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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Journal of Health Science

  • Publisher:
  • FAD Code: JHSC
  • CODEN: JJTHEC
  • ISSN: 1344-9702
  • Abbreviation: J. Health Sci.
  • DOI Prefix: 10.1248/jhs
  • Other Name(s): Eisei Kagaku, Japanese Journal of Toxicology and Environmental Health
  • Language: Japanese
  • Comments: Fulltext from 1999 V45

Citations 5

"Determination Of Isocitrate Using Immobilized Isocitrate Dehydrogenase In A Flow System And Its Application To Analyze The Total Isocitrate Content Of Beverages"
J. Health Sci. 2005 Volume 51, Issue 6 Pages 711-714
Hisakazu Mori, Yukiko Okamoto and Yuko Fujita

Abstract: The quantity of isocitrate was determined using an apparatus containing a reactor with immobilized isocitrate dehydrogenase in a flow line. NADH formed by an enzymatic reaction was fluorometrically detected. The optimal concentration of NAD+ in the carrier was determined. The maximum peak areas due to NADH were observed at pH 8.0 when the pH of the carrier consisting of Tris buffer ranged from 6.0 to 8.6. Various buffer types were also examined as carrier mediums at pH 8.0. In contrast to 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) and triethanolamine buffers which afforded comparable peak areas with that of Tris buffer, phosphate buffer showed a reduced peak area. This peak area decreased with an increase in the pH of the phosphate buffer from 6.5 to 8.5, suggesting the inhibitory effect of phosphate dianions upon the binding of adenosine-5?-monophosphate (AMP) to the binding site of the enzyme. When the carrier composed of Tris buffer (0.1 M, pH 8.0) was used, the calibration curve for isocitrate was linear in the range of 0.1-50 ?M (r = 1.000). The detection limit (S/N = 3) was 0.07 ?M. Relative standard deviations of the peak area at 1 and 10 ?M were 4.0% (n = 7) and 2.8% (n = 7), respectively. Thirty samples of isocitrate (2 ?M) were analyzed for 1 hr. This method was applied to the analysis of total isocitrate in several beverages. The recovery tests for the isocitrate added to samples indicated the reliability of the present method.

"Simultaneous Determination Of Ethanol And Acetaldehyde In Liquor Using A Flow System Composed Of Two Enzyme Reactors And An Octadecylsilica Column"
J. Health Sci. 2003 Volume 49, Issue 1 Pages 55-58
Hisakazu Mori, Yumiko Sekine and Yoshiko Takahashi

Abstract: Simultaneous determination of ethanol and acetaldehyde was performed with an apparatus consisting of two enzyme reactors placed either side of an octadecylsilica column in a single flow line. The enzymes used were alcohol dehydrogenase for ethanol analysis, and aldehyde dehydrogenase for acetaldehyde analysis. The most favorable concentration of NAD+ in the carrier for the simultaneous determination of ethanol and acetaldehyde was studied in wine or sake, in which the ethanol concentration was much higher that that of acetaldehyde. An NAD+ concentration of 0.1 mM was adopted. To distinguish between NADH formed due to ethanol and that formed due to acetaldehyde, several buffers (pH 7.8) were also examined for use as the carrier medium, and triethanolamine buffer was found to be the most favorable. The calibration curve for acetaldehyde was linear (r2 = 1.000) in the range of 0.2-100 ?M. With respect to ethanol, plotting the logarithm of the peak area versus that of the concentration gave a linear relationship (r 2 = 0.997) in the range of 0.04-100 mM. This method was applied to the simultaneous analysis of ethanol and acetaldehyde in several types of liquor. The results were similar to those obtained using a commercially available test-kit method, suggesting the reliability and practicality of this method in analyzing real samples.

"Direct Determination Of Nitrate Using Nitrate Reductase In A Flow System"
J. Health Sci. 2000 Volume 46, Issue 5 Pages 385-388
Hisakazu Mori

Abstract: Nitrate was determined using nitrate reductase (NR) in a flow system. A merging zone method was applied in the system in which a zone of NR and that of nitrate in separate streams were merged to react. The NADPH decreased by the enzymatic reaction was detected at 340 nm. The length of the reaction coil used for the enzymatic reaction was 250 cm. Of the concentrations from 0 to 0.6 mM NADPH in a carrier, 0.02 mM gave the maximum peak area due to decreased NADPH, suggesting that NR may be inhibited by a coenzyme, NADPH. The buffer of pH 7.5 was found to be optimum in the pH range from 6.5 to 8.0 of the buffer used as a carrier medium. Of the various buffer types (pH 7.5) used as the medium of carriers, piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) buffer afforded the maximum peak area. The elevated temperatures of the water bath for enzymatic reaction gave reduced peak areas and the maximum peak area was observed at 32?C. Under the optimum conditions, a linear calibration curve (r = 0.996) was obtained in the nitrate concentration range from 5 to 100 ?M and detection limit (S/N = 3) was 1.8 ?M. The relative standard deviation of the peak area at 20 ?M nitrate was 4.2% (n = 7). The method was applied to the determination of nitrate in samples of natural water. Nitrate content obtained by the present method agreed well with that determined by the JIS method.

"Determination Of Glucose In Plasma Using Immobilized Enzymes In A Flow System"
J. Health Sci. 2000 Volume 46, Issue 3 Pages 219-222
Hisakazu Mori*, Miyuki Ando, Yoshie Sugi, Miyako Kimura, and Katsuhiko Machida

Abstract: Glucose was determined using an apparatus containing an enzyme reactor in a flow line. The enzymes used for glucose assay were hexokinase and glucose-6-phosphate dehydrogenase. NADH formed by enzymatic reactions was fluorometrically detected. The calibration curve for glucose was linear within the range of 0.250 ?M (r=0.9996), and the detection limit was 0.1 ?M (S/N=3). This method was applied to the analysis of glucose in plasma. No influence of sodium fluoride on the activities of immobilized enzymes was observed. Recovery of glucose added to plasma or serum was found to be in the range of 98 to 106%. We investigated the correlation between the glucose content determined by other methods and that determined by the present method. A linear relationship was observed between that determined by the present method and that by both the glucose oxidase-electrode method (y=0.986x-4.6, r=0.9993) and the soluble enzyme method (y=1.004x+1.3, r=0.9982).
Glucose

"Determination Of Acetaldehyde Using Immobilized Aldehyde Dehydrogenase In A Flow System And Application To Analysis Of Acetaldehyde Content In Liquors"
J. Health Sci. 2000 Volume 46, Issue 2 Pages 146-148
Mori Hisakazu

Abstract: The quantity of acetaldehyde was determined using an apparatus containing a reactor with immobilized aldehyde dehydrogenase in a flow line. NADH formed by an enzymatic reaction was fluorometrically detected. The optimal concentration of NAD+ in the carrier was determined. Various buffer types were examined as a carrier medium. When the pH of the carrier was 7.8, great peak areas due to NADH were observed for buffers of phosphate, pyrophosphate, HEPES, PIPES-(piperazine-N,N'-bis(2-ethanesulfonic acid)) and triethanolamine, compared with that for Tris buffer. In the pH range from 7.0 to 8.0, the peak area due to NADH increased with the increase of pH in the case of phosphate buffer, in contrast to the case of Tris buffer in which peak area decreased with the increase of pH. When the carrier composed of phosphate buffer (0.1 M, pH 7.8) was used, the calibration curve for acetaldehyde was linear in the range of 0.2-10 ?M (r=0.9994). Detection limit (S/N=3) was 0.1 ?M. Relative standard deviation of peak area at 2 ?M was 2.6 % (n=7). The sampling rate was 40 samples h-1. This method was applied to the analysis of aldehyde in several liquors, and aldehyde content determined by the method agreed with that determined by a commercially available test-kit method.