nM) relaxation in phenylephrine-precontracted strips (pD(2) 8.4+/-0.5). The vasorelaxant potency of hAM-gly was comparable to that of hAM (pD(2) 8.6+/-0.2) but hAM-gly took a significantly (P<0.01) longer time to reach the maximal relaxation compared with hAM (T(max) 23+/-4 vs. 5+/-2 min). Vasorelaxant responses to hAM-gly were abolished by endothelial removal. N(omega)-nitro-L-arginine (L-NNA) and AM(22-52) significantly (P<0.01) reduced the vasodilator effect of hAM-gly. Furthermore, 4-phenyl-3-butenoic acid (PBA), an alpha-amidation enzyme inhibitor, significantly (P<0.05) inhibited the vasorelaxant responses to hAM-gly without any effect on the hAM-induced relaxation, suggesting the possible process of amidation in the rat aorta. We further clarified that the aorta has the ability to convert exogenous hAM-gly to mature hAM and the conversion is inhibited by PBA. These results suggest that the circulating AM-gly may play a role in regulating vascular tone and increased plasma AM-gly may be involved in the pathophysiology of cardiovascular diseases.
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http://dx.doi.org/10.1016/s0167-0115(03)00002-8DOI Listing
May 2003

Direct measurement of glycine-extended adrenomedullin in plasma and tissue using an ultrasensitive immune complex transfer enzyme immunoassay in rats.

Hypertens Res 2003 Feb;26 Suppl:S45-53

First Department of Internal Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.

The mature form of the vasodilator peptide adrenomedullin (AM-m) is synthesized from a glycine-extended precursor (AM-Gly) by enzymatic amidation. We have developed a highly sensitive enzyme immunoassay (Immune Complex Transfer Enzyme Immunoassay; ICTEIA) that enables us to measure levels of AM-Gly in plasma and tissue directly. The detection limit of this assay is 1 amol/assay, and the intra- and inter-assay precision are 4.5-14.1% and 9.9-20.5%, respectively. Dilution curves for plasma samples showed good linearity, and the analytical recovery was 107-116.6%. Using ICTEIA, we determined that the plasma concentration of immunoreactive AM-Gly is substantially higher than that of AM-m (5.22 +/- 2.56 vs. 1.21 +/- 0.79 fmol/ml). In contrast, levels of AM-Gly were much lower than those of AM-m in the lung, heart, kidney, adrenal gland and liver. We also evaluated AM-Gly and AM-m levels in rats in a morbid state induced by intraperitoneal administration of lipopolysaccharide (LPS). In most tissues, levels of AM-m and AM-Gly were both increased by LPS; however, AM-Gly/AM-m ratios were not significantly affected, which suggests that AM-Gly is rapidly converted to AM-m in tissue.
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http://dx.doi.org/10.1291/hypres.26.s45DOI Listing
February 2003

Rapid phenotypic assay for human immunodeficiency virus type 1 protease using in vitro translation.

J Virol Methods 2002 Oct;106(1):25-37

Department of Internal Medicine II, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1601, Japan.

A rapid in vitro phenotyping method for human immunodeficiency virus type 1 (HIV-1) protease was developed. In this system, both HIV-1 protease and substrates are prepared using a rabbit reticulocyte based coupled in vitro transcription/translation system. The activity of protease is evaluated by the amount of cleaved substrate measured by ELISA. In this system, wild-type protease derived from strain HXB2 was specifically inhibited in a dose-dependent manner by the protease inhibitors, indinavir and nelfinavir. Three drug-resistant proteases carrying a single mutation, D30N, L90M, and V82F, were analyzed in the absence of the inhibitors. Reflecting their impaired fitness, they exhibited decreased protease activity compared with the wild type. The apparent protease activity was greater for a Gag-Pol substrate encompassing the Gag-protease-reverse transcriptase junctions than for a substrate only covering the Gag region. Using the Gag-Pol substrate as the target, the indinavir-resistant mutant V82F was evaluated further. V82F showed 9-fold resistance to its cognitive protease inhibitor, indinavir; however, it manifested only moderate (2-fold) resistance to a non-cognitive inhibitor, nelfinavir. This simple and rapid method may be useful for phenotyping of drug-resistant HIV-1 protease as well as for screening new inhibitors of HIV-1 protease.
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http://dx.doi.org/10.1016/s0166-0934(02)00133-7DOI Listing
October 2002

Immune complex transfer enzyme immunoassay for anti-ovalbumin IgA in serum.

Ann Clin Biochem 2002 Sep;39(Pt 5):482-6

Division of Maternal and Child Health Science, National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku, Tokyo 162-8636, Japan.

Background: An immune complex transfer enzyme immunoassay for antiovalbumin immunoglobulin A (IgA) was developed.

Methods: Serum-specific antibody was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-ovalbumin conjugate and ovalbumin-beta-D-galactosidase conjugate. The complex formed from the three components was trapped onto polystyrene balls coated with anti-2,4-dinitrophenyl group immunoglobulin G, eluted with epsilonN-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with anti-human IgA alpha-chain. Bound beta-D-galactosidase activity was determined by fluorometry.

Results & Conclusions: The detection limit of this method for the measurement of specific anti-ovalbumin IgA was 9 fmol/tube, which was 20-fold lower than that of the enzyme-linked immunosorbent assay (ELISA). Because serum interference with this method was lower than that with the ELISA, the detection limit of this method was 300-fold lower than that by the ELISA. Anti-ovalbumin IgA was detected in 100% of healthy subjects, which was confirmed by pre-incubation with an excess amount of ovalbumin.
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http://dx.doi.org/10.1258/000456302320314494DOI Listing
September 2002

Concentration of egg white lysozyme in the serum of healthy subjects after oral administration.

Clin Exp Pharmacol Physiol 2002 Jan-Feb;29(1-2):79-83

Department of Biochemistry, Miyazaki Medical College, Kiyotake, Miyazaki, Japan.

1. While the egg white lysozyme preparation ER0068 (Neuzym; Eisai, Tokyo, Japan) is widely used clinically, no studies have been performed on its pharmacokinetic properties at clinically relevant doses. In the present study, we used a highly sensitive two-site enzyme immunoassay in order to determine the pharmacokinetic properties of egg white lysozyme after oral administration of two doses within the clinical range, paying particular attention to the effects of food intake. 2. A total of 22 healthy male subjects aged 20-45 years participated in the study. All subjects had been screened for egg white allergy and non-specific lysozyme inhibitors in their serum. Subjects who received 90 mg ER0068 after an overnight fast reached a maximum serum concentration of 1700 pg/mL within 1 h, compared with non-detectable levels in untreated controls. In a second experiment, subjects received 30 and 90 mg ER0068 after an overnight fast and 90 mg in the non-fasted state and exhibited maximum serum levels of 37, 360 and 49 pg/mL, respectively. Egg white lysozyme concentrations in serum returned to undetectable levels after a maximum of 48 h. 3. We conclude that clinically relevant concentrations of egg white lysozyme are absorbed in significant amounts, despite its high molecular weight. However, food intake considerably reduces the amount of enzyme absorbed.
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http://dx.doi.org/10.1046/j.1440-1681.2002.03605.xDOI Listing
June 2002