Evaluation of a new automated, enzymatic inulin assay using D-fructose dehydrogenase.
BACKGROUND
Inulin clearance (Cin) is widely considered to be a gold-standard assessment of glomerular filtration rate (GFR). Although inulin concentrations are commonly determined by the anthrone method, this method has several disadvantages, including a hazardous heating procedure using a strong acid, as well as a nonspecific reaction and being influenced by other saccharides such as glucose.


METHODS
Here, we report the development of a new automated, enzymatic inulin assay. This method uses D: -fructose dehydrogenase (EC1.1.99.11), which does not require NAD or NADP as the electron acceptor, and utilizes oxygen transfer from hydrogen peroxide to an electron acceptor in a sensitive chromophoric system. This method allows rapid and accurate determination of inulin concentrations, and takes only 15 min on automated analyzers. We evaluated this new assay and compared it with the anthrone method.


RESULTS
The detection limit value and linearity of the new method were 5 mg/l and up to 300 mg/l, respectively. The within-run precision coefficient of variations (CVs) for serum and for urine were 2.05 +/- 0.79% (mean +/- SEM; n = 3) and 0.92 +/- 0.24%, respectively, and the between-run precision CVs for serum and for urine were 1.91 +/- 0.72% and 1.86 +/- 0.17%, respectively. No influence of glucose was observed with the new method, while a positive influence of 38.7% was observed with the anthrone method. However, neither method was influenced by fructose (3.0 mg/l in serum or 30 mg/l in urine). The analytical recovery rates of inulin were 97.8 +/- 0.7% (mean +/- SEM; n = 6) in serum and 99.0 +/- 0.4% in urine using the enzymatic method. We compared results for the enzymatic method and the anthrone method using 46 serum samples and 46 urine samples. Bland-Altman plots showed no significant difference between the two methods for the serum samples. However, inulin recovery in the urine samples measured by the enzymatic method was about 5% higher than that obtained by the anthrone method.


CONCLUSIONS
The new enzymatic assay offers a more convenient and more accurate measurement of inulin and may be suitable for routine procedures by automated analyzers in clinical laboratories.
Kimata
Shinsuke
S
Tsuruga Institute of Biotechnology, Toyobo Co. Ltd., 10-24 Toyo-Cho, Tsuruga, Fukui, 914-0047, Japan. shinsuke_kimata@toyobo.jp.
Mizuguchi
Katsuhiko
K
Diagnostic System Department, Toyobo Co. Ltd., 2-2-8 Dojima-hama, Kita-ku, Osaka, 530-8230, Japan.
Hattori
Shizuo
S
Diagnostic System Department, Toyobo Co. Ltd., 2-2-8 Dojima-hama, Kita-ku, Osaka, 530-8230, Japan.
Teshima
Shinichi
S
Diagnostic System Department, Toyobo Co. Ltd., 2-2-8 Dojima-hama, Kita-ku, Osaka, 530-8230, Japan.
Orita
Yoshimasa
Y
Osaka Jikei Research Center, 1-2-8 Miyahara, Yodogawa-ku, Osaka, 532-0003, Japan.
D000873:Anthracenes / Q000737:chemistry; D001322:Autoanalysis; D001681:Biological Assay / Q000592:standards; D001786:Blood Glucose / Q000378:metabolism; D002138:Calibration; D002237:Carbohydrate Dehydrogenases / Q000378:metabolism*; D005632:Fructose / Q000097:blood / Q000652:urine; D005919:Glomerular Filtration Rate; D006801:Humans; D007275:Injections, Intravenous; D007444:Inulin / Q000008:administration & dosage / Q000097:blood / Q000652:urine; D007668:Kidney / Q000378:metabolism / Q000502:physiology*; D015203:Reproducibility of Results; D013053:Spectrophotometry; D013997:Time Factors; D000095322:Anthrones
D000873:Anthracenes; D001786:Blood Glucose; D002237:Carbohydrate Dehydrogenases; D005632:Fructose; D007444:Inulin; C028761:D-fructose 5-dehydrogenase; C004522:anthrone; D000095322:Anthrones