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. METHOD: 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. CONCLUSION: 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.

[Estimation of glomerular filtration rate by inulin clearance: comparison with creatinine clearance].

Inulin clearance (Cin) is widely believed to be the gold standard of the glomerular filtration rate (GFR). However, in Japan, Cin has not been officially recognized by the Ministry of Health, Labour and Welfare of Japan for clinical use. Creatinine clearance (Ccr) has been used to estimate the renal function of patients, but there have been many studies in which Ccr estimates were GFR falsely high because the metabolism and tubular excretion of creatinine widely varied according to the pathophysiological state of the patient. In the present study, we determined Cin and Ccr simultaneously in 116 adult patients with renal diseases and diabetic mellitus. The clearance study was performed by the modified Wesson's method. The inulin preparation was FFI-1010 (Fuji Yakuhin Co. Ltd.). Inulin in serum and urine was determined by the newly devised enzymatic assay (Toyobo Co. Ltd.), which is specific for inulin. The mean Cin was 35.0 +/- 14.4 ml/min/1.73 m2. The mean Ccr(the enzyme assay) was 63.6 +/- 24.1 ml/min/1.73 m2 and that of the kinetic Jaffe assay was 55.3 +/- 19.3 ml/min/1.73 m2. Mean Ccr/Cin was 1.93 +/- 0.73, 1.69 +/- 0.62, respectively. This ratio was significantly different(p < 0.05) in the degree of reduction of Cin, with values of 2.07 +/- 0.82 (Cin < 40 ml/min/1.73 m2) and 1.64 +/- 0.32(40 < Cin < 80 ml/min/1.73 m2), respectively. Only 8 patients were classified into the same degree of reduced renal function (the Guideline of Japanese Society of Nephrology). The findings of this study suggest that the GFR determined by Ccr could misjudge the renal function of patient and delay the administration of proper treatment of the patient. Introduction of Cin into the clinical field is necessary to avoid this delay.

[Diuretics; their characteristics and future development].

We review the diuretics regarding the mechanism of action, way of clinical use and their adverse effects. Recent progress of molecular biology revealed the molecular target of diuretics and thereby, the molecular mechanism of diuretic action became clear to understand. Loop diuretics and thiazides are the most widely used diuretics and the physiologic adaptation to their prolonged use are mentioned. Carbonic anhydrase inhibitors are not used as diuretics but for correction of metabolic alkalosis and treatment of glaucoma. Potassium-sparing diuretics have modest natriuresis but the combination with loop diuretics or thiazides results in strong natriuresis. Adenosine type 1 receptor antagonist has been developed for treatment of edema in chronic heart failure. Vasopressin type 2 receptor antagonists are developed for a new type of diuretics to increase the free water clearance in cases of chronic heart failure and SIADH. The recombinant atrial natriuretic polypeptide is recently used as diuretics in acute heart failure. Knowledge of pharmacological action of diuretics could help the appropriately clinical use of diuretics, in particular the diuretics-resistant edema.

Skim soy protein enhances GFR as much as beefsteak protein in healthy human subjects.

BACKGROUND: It has been reported that an acute load of beefsteak (200 g) significantly enhanced the glomerular filtration rate (GFR; inulin clearance and creatinine clearance) in healthy human subjects compared to that in the same subjects in the fasted state. However, no comparative study of the effects of the same amount of vegetable protein on GFR has been reported to date. METHODS: We attempted to compare changes in the GFR (inulin clearance and creatinine clearance) in six healthy male subjects following consumption of the same amount of beefsteak or baked skim soy with soy sauce (protein, 86.9 g) after fasting. The clearance study was performed by conventional methods. Inulin was measured by the anthrone method. Creatinine was measured by the Jaffe rate assay method. Amino-acid analysis of the beefsteak and baked skim soy with soy sauce was done by acid or hydroxide hydrolysis and an amino-acid analyzer. RESULTS: A significant enhancement of the GFR (both inulin clearance and creatinine clearance) was observed following acute loading with beefsteak or baked skim soy with soy sauce, compared to the GFR in the fasted state. No significant difference was observed between the results with beefsteak and the results with baked skim soy with soy sauce. Amino-acid analysis revealed that the total amount of three amino acids (glycine, alanine, and arginine; or serine, alanine, and proline) was almost identical in beefsteak (animal protein) and baked skim soy with soy sauce (vegetable protein). CONCLUSIONS: The present study demonstrated that vegetable protein with the same amino-acid composition could enhance the GFR in healthy subjects as much as animal protein.