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Eisaku Hokazono Last modified date:2021.10.11

Lecturer / Division of Medical Sciences and Technology
Department of Health Sciences
Faculty of Medical Sciences




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Homepage
https://kyushu-u.pure.elsevier.com/en/persons/eisaku-hokazono
 Reseacher Profiling Tool Kyushu University Pure
Phone
092-642-6737
Fax
092-642-6737
Academic Degree
Doctor of Helth science
Country of degree conferring institution (Overseas)
No
Field of Specialization
Clinical Chemistry
Total Priod of education and research career in the foreign country
00years00months
Research
Research Interests
  • Development of measurement method and laboratory reagents for clinical diagnosis.
    keyword : clinical laboratory, urine protein, analysis of urine constituent
    2005.04.
Academic Activities
Papers
1. Eisaku Hokazono, Eri Ota, Taiki Goto, Saori Fukumoto, YuzoKayamori, Takeshi Uchiumi, SusumuOsawa, Development of a protein assay with copper chelator chromeazurol B, based on the biuret reaction, Analytical Biochemistry, https://doi.org/10.1016/j.ab.2021.114320, https://doi.org/10.1016/j.ab.2021.114320, 2021.07, This study aimed to provide a novel and highly sensitive protein assay based on the biuret reaction and using chromeazurol B, a metal chelate compound. The method consists of two reagents and an automated analyzer. First, a complex of copper and protein (biuret reaction) is formed. Second, a chelating reagent containing chromeazurol B forms a three-dimensional complex of protein, copper, and chromeazurol B at neutral pH, resulting in highly sensitive coloration. The intra-assay (n = 20) variation for the three levels was 3.54 % or lower at each concentration. Each response with α, β-, and γ-globulin was 103.8 % and 104.3 %, respectively, against albumin. The molar absorption coefficient (ε) of the present method was 2.5 × 105 m2/mol against human albumin, higher than that of the commercially available Lowry method (ε = 8.7 × 104 m2/mol), which is based on the same principle. The correlation test for the pyrogallol method with 30 urine samples showed good performance (r = 0.961). The method described here (the Biuret-based CAB method) is a more sensitive and rapid assay than the Lowry method, and it may also be applied to biological samples because of its similar reactivity towards various proteins..
2. A simple method to measure Tamm-Horsfall protein in the urine with 96 well micro-plate.
3. Miki Kawano, Eisaku Hokazono, Susumu Osawa, Shouichi Sato, Takiko Tateishi, Masahiro Manabe, Hirotaka Matsui, Yuzo Kayamori, A novel assay for triglycerides using glycerol dehydrogenase and a water-soluble formazan dye, WST-8, Annals of Clinical Biochemistry, 10.1177/0004563219830715, 56, 4, 442-449, Vol.56(4), 442-449,2019, 2019.01, Background
The GPO-POD chromogenic method is well known that peroxidase is affected by reducing agents, and recently, it has been reported that some materials affect its activity. Moreover, there is a high possibility of non-specific reaction as the method uses many enzymes. Against this background, we developed a simpler assay method for TGs without using peroxidase.
Methods
TGs were hydrolysed to glycerol and fatty acids by LPL followed by the reduction of the glycerol dehydrogenase (GDH) to dihydroxyacetone with simultaneous production of NADH from NAD+. To overcome incomplete conversion of glycerol to dihydroxyacetone by GDH at equilibrium, we added 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) to the reaction mixture to remove NADH, allowing the reaction to complete while showing stoichiometric production of reduced WST-8.
Results
The reaction was linear up to 6.4 mmol/L. The mean intra-assay (n = 20) and inter-assay (n = 20) imprecision, as determined by replicate analysis of three pooled human serum samples with different TG concentrations, were 1.1-2.3% and 1.1-1.5% coefficient of variation (%CV), respectively. No interference by 2.5 g/L haemoglobin, 65 μmol/L free bilirubin, and 359 μmol/L conjugated bilirubin were observed. The equation obtained in comparison with that by the GPO-POD method including endogenous glycerol eliminating step was: y = 1.0002x + 0.0395 mmol/L; r = 0.999; Sy/x = 0.049 mmol/L; n = 97.
Conclusion
Our method is an accurate, yet simpler and more sensitive, alternative method for the quantitative analysis of TGs..
4. Yuka Sato,Masanori Seimiya,Toshihiko Yoshida,Yuji Sawabe, Eisaku Hokazono, Susumu Osawa, Kazuyuki Matsushita, Development of a simple indocyanine green measurement method using an automated biochemical analyser, Annals of Clinical Biochemistry, 10.1177/0004563217745895, 55, 4, 491-495, 2018.07, Background: The indocyanine green retention rate is important for assessing the severity of liver disorders. In the
conventional method, blood needs to be collected twice. In the present study, we developed an automated indocyanine
green method that does not require blood sampling before intravenous indocyanine green injections and is applicable to
an automated biochemical analyser.
Methods: The serum samples of 471 patients collected before and after intravenous indocyanine green injections and
submitted to the clinical laboratory of our hospital were used as samples. The standard procedure established by the
Japan Society of Hepatology was used as the standard method. In the automated indocyanine green method, serum
collected after an intravenous indocyanine green injection was mixed with the saline reagent containing a surfactant,
and the indocyanine green concentration was measured at a dominant wavelength of 805 nm and a complementary
wavelength of 884 nm.
Results: The coefficient of variations of the within- and between-run reproducibilities of this method were 2% or lower,
and dilution linearity passing the origin was noted up to 10 mg/L indocyanine green. The reagent was stable for four
weeks or longer. Haemoglobin, bilirubin and chyle had no impact on the results obtained. The correlation coefficient
between the standard method (x) and this method (y) was r¼0.995; however, slight divergence was noted in turbid
samples.
Conclusion: Divergence in turbid samples may have corresponded to false negativity with the standard procedure.
Our method may be highly practical because blood sampling before indocyanine green loading is unnecessary and
measurements are simple..
5. Shou Terada,Miyuki Sakemoto,Yukiko Kawanobe,Miki Kawano,Takiko Tateishi,Taeko Hotta,Dongchon Kang,Yuzo Kayamori and Eisaku Hokazono, Establishment of a rapid and simple assay for measuring serum trehalase activity, Int J Anal Bio-Sci, 6, 2, 19-24, Vol. 6, No2 (2018), 2018.06, Trehalase (TREH, trehalose-1-glucohydrolase, EC 3.2.1.28) catalyzes the hydrolysis of α,α-trehalose (1-α-D-glucopyranosyl-α-D-glucopyranoside) to form two molecules of glucose. TREH has been identified in the brush border membranes of the kidney, liver, and small intestine of mammals, but its physiological role has not yet been clarified. Some methods for measuring TREH activity have been reported, but they have been time-intensive, and its suitability for quantitative analysis could not be ascertained. Therefore, we developed a simple and rapid enzymatic assay for measuring serum TREH activity using a Hitachi 7600 type automated analyzer and evaluated the assay performance. The precision, linearity, detection limit, and recovery test were good. This new method effectively measures the serum TREH activity and can be easily integrated with an automated analyzer for rapid, high performance assays. This method can also support further research on understanding TREH activity..
6. Eri Ohta, Eisaku Hokazono, Takiko Tateishi, Miki Kawano, Yuzo Kayamori, Development of an enzymatic assay for ethanolamine in plasma, Int J Anal Bio-Sci, 4, 4, 110-116, Vol. 4, No 4 (2016), 2016.12, Ethanolamine (Etn) contributes to the generation of phosphatidyl ethanolamine (PE) in vivo. However, its plasma concentration and clinical significance in humans are still unclear. Therefore, we developed a simple, rapid enzymatic method using an amine oxidase and copper from Arthrobacter sp. (AAO) (EC 1.4.3.6). We found a strong correlation between the values obtained with this method and those obtained with the HPLC (high performance liquid chromatography)-based method (r = 0.89). Thus, it is suitable for routine clinical use in the labora- tory. Moreover, we would like to examine the clinical significance of Etn..
7. New measurement method with sodium periodate for indocyanine green (ICG) test adapted to an automated analyzer .
8. Development of the Enzymatic Method for Maltose Permeability Test of Gastric Mucosa using the Oral Glucose Tolerance Samples.
9. Hokazono, E., Tamezane, H., Hotta, T., Kayamori, Y. and Osawa, S, Enzymatic Assay of Phosphatidylethanolamine in Serum using Amine Oxidase from Arthrobacter sp., Clinica Chimica Acta, Vol.412, 1436-1440, 2011.07.
10. Development of the enzymatic method of plasma choline for new biomarker in ischemic heart disease
.
11. Examination of new urine staining solution with xanthine pigments - PartⅠ: Selection of staining pigments -
.
12. Evaluation of the Measurement Reagent “MicroTP-AR(2)” for Total protein in Urine.
13. Development of new measurment reagent for serum calcium that avoids influences of gadolinium contrast agent for MRI.
14. Evaluation of the measurement for serum calcium using small automated analyzer (S40).
15. Hokazono, E., Osawa, S., Nakano, T., Kawamoto, Y., Oguchi, Y., Hotta, T., Kayamori, Y., Kang, D., Cho, Y., Kiyoko, S. and Sato, K. , Development of a new measurement method for serum calcium with chlorophosphonazo-III, Annals of Clinical Biochemistry, 46, 296-301, 46: 296–301, 2009.07.
16. Establishment of Mail Medical Examination System Using Immediate Plasma Separating Device by the Self-Collection Blood ー The Method of Dilution Ratio Caluculation by Using Internal Standard for the Sample with Different Amount of Collecting Blood ー.
17. Deveropment of new measurment method for serum calcium with chlorophosphonazo-Ⅲ.
18. Analysis of Proteins in Urinary Tract Stones and Urine of Urolithic Patients.
19. Evaluation of Serum Protein Fractions on an Automatic Electrophoresis Analyzer (EPA-696).
Presentations
1. Eisaku Hokazono, Kouki Hosaka, Masaru Akimoto, Noriko Kawamitsu, Miyuki Sakemoto, Miki Kawano, Takiko Tateishi, Yoshihiro Mizoguchi , Masaru Omori, Taeko Hotta, Dongchon Kang, Yuzo Kayamori, Can urinary Tamm-Horsfall protein estimation predict the presence of a urinary cast?
, International Federation of Biomedical Laboratory Science, 2018.09, Background: Tamm-Horsfall protein (THP) was first characterized by Tamm and Horsfall as an inhibitor of hemagglutination. THP is synthesized in the thick ascending limb of the loop of Henle and is the most abundant protein in human urine, excreted at an average rate of 50–100 mg/day. An increase or reduction in the excretion of urinary THP indicates various clinical conditions and diseases. This protein is known as a substrate of urinary casts. The formation of urinary casts suggests various clinical conditions in an individual. We often experience the presence of not a few hyaline casts in approximately 8% of dipstick-negative urine samples: these samples are usually not subjected to microscopic examination. Therefore, the present study aimed to determine whether the estimation of urinary THP could predict the presence of urinary casts.
Method: THP was isolated from urine by salting out: 15% (NH4)2SO4 and 72.5 mmol/L sodium chloride were added to 500 µL of urine sample. THP in the urine sample was detected at Ex/Em=280/325 nm via spectrofluorometry. Urine samples were divided into 2 groups: group A, healthy subject (male: female = 26: 21, Urine protein(-), Cast(-)), group B is disease subject (male: female = 5: 7, eGFR>60, Urine protein(-), Cast(+)). Thereafter, we compared THP levels between groups A and B via the Mann-Whitney U test.
Result: The reference standard interval for urinary THP in healthy female subjects (n = 21) was 28.2–82.6 mg/g•Cre. THP range was significantly higher in women than in healthy men (n = 26; 12.7-55.2 mg/g•Cre) (p<0.01). Furthermore, THP levels were significantly different between groups A and B among men (p<0.05, Area Under the Curve [AUC]: 0.854). THP levels were slightly but not significantly higher in group B than in group A among women not (p= 0.19, AUC: 0.691).
Conclusion: The present results indicate that urinary THP levels tend to increase in accordance with the situation of cast appearance. We intend to determine the cause of differences between men and women in more detail in future studies.
.
2. Miki Kawano, Eisaku Hokazono, Masanori Seimiya, Susumu Osawa, Development of Enzymatic Measuring Method of Ethanolamine Phosphate (part 3), International Federation of Biomedical Laboratory Science, 2018.09, BACKGROUND: Manic-depressive illness is a serious problem in Japan. Therefore, it’s very important to find and treat the disease early. However, because there are no objective diagnostic criteria, it’s difficult. The ethanolamine phosphate (EAP) is newly presented as a major marker of it. The capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) is used to measure serum EAP. However, simpler method is needed because it uses expensive instrument. From the above, we had aimed to make high sensitivity measuring method of EAP using enzymes. We developed an enzymatic assay using ortho-phosphoethanolamine phospho-lyase (PEAlyase). This enzyme acts on EAP to produce phosphoric acid, acetaldehyde, and ammonia. This enzymatic assay had reacted to phosphoric acid and got three molars of hydrogen peroxide from one molar of EAP. The endogenous substances in serum need to be eliminated first. We had used N-Ethyl-N-(2-hydroxy3-sulfopropyl)-3-methylaniline, sodium salt (TOOS) as coloring reagent. As results, the method had shown good linearity, however, the limit of detection and eliminating ability of phosphoric acid had been not enough. In order to solve these problem, we used Sodium-10- (carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine(DA-67) as coloring reagent. METHODS: We examined the linearity, the eliminating ability of phosphoric acid, hypoxanthine, xanthine, and uric acid, and the limit of detection. All examinations were conducted with a Hitachi 7180 automated analyzer. RESULTS: Present method showed linearity to 35 µmol/L. The eliminating ability of phosphoric acid was improved (from 0.7 mmol/L to 2.0 mmol/L). The limit detection was also improved (from 1 µmol/L to 0.4 µmol/L). CONCLUSION: Since DA-67 has higher molar extinction coefficient, we could improve the limit of detection. The same reason also enabled us to reduce sample volume, resulting in improving elimination ability of phosphoric acid. However, these results are not enough because the EAP value of depression patients are lower than 1.5 µmol/L and the phosphoric acid value is easily gets higher in some diseases. In order to solve these problems, we are going to make ultra-high sensitivity detection system using metals and chelating color reagents. .
3. Kouki Hosaka, Noriko Kawamitsu, Masaru Akimoto, Miyuki Sakemoto, Miki Kawano, Takiko Tateishi, Taeko Hotta, Dongchon Kang, Yuzo Kayamori, Eisaku Hokazono, A rapid and simple method to measure Tamm-Horsfall protein in the urine and to
investigate its clinical significance, World Congress of World Association of Societies of Pathology and Laboratory Medicine, 2017.11, Aim: Tamm-Horsfall protein (THP) was characterized by Tamm and Horsfall as an inhibitor of hemagglutination.
THP is the most abundant protein in human urine and the increase or decrease in the urinary excretion of THP is
an indicator of various clinical conditions and diseases, especially renal calculus. This protein readily
aggregates in response to various factors such as salt concentration and osmotic pressure in the urine. THP is
generally measured by an ELISA-based method. However, this method has limitations such as inaccuracy owing to
protein aggregation, and high cost of reagents. Therefore, we developed a rapid and simple method to measure
THP by using cost-effective reagents and studied the clinical significance of THP.
Method: THP was isolated from urine by salting out. For this, 15% ammonium sulfate, 72.5 mmol/L sodium
chloride, and 0.12% formic acid were added to 500 μL of urine sample. This mixture was centrifuged at 20,000 g
for 10 min. The supernatant was discarded, and the pellet was dissolved completely in 500 μL distilled water.
This protocol was repeated twice to completely isolate THP from the urine. Finally, THP in the sample solution
was detected at Ex/Em=280/325 nm with spectrofluorometer.
Result: The detection limit was 3 mg/L, and linearity was maintained in the range of 0-200 mg/L. The within-run
coefficient of variation (CV%) was 2.07% (mean±SD=79.1±1.64). THP recovery rates were good (95-106%).
Finally, the mean THP concentration in five healthy individuals was measured as 57.9 mg/L.
Conclusion: Our THP measurement method is rapid, simple, and cost effective. This simple protocol will enable
further research on THP. We intend to measure more clinical samples and study the clinical significance of THP
in future studies..
4. Shou Terada, Miyuki Sakemoto, Yukiko Kawanobe, Miki Kawano, Takiko Tateishi, Taeko Hotta, Dongchon Kang, Yuzo Kayamori, Eisaku Hokazono, Establishment and clinical utility of a rapid and simple assay for serum trehalase
activity, World Congress of World Association of Societies of Pathology and Laboratory Medicine, 2017.11, Background: Trehalase (TREH, trehalose 1-glucohydrolase, EC 3.2.1.28), which splits trehalose into two
glucose molecules, is found in the brush border membrane of human kidney, liver, and small intestine. Eze et
al. reported that plasma TREH activity is elevated in diabetic patients, although the mechanism for this is not
clear. Current TREH assays are sample- and time-intensive, and require cumbersome pretreatment. Thus, we aim to
establish a rapid, simple assay for serum TREH activity using an automated analyzer and examine its clinical
utility.
Methods: In this method, endogenous glucose is first removed by glucose oxidase (GOD) and catalase. Glucose
produced by TREH is then quantified by a coupled reaction with GOD and peroxidase. This reaction produces a
quinoneimine whose rate of formation is proportional to serum TREH activity and can be measured by absorbance
in a Hitachi 7600 automated analyzer. To examine the assay´s clinical utility, we measured the correlation
between serum TREH activity and each test item in 127 serum specimens submitted for biochemical and tumor marker
tests at Kyushu University Hospital.
Results: The assay was validated by measureing within-run CVs (<2.36%), between-day CVs (<3.41%), linearity (0-
100 U/L), detection limits (2 U/L), and recovery (100<±3%). Endogenous glucose (<200 mg/dL) could be
cleared, and ascorbic acid (<50 mg/dL) had no effect. Serum TREH activity was stable for <1 month at 25°C, <3
months at 4°C, <6 months at -80°C. Serum TREH activity (y) was weakly correlated with HbA1c (r = 0.444,
y=2.25x-1.63) and ChE (r = 0.521, y=0.039x+3.35).
Conclusions: Our assay for serum TREH activity is simple, rapid, and effective. The correlation with HbA1c and
ChE could be relevant for disease and should be examined further..
5. Eisaku Hokazono, Susumu Osawa, Eri Ohta, Miki Kawano, Takiko Tateishi, Masanori Seimiya, Yuzo Kayamori, Preliminary study on a high-sensitivity hydrogen peroxide detection method using the metal chelating reagent, Chromazurol B (CAB), American Association for Clinical Chemistry, 2017.08, Background:
Quantitation of biological sample components and enzyme activities is indispensable for determining the pathological condition of patients. The rapid determination and return of accurate results is critical to clinical chemical analysis for clinical diagnosis. The sensitivity of analytical techniques is vital to ensuring the accuracy of analyses.
Currently, two detection techniques are used for the majority of clinical enzyme activity measurements. The first of these methods, NAD (P) H, has low sensitivity, but is largely unaffected by reducing compounds. Conversely, the hydrogen peroxide-peroxidase method is highly sensitive, but is adversely affected by the presence of reducing species; especially when measuring urinary constituents.
Therefore, in the present study, we developed a novel high-sensitivity measurement system for hydrogen peroxide (H2O2) using a metal chelating reagent, Chromazurol B (CAB).
Methods:
CAB develops an absorption band at 600 nm when chelating Fe3+. In our novel method, Fe2+ is oxidized to Fe3+ by hydrogen peroxide originating from oxidizing enzymes under acidic conditions. Then, the absorbance of the CAB-Fe3+ complex is measured at 600 nm, and the increase in absorbance can be used to determine the quantity of hydrogen peroxide.
In this study, we used a Hitachi Model 7170 and 7600 (P module) automated analyzer to perform a two-point end assay at 37°C. The sample (H2O2, 10 µL) was mixed with 200 µL of reagent 1 (117 µmol/L iron (Ⅱ) sulfate and 0.12 mol/L formic acid buffer (pH 4.0, 25°C)). The mixture was maintained at 37°C for 5 min. After the addition of 24 µL of reagent 2 (975 µmol/L CAB in distilled water), the absorbance was measured at 600/800 (main/sub) nm wavelengths.
Results:
The within-run CVs of the above method using 0.4 and 2.2 µmol/L H2O2 solutions were 3.78 and 1.74%, respectively (n = 20). The results exhibited linearity from 0 to 3.0 µmol/L. The detection limit was 0.2 µmol/L. The molar absorption coefficient, indicating the measurement sensitivity, was 202,160 L·mol−1·cm−1. This is about seven times or over greater than the sensitivity of the current methods.
Conclusion:
The newly-developed method was highly sensitive to hydrogen peroxide, and may be applicable in a wide range of research and clinical laboratories. In the future, we will investigate the effects of reducing substances in biological samples, such as serum and urine, on the sensitivity of this method..
6. Eisaku Hokazono, Yuri Fukuya, Eri Ohta, Yukari Kawamoto, Takiko Tateishi, Miki Kawano, Susumu Osawa, Yuzo Kayamori, Development of the high-sensitivity assay of protein by new principle of three-dimensional complex with protein-copper-Chromazurol B, Asia-Pacific Federation for Clinical Biochemistry, 2016.11, Urine protein measurement has been used for the diagnosis and monitoring of renal disease for many years. In the laboratory, various methods have been used to measure it: Sulfosalicylic acid, Coomassie brilliant blue, Pyrogallol red-molybdate (PR-Mo) and so on. But these methods have different reactivity for each component protein in urine and body fluid.
We applied the copper (Cu)-chelating ligand, Chromazurol B (CAB) to the assay of urine protein based on Biuret reaction in natural pH. The present method used a Hitachi Model 7180 automation analyzer. The assay was performed with a two-point end assay at 37ºC. The calibrator or urine (2 µL) was mixed with 160 µL reagent 1 (4.75 mmol/L Cu and 15 mmol/L potassium sodium tartrate, 0.005% anion surfactant in alkaline buffer), then kept for 5 min at 37ºC. After addition of 80 µL reagent 2 (1.78 mmol/L CAB, 0.36 mmol/L chelating agent in neutral buffer), the absorbance was measured at 660/800 (main/sub) nm wavelength. The PR-Mo method (already commercially available) was performed for comparison. The procedure was performed with designated measurement parameters and the conventional calibrator of manufacturers.
 The within-run CVs of the present method with human serum albumin (HSA) solution (100 and 1000 mg/L) were 4.35% and 0.88%, respectively (n=20). The present method showed linearity from 0 to 3000 mg/L. Each response with α, β-globulin and γ-globulin was 94% and 100%, respectively, based on the reaction for albumin. With regard to the correlation with PR-Mo method (x) and our present method (y), the linear regression formula was y = 1.24 x – 3.13, the correlation coefficient was 0.915 (n=30).
We consider this method can be adapted for not urine samples but also other body fluids (ex. puncture fluid) in a clinical laboratory, because it gives almost the same performance for main proteins, and it also has stabilities and accuracies.
.
7. Eri Ohta, Eisaku Hokazono, Takiko Tateishi, Miki Kawano, Susumu Osawa, Yuzo Kayamori, Development of an enzymatic method for ethanol amine in blood, Asia-Pacific Federation for Clinical Biochemistry, 2016.11, Ethanolamine (EA) is mainly hydrolyzed from phosphatidyl ethanolamine (PE) by phospholipase D (PLD) in vivo. A study repotted that urine EA increased in newborn as Zellweger syndrome that is a congenital metabolic diseased. In recently, a metabolomics study using mass spectrometory reported that EA in saliva of pancreatic cancer patients increased significantly. The blood concentration of EA in healthy volunteer is 11.84 ± 4.15 µmol/L, but its clinical significant in adult were not clarify. Therefore, our purposes are to develop a rapid and simple enzymatic method for EA in blood with an amine oxidase involving cupper from Arthrobacter sp. (AAO) (EC 1.4.3.6) and to examine the clinical meaning of EA in plasma. In our measurement method, regent 1 (R-1) was contained of 0.1 mol/L HEPES buffer (pH 8.0 at 25ºC), 1.5 mmol/L TOOS, 5.0 kU/L POD, 10.0 kU/L L-ascorbate oxidase (ASOD), and regent 2 (R-2) was contained of 0.1 mol/L HEPES buffer (pH 8.0 at 25ºC), 15.0 kU/L AAO, 0.3 mmol/L 4-aminoantipyrine, 90.0 µmol/L potassium ferrocyanide. The assay used a Hitachi 7600 type analyzer. A 30 µL specimen was mixed with 180 µL R-1; after incubation for 5 min at 37 ºC, 90 µL R-2 was added; after another 5 min, the mixture was measured using a 2-point end assay performed at 37 ºC, with wavelengths of 800/546 (sub/main wavelength). The within-run CVs of the present method with kinds of EA solutions were % (n=10). The standard curve showed the linearity from 0 to 100 µmol/L. Analytical recovery was 89.5%. We succeeded in the development of a new enzymatic assay for EA in blood. And our present method will facilitate further research on the physiologic role of EA.
The present method is comprised of two reagents: one is for making β-glucose-1-phosphate by maltose phosphorylase and in the other, measuring NADPH produced by β-phosphoglucomutase and glucose-6-phosphate dehydrogenase. The enzymatic assay is used with a Hitachi 7600 automated analyzer. Plasma maltose was measured based on a 2-point end assay.
The average within-run CVs with maltose solutions (10 and 30 μmol/L) were 3.6% and 2.2%, respectively (n=10), while the recovery test was 95%. We consider that maltose has a potential that is available as a test of damaged gastric mucosal instead of sucrose. We address the relationship between the severity of gastric mucosa disorder and the maltose concentration. Method: < Enzymatic method > This method is comprised of two reagents: one is for removal of endogenous glucose by GOD and in the other α-Glucosidase acts for maltose and the formed glucose is detected. The enzymatic assay is used with a Hitachi 7600 automated analyzer. Plasma maltose was measured based on a 2-point end assay. The following conditions were used. Column; GL InertSustain C18 50×2.0 mm l.D. Eluent; acetonitrile and 40 mmol/L acetic acid/sodium acetate buffer (pH 4.2). Flow rate; 0.3 mL/min. Column temperature; 50ºC. UV detect; at 271 nm. Injection volume; 5 µL. Result: Basic performance evaluations (within-run CVs, recovery test, linearity and detection limits) were good. Maltose was retained at 3.7 min. The present method was sufficient to detect maltose without interference from other components in plasma. The correlation of the two methods is also good. Conclusions: Our enzymatic assay was sensitive enough to monitor plasma maltose concentrations during an oligosaccharide permeability test. We consider Maltose has a potential that is available as the test of damaged gastric mucosal instead of sucrose. We address the relationship between the degree of gastric mucosa disorder and maltose concentration..
8. Yoko Hashida, Akari Umemura, Eisaku Hokazono, Eri Ohta, Takiko Tateishi, Miki Kawano, Susumu Osawa, Yuzo Kayamori, Development of an enzymatic method with maltose phosphorylase for maltose permeability test of gastric mucosa using oral glucose tolerance samples
, Asia-Pacific Federation for Clinical Biochemistry, 2016.11, The examination of gastric mucosal damage is performed by a gastrointestinal test contrasting with X-rays and pepsinogen test in serum. Additionally, sucrose permeability has been suggested as a simple and noninvasive marker of gastric mucosal damage in human subjects. Our objective was to develop a maltose permeability test of gastric mucosa using oral glucose tolerance samples instead of sucrose. We earlier reported a method for maltose measurement using α-glucosidase, but this method had some problems including low specificity to substrate and the necessity to erase endogenous glucose in plasma. Therefore, we aim to develop an enzymatic method using maltose phosphorylase to measure maltose directly and specifically.
The present method is comprised of two reagents: one is for making β-glucose-1-phosphate by maltose phosphorylase and in the other, measuring NADPH produced by β-phosphoglucomutase and glucose-6-phosphate dehydrogenase. The enzymatic assay is used with a Hitachi 7600 automated analyzer. Plasma maltose was measured based on a 2-point end assay.
The average within-run CVs with maltose solutions (10 and 30 μmol/L) were 3.6% and 2.2%, respectively (n=10), while the recovery test was 95%. We consider that maltose has a potential that is available as a test of damaged gastric mucosal instead of sucrose. We address the relationship between the severity of gastric mucosa disorder and the maltose concentration. Method: < Enzymatic method > This method is comprised of two reagents: one is for removal of endogenous glucose by GOD and in the other α-Glucosidase acts for maltose and the formed glucose is detected. The enzymatic assay is used with a Hitachi 7600 automated analyzer. Plasma maltose was measured based on a 2-point end assay. The following conditions were used. Column; GL InertSustain C18 50×2.0 mm l.D. Eluent; acetonitrile and 40 mmol/L acetic acid/sodium acetate buffer (pH 4.2). Flow rate; 0.3 mL/min. Column temperature; 50ºC. UV detect; at 271 nm. Injection volume; 5 µL. Result: Basic performance evaluations (within-run CVs, recovery test, linearity and detection limits) were good. Maltose was retained at 3.7 min. The present method was sufficient to detect maltose without interference from other components in plasma. The correlation of the two methods is also good. Conclusions: Our enzymatic assay was sensitive enough to monitor plasma maltose concentrations during an oligosaccharide permeability test. We consider Maltose has a potential that is available as the test of damaged gastric mucosal instead of sucrose. We address the relationship between the degree of gastric mucosa disorder and maltose concentration..
9. Development of an enzymatic method with maltose phosphorylase for maltose permeability test of gastric mucosa using oral glucose tolerance samples.
10. Yui Nakano, Eisaku Hokazono, Eri Ohta, Miki Kawano, Takiko Tateishi, Susumu Osawa, Yuzo Kayamori, Examinationofthemaltosedetectionmethodastheexaminationofgastricmucosa disorder
-Development of the enzymatic method and UHPLC method for maltose permeability test of gastric mucosa using oral glucose tolerance samples-, International Federation of Biomedical Laboratory Science, 2016.09, Background: Early detection and treatment of gastric cancer are important. An easily and noninvasive screening test is required for detection in the early stage of gastric cancer. Seimiya et al. reported the sucrose permeability test as a simple and noninvasive marker of gastric mucosal damage in human subjects. Therefore, we aim to develop an enzymatic method for a maltose permeability test of gastric mucosa using oral glucose tolerance samples instead of sucrose. Moreover, we developed an Ultra High-Performance Liquid Chromatography (UHPLC) method for measuring maltose as a reference method and estimated the present enzymatic method with it.

Method: < Enzymatic method > This method is comprised of two reagents: one is for removal of endogenous glucose by GOD and in the other α-Glucosidase acts for maltose and the formed glucose is detected. The enzymatic assay is used with a Hitachi 7600 automated analyzer. Plasma maltose was measured based on a 2-point end assay. The following conditions were used. Column; GL InertSustain C18 50×2.0 mm l.D. Eluent; acetonitrile and 40 mmol/L acetic acid/sodium acetate buffer (pH 4.2). Flow rate; 0.3 mL/min. Column temperature; 50ºC. UV detect; at 271 nm. Injection volume; 5 µL.

Result: Basic performance evaluations (within-run CVs, recovery test, linearity and detection limits) were good. Maltose was retained at 3.7 min. The present method was sufficient to detect maltose without interference from other components in plasma. The correlation of the two methods is also good.

Conclusions: Our enzymatic assay was sensitive enough to monitor plasma maltose concentrations during an oligosaccharide permeability test. We consider Maltose has a potential that is available as the test of damaged gastric mucosal instead of sucrose. We address the relationship between the degree of gastric mucosa disorder and maltose concentration..
11. Kouki Hosaka, Miki Kawano, Eri Ohta, Takiko Tateishi, Yuzo Kayamori, Eisaku Hokazono, Examination of purification of Tamm-Horsfall Protein in urine ーImprovement of recovered amount and working efficiency in purification of Tamm-Horsfall Protein in urineー, International Federation of Biomedical Laboratory Science, 2016.09, Aim: Tamm-Horsfall Protein (THP) is synthesized in the thick ascending limb of the loop of Henle and is the most abundant protein in human urine. The increase and decrease of the quantity of this urinary excretion are related to the various clinical conditions and the course of disease, especially renal calculus. This protein has the characteristic that is easy to aggregate by various factors (salt concentration, osmotic pressure in urine etc.). This brings about some problems in purification for measuring THP, especially enzyme-linked immunosorbent assay (ELISA)-based methods. Therefore, we aimed to establish an efficient protocol for purification of THP in urine for a standard, which is used on analyzing THP.

Method: We tried to arrange the method with the diatomaceous earth filtration by Franca et al. to purify from pooled urine of healthy volunteers. We evaluated our present method with imitated urine as the purity and quantity of separation (The evaluation of purity used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and THP concentration was measured by absorbance in 277 nm.).

Results: Compared with the conventional method, our method was able to obtain 1.4 times the amount of THP. And the purified component did not include most proteins in urine except THP. In addition, the present method was able to shorten the operation time.

Conclusion: The present method can purify more THP in urine more easily in a shorter time. Not only are more problems avoided in THP measurement, but also our simple protocol will facilitate further research on the physiologic role of THP..
12. Eisaku Hokazono, E. Ota, S. Osawa, S. Kiuchi, M. Akimoto, T. Tateishi, Yuzo Kayamori, Development of the high sensitive assay of protein by new principle of three-dimensional complex with protein-cupper-Chromazurol B, IFCC WORLDLAB ISTANBUL , 2014.06, BACKGROUND
Urine protein measurement has been used for the diagnosis and monitoring of renal disease for many years. In the laboratory, various methods have been used to measure it: Sulfosalicylic acid method, Coomassie brilliant blue method, Pyrogallol red-molybdate method (PR-Mo) and so on. But their methods have different reactivity for each component protein in urine and body fluid.
METHODS
We applied the copper (Cu)-chelating ligand, Chromazurol B (CAB) to the assay of urine protein based on Biuret reaction in natural pH. The present measurement method used a Hitachi Model 7170 automation analyzer. The assay was performed with a one-point end assay at 37ºC. The calibrator or urine (3 µL) was mixed with 35 µL reagent 1 (R-1: 0.1 mol/L NaOH solution containing 12 mmol/L Cu and 50 mmol/L potassium sodium tartrate), then kept for 5 min at 37ºC. After addition of 175 µL reagent 2 (R-2: pH 5.3, 0.1 mol/L 2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) solution containing 0.5 mmol/L CAB), the absorbance was measured at 600/800 (main/sub) nm wavelength. PR-Mo method (already commercially available) was performed for comparison. The procedure was performed with designated measurement parameters and the calibrator of manufacturers. Urine specimens were collected from in- and outpatients in Kyushu University Hospital.
RESULTS
The within-run CVs of the present method with human serum albumin (HSA) solution (150 and 1000 mg/L) were 2.45% and 0.49%, respectively (n=20). The present method showed linearity from 0 to 4000 mg/L. Analytical recovery was 106%. Each response with α, β-globulin and γ-globulin was 98% and 97%, respectively, based on the reaction for albumin. With regard to the correlation with PR-Mo method (x) and our present method (y), the linear regression formula was y = 1.24 x + 30.9, and the correlation coefficient was 0.95 (n=48).
CONCLUSION
We consider this method can be adapted for not urine samples but also other body fluids (ex. marrow liquid) in a clinical laboratory, because it gives the almost same performance for main proteins, and it also has stabilities and accuracies.
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13. Eri Ohta, Eisaku Hokazono, Yuzo Kayamori, Development of the enzymatic method of serum ethanolamine and examination of its clinical
utility
, the Asian Society of Clinical Pathology and Laboratory Medicine, 2012.11, Ethanolamine (EA) is mainly hydrolyzed from phosphatidyl ethanolamine (PE) by phospholipase D (PLD) in vivo. A study reported that urine EA increased in newborns as Zellweger syndrome, a congenital metabolic disease, but its concentration in blood and clinical significance in adults were not clarified. Recently, a metabolomics study using mass spectrometry reported that EA in saliva of pancreatic cancer patients increased significantly. Therefore, our purposes are to develop a rapid and simple enzymatic method with an amine oxidase involving copper from Arthrobacter sp. (AAO) (EC 1.4.3.6) and to examine the clinical meaning of EA in serum. In our measurement method, reagent 1 (R-1) contained 0.1 mol/L HEPES buffer (pH 8.2 at 25ºC), 1.6 mmol/L TOOS, 5.0 kU/L POD, 1.12 mmol/L N-ethylmaleimide, 16.8 kU/L L-ascorbate oxidase (ASOD) and 7.47 mmol/L NaN3. Reagent 2 (R-2) contained 0.1 mol/L HEPES buffer (pH 8.2 at 25ºC), 20.0 kU/L AAO and 0.4 mmol/L 4-aminoantipyrine. The assay used a Hitachi 7170 type analyzer. A 10 µL specimen was mixed with 180 µL R-1; after incubation for 5 min at 37 ºC, 90 µL R-2 was added; after another 5 min, the mixture was measured using a 2-point end assay performed at 37 ºC, with wavelengths of 700/546 (sub/main wavelength). The within-run CVs of the present method with three kinds of EA solutions ranged 0.43-7.13% (n=10). The standard curve showed linearity from 0 to 800 µmol/L. Analytical recovery was 98.4%. The reference interval of EA in normal was 8.17 ± 4.83 µmol/L (n=19). EA in hepatocellular carcinoma patient sera was significantly higher than normal. In conclusion, our method for serum EA is suitable for routine clinical use in the laboratory for determination of accuracy and simplicity. We consider EA in serum may be a new biomarker of hepatocellular carcinoma..
Membership in Academic Society
  • Japanese Journal of Clinical Chemistry
  • The Society of Analytical Bio-Science
  • Japanese Journal of Medical Technology
  • The Functional Food Consultant Certification Institute
  • Japanese Society of Laboratory Medicine
  • Japanese Journal of Clinical Laboratory Automation
  • Japanese Society of Sonographers
  • American Association for Clinical Chemistry