1. Urine Protein Measurement Issues Greg Miller, PhD Professor of Pathology Virginia Commonwealth University Richmond, VA (USA)

2. Outline Albumin molecules in urine Albumin measurement Total protein measurement

3. Albumin information is based on a conference in March 2007 to address standardization issues for urine albumin/creatinine measurement and reporting

4. Molecular forms of albumin Conformation is influenced by ligand binding Urine concentrates many ligands Unpaired cysteine-34 can form albumin dimers and other covalent modifications (plasma or urine) 1-10% is glycated in plasma (higher in diabetes) Glycated forms are a greater proportion in urine - attributed to differential uptake in tubules Large (>5 kDa) and small (500-5000 Da) fragments have been identified in plasma and urine C and N terminal truncation occurs

5. Albumin molecules in urine Conformation changes in plasma may influence the filtration rate at the glomerulus Fragmented forms in plasma should be more easily filtered by the glomerulus Tubular uptake is receptor mediated and may influence enrichment of modified forms in urine Proteases and chemical modifications occur in the urinary tract and in the urine after collection Influence of pH, osmolality, contact with sediment, adsorption to containers, and other sample handling factors are not well understood

6. Outline Albumin molecules in urine Albumin measurement Total protein measurement

7. Albumin measurement procedures Immunoassay Influenced by the albumin epitope(s) recognized by the antibody, and by reactivity with modified forms of albumin Evidence that polyclonal immunoassays are reactive with some modified forms HPLC Albumin may not be resolved from other urine proteins (causing overestimation) Hypothesis of non-immunoreactive albumin may be related to non-specificity

8. Measurement issues Influence of urine matrix variability and molecular forms of albumin are not well understood No urine albumin reference material No reference measurement procedure Calibration traceability is to diluted CRM 470 (ERM DA470) serum protein reference material Details of traceability design, dilution protocols, and measurement implementation are not standardized and appear to influence calibration uniformity between methods Some methods use the molar absorptivity of albumin in solution for calibration

9. Current status of albumin measurement Proficiency Testing suggested: A range of results for the same sample Influenced by non-commutability of samples Urine dipstick results were highly variable A range of imprecision; with some methods having acceptable imprecision A variety of reporting units for albumin concentration, excretion rate, and albumin/creatinine ratio

10. PT example: CAP urine albumin (USA) pooled human urine supplemented with albumin, creatinine and other substances, liquid, within method comparison mg/LCV, %±2 SD 113-268-15 265-1021-31 873-874-100

11. PT example: Finland, Norway, Canada pooled or single donation human urine (may be supplemented with albumin and creatinine), liquid, among lab/method comparison MethodNMeanCV±2SD range mg/L%mg/L Finland/Norway1361915.414-25 Norway (GPs)10123512.127-44 Canada 282016.514-26

12. Current status of albumin measurement Within subject biological variability (CV i ) 30-40% Limitation: the biological variability data was not uniformly acquired nor analyzed; CV i is difficult to define for disease conditions. Many methods have adequate imprecision (CV); some do not (based on PT samples with a uniform molecular species) Reliable information on bias among methods is lacking Calibration uniformity among methods needs improvement The albumin molecular species that are being measured is not well understood

13. Recommendations: NKDEP/IFCC UA Conf. Albumin/Creatinine ratio should be reported –“mg/mmol” or “mg/g” should be used uniformly in a country or region Albumin concentration (mg/L) is difficult to interpret and should not be reported alone First morning urine has lower biologic variability than a random collection Albumin should be measured on fresh (non- frozen) urine “urine albumin” should replace “microalbumin”

14. Further investigations needed: UA Conf. Clarify sample collection and handling requirements for minimum biologic variability Clarify the measurand in a urine sample; and the variability of the urine matrix Clarify reference intervals and decision points related to risk for kidney damage by age, gender, ethnicity, and concomitant disease Develop a reference system –ID/MS measurement procedure at Mayo Clinic (USA) –Secondary urine reference material from Japan (JSCC and JCCLS)

15. Outline Albumin molecules in urine Albumin measurement Total protein measurement

16. Proteins in Urine Albumin Others Immunoglobulins Bence-Jones Tamm-Horsfall Lysozyme Myoglobin Hemoglobin Bacterial origin Peptides

17. Quantitative urine protein methods In order of clinical lab market share in USA: Pyrogallol red (dye binding) Pyrocatechol violet (dye binding) Benzethonium chloride (denaturation/turbidimetry) Biuret with precipitation (reference) Coomassie blue (dye binding)

18. Issues with urine protein methods Different measurement signal with different proteins Different chemical reaction procedures have different response ratios to different proteins No uniform calibration standard

19. Benzethonium chloridePyrocatechol violet Pyrogallol red #2Pyrogallol red #1 Dube et al. Clin Biochem 2005;38:479-485. Normal urines supplemented with: albumin, gamma-globulins, polypeptides, mixed protein

20. Imai, Clin Chem 1995;32,1986. Mean total protein of 12 urine samples measured by 7 methods and using 3 standard materials Methods: SSA – sulfosalicylic acid SSA-SS - sulfosalicylic acid sodium sulfate TCA – trichloroacetic acid BC – benzethonium chloride CBB – comassie brilliant blue PR-M – Pyrogallol red molybdenum TCA-B - Trichloroacetic acid precipitation biuret Patients: (3) nephrotic syndrome (1)diabetic nephropathy (1) systemic lupus (1)acute glomerulonephritis (2)multiple meyoloma (4) cancer

21. Interfering substances with urine protein methods A non-inclusive list: Creatinine Aminoglycosides Phenothiazines Amino acids Organic acids Peptides Other substances

22. Conclusions Urine albumin measurements are less variable than urine total protein measurements Clinical trials should use a central lab Data from different clinical trials may be difficult to aggregate

23. Questions Comments