透析アミロイドーシス (DRA) の原因蛋白は不可逆性にunfoldしたアミロイド化β2ミクログロブリン (A-β2M) であるが, そのアミロイド化の過程で部分的にunfoldした中間体の存在が確認されている. 今回, 血液透析患者の血清中β2MをLC/MS分析で解析した. 保存期腎不全患者, 導入期血液透析患者, 長期血液透析患者の血清β2Mでは＋7価から＋10価までの4個の荷電スペクトラムが確認された. 一方, 正常β2Mでは＋7価, ＋8価に, A-β2Mでは＋9価, ＋10価にピークを認めたため, (9価＋10価) / (7価＋8価) をMS Index (MS-I) として検討した. MS-Iは保存期1.70±0.13, 導入期1.90±0.13, 長期1.79±0.25と各患者群で差を認めなかった. アミロイド組織ではMS-Iは4.15, 7.33と高値であった. LC/MS分析でも血液透析患者の血清中にアミロイド原性を有するA-β2Mは認められず, 血管外に移行した中間体β2Mの一部でunfoldingが進行し, これが蓄積してDRAが発症するというshuttle仮説に合致する結果であった.

As well approved in current experimental studies over 20years or more, the intermediate molecules, i.e., the conformational variants of globular native protein, had been confirmed in the transitional process of the in vitro amyloidogenesis [1]. The presence of this molecule in hemodialysis (HD) setting had firstly reported in amyloid tissue from a femoral bone cyst in patient with the DRA by Bellotti’s group [2]. Then, we had identified this intermediate β2microglobulin (I-β2M) using with capillary electrophoresis (C.E) in serum not only from HD patients but also the chronic kidney disease (CKD) patients and healthy persons, then, we proposed “β2M shuttle hypothesis “ as amyloidogenic concept in clinical setting of HD [3,4]. This concept is based upon 5 evidences as follows:

Dialysis-related amyloidosis (DRA) is characterized by accumulation of amyloid β2- microglobulin (β2m) in the interstitial matrix. Matrix substances such as heparin have reportedly been strongly implicated in the pathogenesis of dialysis-related amyloidosis. In clinical setting of hemodialysis, two types of heparin, i.e., high and low molecular heparin (H.M.H. and L.M.H.) have been routinely used. Still commonly used is H.M.H., followed by L.M.H. preparations with distinct advantages. Here, we studied that the interaction of native and two amyloidogenic β2m variants: ΔN6β2m and D76N β2m with H.M.H. and L.M.H. We also investigated whether heparin could induce β2m to have an amyloidogenic conformation. Biolayer interferometry revealed that ΔN6β2m had a strong reaction and D76N β2m had a moderate reaction with H.M.H.. Furthermore,H.M.H. induced the C-terminal unfolding in a native β2m. By contrast, L.M.H. showed no reaction even with ΔN6β2m. This study showed firstly a direct binding of β2m with H.M.H.. H.M.H. would provoked a C-terminal unfolding of β2m, which indicated production of an amyloidogenic intermediate, i.e., β2m92-99. In addition, our findings also suggest that L.M.H. may provide beneficial effects against the development of the DRA.

Objectives: A beta(2)-microglobulin (beta(2)m) fragment that lacks the first six amino acids, i.e., Delta N6 beta(2)-microglobulin (Delta N6 beta(2)m), is an endogenous, proteolytically derived, amyloidogenic fragment of beta(2)m, the precursor protein in A beta M-2 amyloidosis (dialysis-related amyloidosis). As reports suggest the importance of C-terminal unfolding for the amyloidogenicity of beta(2)m, in this study we aimed to investigate conformational characteristics of Delta N6 beta(2)m related to amyloidogenicity. We also measured the concentration of an amyloidogenic intermediate of beta(2)m with C-terminal unfolding (beta(2)m92-99) in serum samples from 10 patients undergoing hemodialysis (HD).Methods: We utilized capillary electrophoretic analysis, surface plasmon resonance and enzyme-linked immunosorbent assay.Results and conclusions: We confirmed the normal core structure of Delta N6 beta(2)m with a commercial monoclonal anti-beta(2)m antibody. In addition, using the specific monoclonal antibody for the C-terminal peptide, i.e. mAb 92-99, we confirmed unfolding in the C-terminal region of Delta N6 beta(2)m. On the basis of these findings, we established an ELISA to measure beta(2)m92-99 using Delta N6 beta(2)m as a standard molecule in circulation. However, we did not detect beta(2)m92-99 in serum from 10 HD patients, despite the absence of uremic inhibitors in the serum.

Among subfractions of low-density lipoprotein cholesterol (LDL-C), small dense LDL-C (SdLDL-C) has been highlighted as the most atherogenic lipoprotein cholesterol. The present study aimed to compare the relationship of SdLDL-C with blood viscosity, a surrogate marker for cardiovascular disease, with that of other lipid fractions with blood viscosity in essential hypertensives (EHTs). In 128 untreated, early-stage EHTs, blood viscosity was measured with a falling-ball microviscometer, and serum levels of lipid fractions were determined. Blood and plasma viscosity was significantly higher in 49 patients with dyslipidemia (fasting serum level of LDL-C >140 mg dl−1, triglyceride >150 mg dl−1 or high-density lipoprotein cholesterol (HDL-C)<40 mg dl−1) compared with 79 patients without dyslipidemia, although hematocrit and RBC rigidity index ‘k’ did not differ between the two groups. Together, SdLDL-C, LDL-C, triglyceride and large LDL-C were positively correlated with blood viscosity, but for HDL-C, the correlation was negative. After adjusting for non-lipid variables that correlated with blood viscosity (that is, the age, body mass index, resting diastolic blood pressure, sex, hematocrit, plasma viscosity and homeostasis model of assessment of insulin resistance), SdLDL-C was most strongly associated with blood viscosity among the lipid fractions. These data suggest that SdLDL-C could strongly increase blood viscosity in EHTs.

Human amniotic membrane (AM) has been used widely as graft biomaterial for a variety of clinical applications. But, there are some persistent problems related to the preparation, storage, and sterilization. To resolve these problems, we developed hyperdry AM (HD-AM) using far-infrared rays, depression of air, and microwaves and then sterilized by γ-ray irradiation. To elucidate the benefit of HD-AM as biological materials, compare with the physical and histological properties of HD-AM with a freeze-dried AM (FD-AM) as typical freeze-dried methods, evaluate the safety of HD-AM in vivo experiment used nude mice, and demonstrate the feasibility of HD-AM transplant in pterygium. The water permeability and the sieving coefficient of HD-AM were significantly lower than that of FD-AM. HD-AM has kept the morphological structure of epithelium and connective tissues. At 18 months after transplanted, single and multilayers of HD-AM in the intraperitoneal cavity was degraded without any infiltrated cells. For clinical treatment, recurrence of pterygium and regrowth of the subconjunctival fibrosis were not observed during the 6-month follow-up periods after the surgery. It was proposed that HD-AM was a safe and effective new biological material for clinical use including treatment for recurrent pterygium.

We previously identified an intermediate β2‐microglobulin (I‐β2m), which is an amyloidogenic β2m variant, via capillary electrophoresis (CE) and reported hemodialysis (HD)‐associated variations in the serum concentrations of each β2m component, including that found in the rebound phase. Recent research has indicated that I‐β2m can bind, via the SO3‐ moiety, with glycosaminoglycan or proteoglycan, which are major components of interstitial tissue. Because alterations in I‐β2m are likely to be important in view of the possible accumulation of amyloidogenic precursor proteins in the interstitial space, we studied the I‐β2m profile as related to HD. We used CE to determine the I‐β2m profile both at the start and at the end of HD and during the rebound phase in 12 HD patients. We found both an unfolded β2m and a destructured I‐β2m. More important, two peaks appeared in the rebound phase, one suggesting a refolding and one suggesting an irreversible destruction. Given that the intercompartmental transfer coefficient for β2m is 1.0, our results indicated concomitant processes occurring after HD: refolding of the β2m conformation and trapping of destructured I‐β2m in the extravascular space. Because the trapping of destructured I‐β2m supposedly leads to accumulation of β2m in the interstitial space, we have proposed a new concept—a “shuttle” concept—for amyloid formation from β2m in the HD setting.

Objectives: Recently, small dense low-density lipoprotein cholesterol (sdLDL-C) has been highlighted as the most atherogenic lipoprotein in cardiovascular disease. In this study, the relations of sdLDL-C and other lipid parameters to surrogate markers of atherosclerosis were investigated in essential hypertensives (EHT) with normal renal function. Design and Methods: In 137 untreated EHT with GFR≥60 mL/min, plasma levels of sdLDL-C (using assay kit supplied by Denka Seiken Co., Ltd., Niigata, Japan), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), adiponectin and norepinephrine, and blood sugar were measured. Intima-media thickness (IMT) and stiffness index β (β) of carotid artery (i.e., index for wall thickness and arterial stiffness of large artery, respectively) was also evaluated by B-mode ultrasonography and ultrasonic phase-locked echo-tracking system, respectively. Results: Although LDL, TG and HDL-C did not associate with IMT, sdLDL-C and ratio of sdLDL-C to LDL significantly correlated with IMT (r = 0.226, 0.181, p < 0.05 of each). Multiple regression analysis demonstrated that sdLDL-C was an independent determinant for IMT together with age, blood sugar and plasma levels of adiponectin and norepinephrine (r2 = 0.323, P < 0.05). In contrast, sdLDL-C and the other lipoproteins did not relate to β. In addition, either blood pressure or pulse rate did not associate with IMT and β. Conclusions: These results indicate that in EHT with normal renal function, sdLDL was the best marker for large arterial wall thickness among lipid parameters, and suggest that quantitative measurement of sdLDL could give the useful information for atherosclerosis compared with systemic hemodynamics.

Heparin, one of the essential molecules called glycosaminoglycans (GAGs), is the anticoagulant that is commonly used in regular hemodialysis, during which dialysis-related amyloidosis (DRA) may develop. The pathogenic protein, i.e. precursor protein, in DRA is β(2)-microglobulin (β(2)m). Recent studies defined amyloidosis as a protein misfolding disease of precursor proteins including β(2)m. Because the analytic technique capillary electrophoresis can identify molecular variants of the folded β(2)m, i.e. conformational variants, we utilized it to investigate the effect of heparin on β(2)m conformation and thus determined whether heparin can promote DRA development by inducing a conformational change in the amyloidogenic β(2)m molecule. Heparin had a slight but significant effect on intermediate β(2)m conformation but no effect on native β(2)m conformation and on conversion of native to intermediate β(2)m. Our findings thus suggest a possible association of β(2)m with GAGs containing a sulfate moiety, including heparin, in HD patients.

In recent years, genetic diagnostics of pathogenic splicing abnormalities are increasingly recognized as critically important in the clinical genetic diagnostics. It is reported that approximately 10% of pathogenic mutations causing human inherited diseases are splicing mutations. Nonetheless, it is still difficult to identify splicing abnormalities in routine genetic diagnostic settings. Here, we studied two different kinds of cases with splicing abnormalities. The first case is a protein S deficiency. Nucleotide analyses revealed that the proband had a previously reported G to C substitution in the invariant AG dinucleotide at the splicing acceptor site of intronl/exon2, which produces multiple splicing abnormalities resulting in protein S deficiency. The second case is an antithrombin (AT) deficiency. This proband had a previously reported G to A substitution, at nucleotide position 9788 in intron 4, 14 bp in front of exon 5, which created a de novo exon 5 splice site and resulted in AT deficiency. From a practical standpoint, we discussed the pitfalls, attentions, and screening approaches in genetic diagnostics of pathogenic splicing abnormalities. Due to the difficulty with full-length sequence analysis of introns, and the lack of RNA samples, splicing mutations may escape identification. Although current genetic testing remains to be improved, to screen for splicing abnormalities more efficiently, it is significant to use an appropriate combination of various approaches such as DNA and/or RNA samples, splicing mutation databases, bioinformatic tools to detect splice sites and cis-regulatory elements, and in vitro and/or in vivo experimentally methods as needed.

Background/Aims: A misfolded β2-microglobulin (β2m) is a principle component in dialysis-related amyloidosis. However, no such conformational variant of β2m has yet been reported in a clinical setting. Capillary electrophoresis is a tool that can identify the conformational variant of β2m. Methods: Capillary electrophoresis was used to measure a transitional intermediate from native β2m (N-β2m) to the amyloid β2m. This technique was utilized to assay for intermediate β2m (I-β2m) in serum from 31 hemodialysis (HD) patients before and after HD, 5 patients with non-dialysis chronic renal failure (CRF), and 5 healthy persons. Results:The predialysis values of serum I-β2m and N-β2m were 2.7 ± 1.4 and 29.4 ± 6.8 mg/l, respectively, in the HD patients. The presence of serum I-β2m correlated weakly with the total serum β2m concentration in all HD patients. The serum N-β2m concentration decreased significantly during two types of dialysis treatment: by 32.8% on HD using a polymethylmethacrylate (PMMA) membrane and by 71.2% on online hemodiafiltration (HDF) with a polysulfone (PS) membrane. On the other hand, a dialysis-associated change in serum I-β2m varied from –36.4 to +203.5% in HD patients using PMMA and from –70.8 to +62.5% in online HDF patients using PS. Moreover, a rebound β2m profile suggested that I-β2m might be immobilized in the extracellular space. Conclusion: This study demonstrated that two or three conformational isomers of β2m were probably ubiquitously recognized in human serum. Though no progressive increase in serum I-β2m concentration could be found along with HD, this study shows a significantly poor removal of I-β2m in comparison to N-β2m in patients receiving ongoing dialysis treatment, even with online HDF.

Quality, cost, risk, and knowledge control are essential in clinical laboratory management. In addition, a benchmark set by a third party is important. However, it is very difficult to develop these systems without a guideline and an authoritative body to set the benchmark. ISO15189 issued in 2003 is an international standard of "medical laboratories--particular requirements for quality and competence". This international standard is superior as a guideline. ISO15189 specifies quality and competence particular to medical laboratories. Also, it can be used by medical laboratories to develop their quality management systems and assess their own competence, and by accreditation bodies to confirm or recognize the competence of medical laboratories. In Japan, the Japan Accreditation Board for Conformity Assessment (JAB) is an accreditation body of ISO15189. We were awarded ISO15189 accreditation on July 31, 2008. In this report, we summarize the preparations for ISO15189 accreditation and the effects on our laboratory.