| 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) (pConclusion: 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.
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| 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 (100 U/L), detection limits (2 U/L), and recovery (100cleared, and ascorbic acid (months at 4°C, 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. |
Fumi Yoshitomi, Miki Kawano, Takiko Tateishi, Eisaku Hokazono, Yuzo Kayamori, Hayato Okurano, Sonoko Yoshihiro, New colorimetric method for estimation of the redox state of human serum albumin., World Congress of World Association of Societies of Pathology and Laboratory Medicine, 2017.11, [Background and Purpose]
Human serum albumin (HSA) consists of three components: human mercaptoalbumin (HMA) with a free thiol group on Cys-34 having reducing power, reversibly oxidized human non-mercaptoalbumin-1 (HNA-1) with Cys-34 bound to cysteine or glutathione by a disulfide bound, and strongly oxidized human non-mercaptoalbumin-2 (HNA-2) with Cys-34 oxidized to a sulfenic, sulfinic, or sulfonic acid group.
To date, several high-performance liquid chromatographic (HPLC) methods have been developed for the measurement of the redox status of HSA. However, such methods may involve time-consuming procedures, making them unsatisfactory for routine use in the clinical laboratory. Thus, we tried to measure the redox status of HSA using the absorbance measured by the bromocresol purple (BCP) method and the modified BCP method, because HNA is more reactive than HMA in the BCP method, unlike the modified BCP method.
[Principal]
・Measurement of all albumin (modified BCP)
ALB-SH + R-S-S-R’ → ALB-S-S-R + HS-R’
BCP + ALB → BCP-ALB complex
・Measurement of the estimated HNA and HMA (BCP)
BCP + ALB → BCP-ALB complex
[Materials and Methods]
Two samples which were prepared at high HMA and HNA levels artificially were analyzed by the BCP method using a Hitachi automatic analyzer and by HPLC. The proportion of HMA or HNA in the samples was estimated using a plot of absorbance from the BCP method vs. redox% from HPLC and a regression analysis.
[Conclusions and Perspective]
An estimation of HMA and HNA levels is possible using spectrophotometry and HPLC for validation, but optimization of the BCP reaction conditions and a larger sample size is required to improve the method's accuracy. In the future, the method will be used to estimate the redox state of HSA in patient samples.
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| 6. |
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.. |
| 7. |
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.
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| 8. |
Hayato Okurano, Mai Sakamoto, Sonoko Yoshihiro, Miki Kawano, Eri Ohta, Takiko Tateishi, Eisaku Hokazono, Yuzo Kayamori, Separation of Nonmercaptalbumin and Mercaptalbumin with High-Performance Liquid Chromatography, Asia-Pacific Federation for Clinical Biochemistry, 2016.11, Human serum albumin is a simple protein with 585 amino acid residues. Albumin has a variety of physiological functions. One of them is its oxidation-reduction buff-ering capacity. In addition, the albumin has a thiol group from the N-terminal 34th cysteine (Cys-34) called reduced albumin (human nonmercapt-albumin), and the albu-min by which the thiol group from Cys-34 is oxidized is called oxidized albumin (hu-man mercapt-albumin). Moreover, HNA can be classified into two types. One that formed an S-S bond with sulfur-containing amino acid and can be reversibly oxidized, called HNA-1. Another is a thiol group that has irreversibly oxidized to –SOH or -SOH2, -SOH3 by such as an active oxygen species (ROS), called HNA-2. In vivo, HMA shows antioxidant activity to be oxidized to HNA. Albumin's role is very im-portant because it maintains homeostasis of the extracellular environment with low mo-lecular and contains a higher concentration than other ingredients.
In 1984, Sogami and others developed a method to separate HMA and HNA using high-performance liquid chromatography (HPLC), and research on the pathophysiolog-ical significance of each disease began. For example, their studies showed a connec-tion with liver disease and kidney disease, aging and so on. For the above reasons, examining the ratio of HNA (%HNA) is expected to be useful as an indicator of effec-tive diagnosis and therapy.
The objective of the present study is to show the usefulness of measuring %HNA as an oxidative stress marker. Therefore, this research’s target samples are sera before and after hemodialysis of kidney disease patients. We will measure %HNA of their sam-ples and we will analyze the correlation between %HNA and kidney function test and the relation between %HNA and other oxidative-stress markers by using statistical methods. Therefore, this research is expected to show the usefulness of measuring %HNA as an oxidative stress marker.
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| 9. |
Miki Kawano, Kazuya Yukishita, Susumu Osawa, Masanori Seimiya, Eri Ohta, Takiko Tateishi, Eisaku Hokazono, Yuzo Kayamori, Preliminary Study on a High-Sensitivity NADH Detection Method Using the Metal Chelating Reagent, Nitroso-PSAP, Asia-Pacific Federation for Clinical Biochemistry, 2016.11, NAD(P)H is often used as the detection agent in enzyme assays in the field of medical technology. It is the co-enzyme of a number of dehydrogenases, and, thus, is applied to indirectly measure the concentrations of substrates or activities of enzymes. Although this method is highly specific, its sensitivity is very low. Therefore, we have been conducting research with the aim of overcoming this limitation. Nitroso-PSAP is a chelating agent that combines with many heavy metals. It forms an Fe2+-Nitroso-PSAP chelating compound with Fe2+, with color development showing absorption at 750 nm. We herein report a high-sensitivity NAD(P)H detecting method using Nitroso-PSAP. In this method, Fe3+ is reduced to Fe2+ by NAD(P)H and increases in absorbance are measured according to the generation of the Fe2+-Nitroso-PSAP chelating compound at 750 nm.
The ultimate goal of this research is to detect the Fe2+-Nitroso-PSAP chelating compound formed in reactions between substances and dehydrogenases; however, since the present study is still in the primary stage, we used NADH as a sample. This method is constructed by 2 reagents. Reagent 1 contains 75 µmol/L Nitroso-PSAP, 50 µmol/L potassium sodium tartrate at pH 8.0, and 0.2 mol/L TAPS buffer, while reagent 2 contains 50 µmol/L ferric chloride, 6.75 µmol/L 1-Methoxy PMS at pH 3.0, and 0.05 mol/L acetate-sodium acetate dilution. All concentrations are shown as final concentrations. Using this method, we confirmed linearity, reproducibility, minimum detectable sensitivity, and molecular absorptivity. We then investigated the effects of substances potentially included in urine (e.g. albumin, glucose, oxalic acid, calcium chloride, urea, phosphoric acid, uric acid, creatinine, citric acid, magnesium chloride, and ascorbic acid) by applying this detection method to urine samples.
Most validation tests achieved good results; however, the reproducibility of low concentrations of samples was not sufficient. This method was affected by the large number of substances in urine, and, thus, further studies are needed.
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| 10. |
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: 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. |
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: 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.. |
| 12. |
Development of an enzymatic method with maltose phosphorylase for maltose permeability test of gastric mucosa using oral glucose tolerance samples. |
| 13. |
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: 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.. |
| 14. |
Akito Mominoki, Eri Ohta, Miki Kawano, Takiko Tateishi, Eisaku Hokazono, Yuzo Kayamori, Highsensitivity double-kinetic assay of creatinine in serum with enzyme cycling method ーPreliminary study part 1ー, International Federation of Biomedical Laboratory Science, 2016.09. |
| 15. |
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.. |
| 16. |
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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| 17. |
Susumu Osawa, Sachiko Kiuchi, Yoshiko Kudo, Eisaku Hokazono, Development of the enzymatic method for maltose permeability test of gastric mucosa using oral glucose tolerance samples, 13th Asia-Pacific Federation for Clinical Biochemistry and Laboratory Medicine Congress, 2013.10, The examination of gastric mucosal damage is performed by the 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. To develop an oligosaccharide permeability test of gastric mucosa using oral glucose tolerance samples instead of sucrose. Endogenous glucose was converted to glucose-6-phosphate by hexokinase involving an enzymatic method for oligosaccharide. Oligosaccharide absorption from the gastric mucosa was determined using the α-glucosidase-glucoseoxidase-peroxidase-Trinder’s reagent detection system. This enzymatic assay was used with a Hitachi 7050 automated analyzer. The oligosaccharide concentration was estimated as the concentration of maltose in this system. Serum maltose was measured based on a 2-point end assay. The average within-run and between day CVs were 0.4 and 2.3% at 200 μmol/L of maltose respectively, while the mean recovery was 93%. The serum maltose concentration rose increased by 50% in healthy subjects with a gastric mucosa disorder on oral alcohol loading. The assay was sensitive enough to monitor serum oligosaccharide concentrations during the oligosaccharide permeability test. This automated assay might be applicable to routine analysis in a large number of subjects as a simple screening test for gastric mucosa disorder.. |
| 18. |
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.. |
