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Letters to the Editor |
Department of Clinical Chemistry, University Hospital, Ghent, Belgium
aAddress correspondence to this author at: Department of Clinical Chemistry, University Hospital Ghent, De Pintelaan 185, B 9000 Ghent, Belgium, Fax 32-9-3324985, E-mail Joris.Delanghe{at}ugent.be
To the Editor:
Creatinine is the most commonly used marker for the estimation of glomerular filtration rate (GFR) in routine clinical use (1), but serum concentrations of low molecular mass proteins can provide an interesting alternative for estimating renal function. Serum concentrations of low molecular weight proteins are primarily determined by GFR, and an ideal marker should have a constant production rate and not vary in concentration during an acute-phase reaction. Cystatin C (13 kDa) and β-trace protein (23–29 kDa) share these properties (1)(2)(3). Cystatin C appears to be superior to creatinine for estimation of GFR, especially in the so-called creatinine-blind range. As a screening test for detecting decreased GFR, cystatin C has fewer inherent limitations than serum creatinine. Moreover, unlike creatinine, cystatin C reflects renal function independent of age, sex, height, and body composition (1). Formulae have been developed allowing estimation of GFR based on cystatin C (1). However, standardization for cystatin C assays is still lacking. Recently, several caveats have been reported: cystatin C concentrations appear to be affected by upregulation in certain tumors and drug treatments (e.g., glucocorticoids). In addition, thyroid dysfunction affects cystatin C concentrations, possibly influencing the production rate of the protein (1).
We were struck by discrepant results between serum creatinine and cystatin C in a 63-year-old female patient (height 1.60 m, weight 49 kg) presenting with biliary obstruction. The patient’s medical history included breast cancer, arterial hypertension, hypercholesterolemia, osteoporosis, and autoimmune hemolytic anemia. During recent treatment with corticoid therapy the patient was observed to have diabetes. At presentation, she was found to have high levels of liver enzyme activity, particularly 
-glutamyl transferase (596 U/L; reference range, 9–36 U/L) and alkaline phosphatase (403 U/L; reference range, 30–120 U/L). C-reactive protein concentrations were very high (293 mg/L; reference range, <5 mg/L). Serum creatinine was very low (29.2 µmol/L; reference range, 49–85 µmol/L), corresponding to an estimated GFR of >90 mL/min (Modification of Diet in Renal Disease formula) and a creatinine clearance of >120 mL/min (Cockroft-Gault formula). After 2 months of hospitalization, the patient’s inherent muscle wasting made a creatinine-based GFR estimation less reliable. Serum β-trace protein was low (0.393 mg/L; reference range, 0.20–0.72 mg/L), corresponding to an estimated GFR of 101 mL/min (3). In contrast, serum and EDTA cystatin C, measured using a serum cystatin C assay (Tina-quant cystatin C, Roche), were strongly increased to 2.5 mg/L and 5.3 mg/L, respectively (reference range, 0.63–1.44 mg/L), which corresponded to an estimated GFR of only 30 mL/min (1). Serum electrophoresis of alkaline phosphatase revealed the presence of a macromolecular fraction (4). Further cystatin C analysis using an alternative method [latex particle–enhanced fixed-time immunonephelometry on a BN II analyzer, (Dade Behring)] based on rabbit antihuman cystatin C antibodies (2) revealed a moderately increased serum cystatin C value (1.14 mg/L, reference range, 0.53–0.95 mg/L). Patient serum (30 µL) was injected into a Waters 650E advanced protein purification system (Millipore) and subjected to high-pressure gel permeation chromatography (HPGPC). A 300-mm Protein PAK glass 300SW column (Waters Nihon) was used with a mobile phase of PBS (0.1 mol/L, containing 137 mmol/L NaCl, 2.7 mmol/L KCl, 8.1 mmol/L Na2HPO4, 1.47 mmol/L KH2PO4, pH 7.3). The flow rate was 1.0 mL/min. Concentrations of the various cystatin C–containing fractions were confirmed nephelometrically. The chromatogram showed a bimodal distribution: macromolecular cystatin C complexes (36%) were separated from an excess of free cystatin C (47%). The macromolecular cystatin C fraction was characterized by an elution volume very close to the void volume of the HPGPC column. After extraction of the patient’s serum with n-butanol, the macromolecular fraction disappeared on HPGPC. Fig. 1
illustrates the unique molecular mass distribution of the patient’s cystatin C.
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The present case demonstrates that when biliary obstruction occurs, serum cystatin C may be present not only as a free 13-kDa molecule but may also be present in a macromolecular form. In biliary obstruction, the presence of triple-layered vesicles (containing plasma membrane–bound enzymes) in serum is a common finding (4). The n-butanol experiment demonstrates that cystatin C is partly associated with these lipid particles, causing an accumulation of cystatin C in serum. In the presence of Ca2+ ions, cystatin C is known to bind brush border membranes (5). This property could explain the observed differences between EDTA and serum samples in the patient we describe. Clinicians should be aware that in these circumstances, estimating GFR using cystatin C–based formulas might be invalid. Methods that do not involve measuring the lipid-associated fractions might underestimate total cystatin C concentrations, but may yield a more reliable GFR measurement.
Acknowledgments
Grant/Funding Support: None declared.
Financial Disclosures: None declared.
Acknowledgments: We thank Roche for providing the necessary reagents.
References
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