University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Hiroyuki Ukeda

Abbrev:
Ukeda, H.
Other Names:
Address:
Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe B-200, Nankoku 783-8502, Japan
Phone:
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Fax:
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Citations 13

"Flow Injection Analysis Of DPPH Radical Based On Electron Spin Resonance"
Talanta 2002 Volume 58, Issue 6 Pages 1279-1283
Hiroyuki Ukeda, Yukiko Adachi and Masayoshi Sawamura

Abstract: In order to construct a rapid and selective determination system of free radicals, we developed an FIA system using an electron spin resonance (ESR) spectrophotometer (flow injection spin analysis) equipped with a flow-through flat cell. In the present investigation, 1,1-diphenyl-2-picrylhydrazyl (DPPH) was used as a model free radical. Using a single line flow system, 0.5 mM DPPH was repetitively injected. When the magnetic field was fixed at 335.3 mT, the largest change in the ESR signal was observed and obtained peak height was proportional to the concentration of DPPH radical. A double line flow system was constructed in which a carrier stream containing 0.15 mM DPPH was fed into the flat cell after confluence with a sample stream. When ascorbic acid was injected as a typical DPPH radical scavenger, a negative peak appeared in proportional to the concentration. Lower detection limit of ascorbic acid was 0.01 mM (S/N = 4), sampling frequency was 13 samples per h, and a satisfactory reproducibility (CV = 3.2%, 0.1 mM, n = 5) was obtained. The present system was also applied to estimate the DPPH radical-scavenging activity of other substances and food samples. (C) 2002 Elsevier Science B.V. All rights reserved.
Ascorbic acid

"Application Of Novel Assay Methods For Superoxide Anion-scavenging Activity To Food Samples"
Nippon Shokuhin Kagaku Kogaku Kaishi 2002 Volume 49, Issue 1 Pages 25-31
Hiroyuki UKEDA, Hironori MORIYAMA, Daisuke KAWANA, Yasuyuki KATAYAMA, Kinichi NAKABAYASHI, Masayoshi SAWAMURA

Abstract: Recently, the authors developed novel assay methods of superoxide dismutase, WST-1 method and chemiluminometric FIA (CL-FIA) method. In the present investigation, those two methods were applied to estimate superoxide anion-scavenging activity (SOSA) of foods and the results were compared with those by ESR method. Red wine, green tea, cocoa and coffee were used as the sample. The first one is the colorimetric method using highly water-soluble tetrazolium salt (WST-1). The dilution ratio giving 50% inhibition (IC50) of each sample by the WST-1 method was as follows : red wine, X 180-230 : green tea, X 3000; cocoa, X 230; coffee, X 210. Although direct reduction of WST-1 with food components was observed without involvement of superoxide anion, the extent of the non-specific reduction was less than 3% in those dilution ratios. The second one is the CL-FIA method using chemiluminescence of lucigenin. The immobilized xanthine oxidase in a reactor was used to generate superoxide anion. This method could be a useful tool for measuring SOSA (60 samples/h) much more rapidly in comparison with the other conventional methods. The following IC50 was obtained : red wine, X 3800-5800; green tea, X 120000-150000; cocoa, X 4300 : coffee, X 4100. The non-specific chemiluminescence was not observed in these dilution ratios. Furthermore, this system showed high stability even during 250 repetitive injections of diluted food samples. A significant linear relationship between these novel methods and the ESR method was recognized, indicating that these methods might be applicable to estimate SOSA of foods.

"Food Analysis By Biosensor Flow Injection Analytical Methods"
Nippon Shokuhin Kagaku Kogaku Kaishi 1995 Volume 42, Issue 10 Pages 849-858
Hiroyuki UKEDA

Abstract: 28 References
Dairy Products Cows Milk Amperometry Sensor Immobilized enzyme Sepharose beads

"Flow Injection Analysis Of Milk Sample"
J. Flow Injection Anal. 1994 Volume 11, Issue 2 Pages 142-153
Ukeda, H.

Abstract: A general review is presented concerning the need for milk analysis and the application of flow injection analysis (FIA) for the determination of milk components such as lactose, lipid, short-chain fatty acids, protein and so on and the microbial and somatic cell counting.
Lactose Lipids Fatty acids Protein Cow Review

"A Microbial Sensor For Determination Of Short-chain Fatty Acids And Its Application To Raw Milk Samples"
J. Flow Injection Anal. 1992 Volume 9, Issue 2 Pages 164-174
Hiroyuki Ukeda*, Gabriele Wagner, Ursula Bilitewski and Rolf D. Schmid

Abstract: The cited sensor comprised of a membrane of Arthrobacter nicotiana immobilized in poly(vinyl alcohol), fixed on a PTFE membrane of an O electrode and covered with a dialysis membrane (cut-off mol. wt. of 12,400; Sigma). The sensor was used to detect short-chain fatty acids in milk after FIA. Sample solution was injected into 0.1 M potassium phosphate buffer (pH 7) carrier solution (1 mL min-1) and transported through a mixing coil to a flow-through cell equipped with the sensor; measurements were carried out at 30°C. The sensor system was highly selective for short-chain fatty acids (C4:0 to C12:0); the calibration graph was rectilinear for 0.11 to 1.7 mM butyric acid. The sampling frequency was 20 h-1. The sensor system response results compared well with those obtained by GC and titrimetry (r = 0.92 and 0.78, respectively).
Acids, fatty Cow Electrode Electrode Sensor Heated reaction Method comparison Dialysis

"Enzymatic Assay Of Dehydrogenase Substrate Based On The Detection Of Superoxide Anion"
Food Res. Int. 2001 Volume 34, Issue 5 Pages 393-399
Ashok K. Sarker, Hiroyuki Ukeda, Daisuke Kawana and Masayoshi Sawamura

Abstract: The oxidation of NADH using NADH oxidase from Bacillus licheniformis or diaphorase from Clostridium kluyveri, was found to produce superoxide anion (O-2(-)). The generated O-2(-) reduced water-soluble tetrazolium salt WST-1 to WST-1 formazan. The formation of WST-1 formazan was monitored as a change in the absorbance at 438 nm. The formation of WST-1 formazan was found to be proportional to the NADH concentration. As a result, a linear curve was obtained within the range of 0.5 muM-50 muM NADH concentration. The concentration of NADH was determined by chemiluminescence using lucigenin specific for O-2(-) instead of WST-1. Application of NADH oxidase from Bacillus licheniformis was found to be 400 times stronger chemiluminescence than that of diaphorase with a lower detection limit of 15 nM. The present method was applied to determine ethanol combined with yeast alcohol dehydrogenase. The ethanol concentrations of various kinds of alcoholic beverages obtained by the present method were compared with those obtained using F-kit method. A good linear correlation was observed between them (r=0.9997) and the slope (1.005) was very close to unity, suggesting that the present method could be applied for the determination of ethanol in practical samples. (C) 2001 Elsevier Science Ltd. All rights reserved.

"Development Of Food Analytical Systems Based On The Use Of Immobilized Biocatalysts"
Bunseki Kagaku 1999 Volume 48, Issue 2 Pages 183-191
Hiroyuki Ukeda

Abstract: A convenient and rapid analytical system for food components responsible for food quality was developed based on the use of immobilized enzymes and microorganisms. First, an analytical system for superoxide dismutase (SOD) activity was constructed using the combination of an immobilized enzyme with flow-injection analysis (FIA). In this system, the immobilized xanthine oxidase played the role of a generator of superoxide anion; the sampling frequency was about 30 samples/h. Next, a novel analytical format was proposed involving a combination of immobilized enzymes with a 96-well microtiter plate. An alternate and repeated deposition of avidin and biotinylated enzyme was useful for enzyme immobilization with high activity and stability. Immobilized glucose oxidase under the optimized conditions was applicable to glucose determination, and the activity was retained during storage for 6 months and 50 successive determinations. The ability of microorganisms to recognize a given group of substances is useful for exploiting novel analytes. An example was indicated by the development of a rapid and convenient analytical system for short-chain fatty acids using immobilized Arthrobacter nicotiana-FIA. A sample throughput of 20 samples/h was achieved, and the obtained response showed a linear correlation with the total concentration of C-4:0 to C-12:0. The response was also partly related to the somatic cell count in raw-milk samples (n = 150), suggesting that the system should be applicable to detecting mastitis milk. The application of immobilized Psuedomonas putida to the determination of organic acids in wine is also described, in which the relationship between the specificity and the carbon sources used in the growth medium was investigated.
Glucose Immobilized enzyme Review

"Flow Injection Determination Of Xanthine Oxidase Inhibitory Activity And Its Application To Food Samples"
Anal. Sci. 2006 Volume 22, Issue 1 Pages 105-109
Le Hoang Lam, Ken Sakaguchi, Hiroyuki Ukeda And Masayoshi Sawamura

Abstract: The enzyme xanthine oxidase (XOD) has been recognized as a key enzyme causing oxidative injury to tissues by ischemia-reperfusion. For this reason, XOD inhibitor, which effectively suppresses this enzyme, plays an important role in the inhibition of many diseases related to reactive oxygen species (ROS). In order to screen XOD inhibitors rapidly and conveniently, a novel assay using flow injection analysis (FIA) was proposed in the present investigation. To optimize the practical FIA system, we studied the effect of the reagent concentrations and the flow condition on the enzymatic reaction, and then selected the optimum condition as follows: 200 mU/mL XOD concentration, 0.5 mM xanthine concentration, 0.5 mL/min flow rate, and 2 m mixing coil length. Under this condition, a typical XOD inhibitor quercetin was determined in the concentration range 0.1 - 1.5 mM at a sampling frequency of 10 samples/h. Using the optimized FIA method, we determined the XOD inhibitory activity of some food samples: onions, apples and teas, which are the high sources of flavonoids known as the potential XOD inhibitors. Among these samples, tea leaves showed the highest activity, the second was onions and the lowest was apples. Based on the result of the assay, not only quercetin, but also other components in investigated samples, contributed to the XOD inhibitory activity.

"Spectrophotometric Flow Injection Analysis For Hypoxanthine Based On The Detection Of Superoxide Anion"
Anal. Sci. 1999 Volume 15, Issue 11 Pages 1141-1144
Ashok K. SARKER), Hiroyuki UKED), Daisuke KAWAN) and Masayoshi SAWAMURA

Abstract: Biosensors consisting of a biological recognition system and a physicochemical transducer have been developed as new sensing instruments. Owing to their technical simplicity, rapidity and high selectivity, the use of biosensors has been introduced to food industries.1-3 Typical examples are biosensors related to fish-freshness monitoring.4-6 After the death of a fish, ATP in the meat is decomposed to uric acid by a series of catabolic enzymes.7 Hypoxanthine is an intermediate of these reactions and accumulates as the storage time increases. Therefore, simple and rapid methods for the determination of hypoxanthine are required in food industries. Hypoxanthine biosensors have been developed using immobilized xanthine oxidase (XO).8-11 The principle of the methods developed so far has mainly been based on the electrochemical detection of consumed oxygen and formed hydrogen peroxide.
Reactor

"Flow Injection Assay Of Superoxide Dismutase Based On The Reduction Of Highly Water-soluble Tetrazolium"
Anal. Sci. 1999 Volume 15, Issue 4 Pages 353-357
Hiroyuki Ukeda, Ashok K. Sarker, Daisuke Kawana And Masayoshi Sawamura

Abstract: A flow injection analysis (FIA) for superoxide dismutase (SOD) activity was developed based on the use of tetrazolium salt, WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt) and an enzyme reactor packed with Sepharose 4B on which xanthine oxidase (XO) and catalase were co-immobilized. WST-1 is highly water-soluble, and no adhesion of the reduced form to the FIA line was observed during continuous operation for 3 months. As optimized conditions, a sample (9 vol) was mixed with a reagent solution (1 vol) containing 3 mM hypoxanthine and 2 mM WST-1, and the mixed solution (20 µl) was injected into a carrier stream of 50 mM carbonate buffer (pH 10.2) at a flow rate of 0.4 ml/min. Under the conditions, the concentration of the SOD preparation giving 50% inhibition (IC50) was 2.7 µg/ml and the sampling frequency was 30 samples/h. When the SOD activity in erythrocytes of rats was determined by the present FIA method, the values were linearly related to those obtained by the conventional nitroblue tetrazolium (NBT) assay (r=0.975; n=18). The enzyme reactor was stable for at least 200 repetitive injections.
Immobilized enzyme

"Microbial Sensor For Estimating Organic-acids In Wine"
Anal. Sci. 1995 Volume 11, Issue 6 Pages 941-945
H. UKEDA, N. YAMAMOTO, M. SAWAMURA and H. KUSUNOSE

Abstract: A microbial sensor based on an oxygen electrode and the microorganism Pseudomonas putida was developed in order to determine fixed acids, such as L-malic acid, succinic acid and L-lactic acid, in wine. The carbon source used in cultivating the microorganism affected the selectivity of the sensor to a great extent. When L-lactic acid was used as the carbon source, the microbial sensor gave a selective response for L-lactic acid. determining L-lactic acid was constructed and applied to commercially available wine samples. The obtained result was a flow injection analysis system for determining L-lactic acid was constructed and applied to commercially available wine samples. The obtained result was comparable to that obtained by the F-kit method. On the other hand, the microbial sensor showed a response for L-malic acid, L-lactic acid, succinic acid and ethanol present in wine when meso-tartaric acid was used in the cultivation. The addition of L-lactic acid (0.05 mM) and ethanol (0.01%) into the carrier solution suppressed the response for L-lactic acid and ethanol, and under the conditions the microbial sensor was thus selective for L-malic acid and succinic acid. The sensor responses for wine samples (n = 6) were linearly related to the sum of the L-malic acid and succinic acid concentrations determined by HPLC with a correlation coefficient of 0.969. (12 references)
Acids, organic l-Lactic acid l-Malic acid Succinic acid Wine Electrode Sensor Immobilized cell Method comparison Interferences

"Flow Injection Chemiluminometric Assay Of Superoxide Dismutase"
Anal. Chim. Acta 2001 Volume 438, Issue 1-2 Pages 137-142
Hiroyuki Ukeda, Ashok K. Sarker, Daisuke Kawana and Masayoshi Sawamura

Abstract: A rapid and sensitive FIA method for superoxide dismutase (SOD) activity was developed using the chemiluminescence of lucigenin, which is specific to superoxide anion (O-2-) The immobilized xanthine oxidase in a reactor was used to generate O-2- and hypoxanthine was selected as the substrate. In the absence of SOD, the maximum amount of lucigenin reduced with O-2- is observed (control). The presence of SOD suppresses the reduction of lucigenin. Therefore, the suppression ratio against the control can be regarded as being the inhibition ratio of each sample. Under the optimum conditions, the concentration of the SOD preparation giving 50% inhibition (IC50) was < 200 ng/ml (about 3.6 ng per injection) and the sampling frequency was about 60 samples/h. No tendency of the response to decrease was recognized by 200 repetitive injections. As another approach, a double-line flow system was constructed in which a carrier stream containing hypoxanthine and lucigenin was fed into the enzyme reactor after confluence with a sample stream. Optimized conditions were examined and the performance was compared with that of the above single line system. When the SOD activity in erythrocytes of rats was determined by the present FIA method, the values were linearly related to those obtained by the conventional nitroblue tetrazolium (NBT) assay (r = 0.980, n = 19).

"Dynamic Analysis Of The Binding Process Of Bovine Serum Albumin On Glutaraldehyde-activated Controlled Pore Glass"
Anal. Chim. Acta 1995 Volume 308, Issue 1-3 Pages 261-268
Hiroyuki Ukeda*, Tohru Ishii, Masayoshi Sawamura and Hirozo Kusunose

Abstract: Twenty microlitre volumes of BSA solution (5 mg/ml) were repeatedly injected into a column (5 cm x 1.68 mm i.d.) packed with aminopropyl-controlled pore glass activated with glutaraldehyde (GA-CPG). The elution profile of BSA was recorded using phosphate buffer eluents (0.5 ml/min) and detection at 280 nm. The results were analyzed using a model based on the assumption that two modes are involved in binding BSA to GA-CPG. Binding process parameters such as bound amounts and binding rate constants were estimated by a curve fitting method. An increase in ionic strength of the carrier solution resulted in a reduction in the total amount of BSA bound. The maximum bound amount occurred at pH 6 with low ionic strength carriers and at pH 7 for higher ionic strength carriers. The reduction of GA-CPG with sodium borohydride reduced the bound amount while blocking treatment with amine had no effect.
Albumin Cow Serum Spectrophotometry Controlled pore glass pH Optimization