Sensitive enzymatic assay for erythrocyte creatine with production of methylene blue

Abstract

We developed a new, highly sensitive enzymatic method for quantifying creatine in erythrocytes, which comprises creatine amidinohydrolase, sarcosine oxidase, and peroxidase. In the present method, an N-methylcarbamoyl derivative of methylene blue, 10-N-methylcarbamoyl-3,7-bis(dimethylamino)phenothiazine (MCDP), was used as a sensitive chromogenic compound. Potassium ferrocyanide was used to prevent nonspecific oxidation of MCDP. The enzymatic method exhibited good analytical performance: precision, within-run CVs <1.0% and between-day CVs <2.0%; average analytical recovery, 99.3% ± 1.8%; detection limit, 1.0 μmol/L in hemolysate; and linearity, at least up to 500 μmol/L as creatine concentration in hemolysate. Excellent agreement was observed between the present method (y) and HPLC (x), y = 1.029x − 0.002 μmol/g hemoglobin, r = 0.9998, Sy‖x = 0.053 μmol/g hemoglobin (n = 110). No significant interference was produced by various compounds, including guanidino compounds, amino acids, and reducing materials. The reference intervals (mean ± 2 SD) for erythrocyte creatine obtained from 60 males and 60 females were (in μmol/g hemoglobin) 1.18 ± 0.52 (0.66–1.70) for males and 1.35 ± 0.49 (0.86–1.84) for females. Using this method, we documented changes in erythrocyte creatine in patients with various hemolytic conditions, including hemolytic anemia, liver cirrhosis, renal insufficiency, and chronic renal failure treated with hemodialysis with or without the administration of erythropoietin. We conclude that the use of MCDP allows sensitive measurement of erythrocyte creatine and that MCDP with potassium ferrocyanide can improve the sensitivity of assays that use peroxidase for detection of H2O2.

Raised erythrocyte creatine in patients with pulmonary arterial hypertension – Evidence for subclinical hemolysis

“…An alternative is to assay Erythrocyte Creatine (EC) levels, since RBC creatine levels decrease as the cell ages. 13 EC levels correlate well with the gold-standard 51-Cr labeling technique. 14 The clinical utility of EC has been established in a cohort of patients with hemolytic anemia and hemolysis caused by mechanical heart valves.…”

“…The EC was performed as per the technique described in detail elsewhere. 13 In brief, plasma and buffy coat are aspirated from the blood sample after centrifugation. A small sample of mixed packed RBCs is then hemolyzed in a four-fold volume of 1 g/L saponin solution over 20 min at room temperature.…”

“…Creatine concentration in the supernatant was measured with the enzymatic assay method involving creatine amidinohydrolase, sarcosine oxidase and peroxidase. 13 Measured data were expressed as micromole creatine per gram of hemoglobin (mcmol/g Hb). Aliquots of serum were collected from centrifuged clotted-blood and stored at À80 C for LDH isoenzyme assay (Hydragel 150 assay, Sebia, Norcross, GA).…”

“…14 The clinical utility of EC has been established in a cohort of patients with hemolytic anemia and hemolysis caused by mechanical heart valves. 13,15 We hypothesized that hemolysis might be present in PAH and this study hypothesis was that EC levels will be higher in PAH patients than in controls.…”

Estimation of the Hemoglobin Glycation Rate Constant

“…This may reduce the reliability of the system. In contrast, in this study creatine was measured using an enzymatic method which was sensitive and specific to creatine in erythrocytes which uses 10-N -methylcarbamoyl-3,7-bis (dimethylamino) phenothiazine (MCDP), an N -methylcarbamoyl derivative of methylene blue, with a high molar absorption coefficient (9.6 × 10 7 L mol -1 cm -1 ) [16], as a chromogen. [10] and Ishii [11] were calculated from blood glucose values using self-monitoring of blood glucose (SMBG).…”