Serum creatinine (Scr) levels are determined by two factors: (1) the rate of production from muscle mass, and (2) the GFR that determine the rate of its elimination. Since its rate of production is related to muscle mass, so that intra-individual concentrations are relatively constant. Therefore, the change in serum creatinine level reflects glomerular filtration function.
The most common method for creatinine measurement is picric acid based. However, it is now well established that picric acid based methods are inaccurate in mice due to the significant contribution of a cross-reacting chromagen in mouse serum that is especially severe in diabetic animals (13, 14, 15). Recently an assay for creatinine using HPLC has been established (16). Study shows that serum creatinine determined via HPLC correlates with inulin clearance as a measure of GFR in mice (16). Compared with creatinine clearance and inulin clearance, serum creatinine is relatively insensitive for detecting small decreases in GFR because of the nonlinear relationship between plasma creatinine and GRF. Also when serum creatinine levels are increased, tubular secretion of SCr increases, leading to an overestimation of GFR in individuals with moderate to severe decreases in GFR (<50 mL/min) . So levels of GFR can be screened with HPLC serum creatinine. Detailed protocol for this assay is provided on the protocols page for this segment.
Preparation of plasma, serum and urine for creatinine measurement by HPLC
For plasma and serum: Mix, and if necessary, squeeze any clot using wooden applicator stick, then centrifuge (minimum 3,000 rpm for 10 minutes). Prepare acidified acetonitrile (ACN) by adding 50 μl of HPLC grade glacial acetic acid to 10 ml of HPLC grade ACN.
For a 25 μl plasma/serum: Use this ratio of 4:1 (ACN to specimen – 100 μl ACN to 25 μl plasma) if less or more specimen is used keep the 4:1 ratio. (3:1 or 5:1 is also OK)
- Transfer 25.0 μl of plasma to each tube containing 100 μl of ACN; vortex about 15 seconds to mix, extracting the creatinine into the ACN. Let sit for about 15 minutes in the minus 20 freezer. This aids in precipitation and freezes the aqueous ppt. avoiding carryover and undissolved particles.
- Centrifuge (minimum 10,000 rpm @ 6º for 10 minutes) at 4 degrees.
- Transfer the supernatant to new clean labeled tube (1.5 or 0.5 ml Starstadt tubes work well).
- The volume should be very close to 125 μl. Careful not to transfer any of the ppt. (button) which should be on the wall or bottom of the tube.
- Evaporate the ACN to dryness using the Speed Vac, Centrivap or stream of dry nitrogen. Mild heat may be used if necessary in Speed Vac. This should take about 30 minutes.
- Remove the tubes and reconstitute to 25.0 μl with filtered Mobile Phase (Solvent A). Mix to dissolve the residue containing creatinine using the pipettor.
- Transfer the liquid containing the creatinine to the special Perkin Elmer tubes for autosampler. Cap with special slit caps and centrifuge (@2,000-3,000 rpm for 10 minutes). Use swinging bucket rotor to get any debris to go to the bottom. Carefully remove autosampler vials from centrifuge and transfer to the Peltier tray. Examine each tube to insure that the liquid is touching the bottom (the V) of the tube.
- Program the autosampler making sure the vials correspond to the numbers and the ID of the specimens. Check twice; they sometimes change.
- With each run, a banked quality control (QC) sample must be run. The QC is a previously assayed pooled mouse plasma that has been aliquoted and stored in a minus 20 freezer.
Assay for creatinine by High Performance Liquid Chromatography (HPLC):
All reagents are HPLC grade. All aqueous buffers are filtered through 0.22 micron filter. Tubing is PEEK 0.005 mm tubing with hand-tight connectors.
- Mobile phase: 5mM sodium acetate adjusted to pH 4.1 ± 0.1 with glacial acetic acid (final solvent strength is about 15 mM after acetic acid is added. Mobile phase was degassed.
- Column: Zorbax SCX, -- strong cation exchange, (Aglient, Wilmington, DE), 50 mm x 2.1 mm, 5μ particle size). Five micron in-line filter and SCX guard column placed in front of analytical column.
- Temperature and flow rate: Column run at 45 ± 0.5¼ C. at a flow of 0.30 ± 0.02 ml/min. Backpressure was around 900 psi. Runtime was 10 minutes.
- Detection: UV (deuterium source) at 225nm (flow cell volume is 12 μl).
- Injection volume: three microliters (3 μl) from auto sampler. Temperature of autosampler tray was 18 ± 0.5¼ C.
- Identification and quantitation: Identification by comparison of retention time to pure standard of creatinine. Average retention time was 3.654 ± 0.022 minutes. Quantitation was achieved by external standard ranging from 0.003 to 1.000 mg/dl by serial dilutions using a weighted 1/χ2 regression line (8-10 points). May also use Excel, Prism or any other software to calculate a regression line and compute the unknowns.
Publications for Serum creatinine (4)
BACKGROUND: The use of endogenous plasma creatinine levels and creatinine clearance as a tool to evaluate renal function in mice has come under scrutiny as prior studies have reported that the Jaffe alkaline picrate method grossly overestimates true plasma creatinine in mice. As members of the NIDDK Animal Models of Diabetic Complications Consortium (AMDCC), we evaluated the performance and feasibility of an alternative high-performance liquid chromatography (HPLC)-based method for standard determination of plasma creatinine and creatinine clearance in mice. Our purpose was to develop a simple method that provides a reliable, reproducible, and sensitive assay for small volumes (<25 microL) of mouse plasma and sera. METHODS: We compared creatinine clearance measured by HPLC with the Jaffe method and HPLC creatinine clearance with inulin clearance [fluoroscein isothiocyanate (FITC) inulin in an osmotic pump implanted in mouse] in C57BL/6J mice. Different groups of mice underwent either one of two protocols. Protocol A included dietary intervention with normal, low salt plus enalapril, or high salt. Protocol B induced diabetes using streptozotocin. RESULTS: First, mean plasma creatinine levels were significantly lower (P < 0.0001) by HPLC (0.128 +/0.026 mg/dL) vs. Jaffe (0.4 +/0.12 mg/dL) for mice on a normal diet. Urine creatinine concentrations measured by HPLC were 10% lower than by Jaffe (P < 0.01). Second, mean creatinine clearance by HPLC for mice on a normal diet was 255 +/68 microL/min. Mice on low salt diet plus enalapril had reduced creatinine clearance (72.8 +/24.2 microL/min) while mice on high salt diet had an elevated creatinine clearance (355 +/105 microL/min). Third, diabetic mice (19 to 24 weeks of diabetes) exhibited hyperfiltration as creatinine clearance was 524 +/214 microL/min whereas nondiabetic age/gender-matched mice showed a mean creatinine clearance of 206 +/41 microL/min. Finally, significant correlation was demonstrated for creatinine clearance by HPLC vs. inulin clearance (R= 0.643; P < 0.001). CONCLUSION: HPLC is highly accurate, much more sensitive and specific than the Jaffe method for plasma creatinine measurements in mice. Creatinine clearance in mice measured by HPLC reflects changes in renal function induced by diet and diabetes.
Kemperman FA, Weber JA, Gorgels J, van Zanten AP, Krediet RT, Arisz L. The influence of ketoacids on plasma creatinine assays in diabetic ketoacidosis. J Intern Med (2000) 248:511-7
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OBJECTIVE: Analysis of the interference of ketoacids on various routine plasma creatinine assays during a clinical episode of diabetic ketoacidosis (DKA). DESIGN: Observational study. Blood samples were drawn before, during and after standard in-hospital treatment. Plasma creatinine was measured with two dissimilar enzymatic assays (creatininase PAP + and creatinine iminohydrolase Serapak), a kinetic alkaline picrate method (Jaffe) and a high-performance liquid chromatography (HPLC) procedure. Acetoacetate and beta-hydroxybutyrate were analysed by enzymatic methods. SETTING: Department of Medicine, University Hospital. SUBJECTS: Nine patients who experienced 10 episodes of DKA. MAIN OUTCOME MEASURES: Agreement of the routine plasma creatinine assays with HPLC and analysis of possible interferents. RESULTS: At presentation, the Jaffe assay gave falsely high values of plasma creatinine (median 99 micromol L(-1)), in contrast to the PAP+ (median 60.5 micromol L(-1)) and HPLC assays (median 67.5 micromol L(-1)). This positive error decreased during treatment. This was due to a decrease in acetoacetate, as the positive error by the Jaffe method correlated with the acetoacetate concentration (r = 0.79, P < 0.0001). In the multiple regression analysis, beta-hydroxybutyrate caused no additional interference by the Jaffe assay, confirmed by in vitro experiments. Analysis of agreement showed that the difference between PAP+ and HPLC creatinine was -4.6 +/3.0 micromol L(-1) (mean +/SD), and 2.0 +/5.3 micromol L(-1) between Serapak and HPLC. This was statistically significant, but clinically negligible. CONCLUSION: Acetoacetate caused severe interference of the alkaline picrate (Jaffe) assay, which might influence therapeutic decisions at the start of diabetic ketoacidosis. Enzymatic assays lack this interference.
Gerard SK, Khayam-Bashi H. Characterization of creatinine error in ketotic patients. A prospective
comparison of alkaline picrate methods with an enzymatic method. Am J Clin Pathol (1985) 84:659-64
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Meyer MH, Meyer RA Jr, Gray RW, Irwin RL. Picric acid methods greatly overestimate serum creatinine in mice: more accurate
results with high-performance liquid chromatography. Anal Biochem (1985) 144:285-90
View abstract View in PubMed
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Last updated on 2013-11-06 Moderated by Jimmy Hao