| Project/Area Number |
60480478
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Laboratory medicine
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| Research Institution | Nagoya University |
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Principal Investigator |
KOSAKA Akira Nagoya University, School of Medicine, 医学部, 講師 (50022794)
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| Co-Investigator(Kenkyū-buntansha) |
NAKASHIMA Nobuo Nagoya University, School of Medicine, 医学部, 講師 (80023810)
TAKEUCHI Jun Nagoya University, School of Medicine, 医学部, 教授 (60064789)
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| Project Period (FY) |
1985 – 1986
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| Project Status |
Completed (Fiscal Year 1986)
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| Budget Amount *help |
¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1986: ¥600,000 (Direct Cost: ¥600,000)
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| Keywords | Unconjugated bilirubin / Conjugated bilirubin / Bili-protein / Delta bilirubin / Bilirubin oxidase / Human serum albumin / Diazo reaction |
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| Research Abstract |
Most clinical laboratories measure bilirubin by diazo cleavage method. Total and direct reacting bilirubion are differentiated by their different rates of diazotization in the presence and absence of an added accelerator such as alcohol or caffeine. However, no accurate methods are available for specific determination of unconjugated bilirubin (Bu), conjugated bilirubin (Bc) and delta fraction of bilirubin (B <delta> ) which was tightly bound to serum albumin. It should be realized that direct and indirect bilirubin are operationally defined and are therefore dependent on the diazo reaction conditions, and do not accurately reflect Bc + B <delta> and Bu, respectively.
Our research project was aimed at the specific determination of bilirubin fractions in serum, and the following results were obtained;
1. A novel HPLC procedure which does not need any prior treatment of serum was developed for the separation of bilirubin fractions in serum using an octadecyl silane-bonded wide pore silica and a specific elution system.
2. B <delta> was highly purified by use of human serum albumin-immobilized affinity column, and its properties were precisely investigated.
3. The reactivity of bilirubin fractions with direct and indirect diazo reagents was investigated. It was found that Bc and B <delta> were not fully diazotized by the direct diazo reagent and Bu was partly diazotized dependent on the reaction conditions.
4. Novel enzymatic methods using bilirubin oxidase were established for the determination of bilirubin fractions in serum. The determination was based on the different pH reactivity of the enzyme to bilirubin fractions. In the absence of an anionic detergent, Bc and B <delta> were oxidized at acidic pH, Bc was oxidized at alkaline pH and Bu was neither oxidized at acidic nor alkaline pH. Total bilirubin was completely oxidized at neutral pH in the presence of the detergent.
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