Urine Protein Measurement Issues

Greg Miller, PhD

Professor of Pathology

Virginia Commonwealth University

Richmond, VA (USA)

Outline

• Albumin molecules in urine

• Albumin measurement

• Total protein measurement

Albumin information is based on a

conference in March 2007 to address

standardization issues for urine

albumin/creatinine measurement and

reporting

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

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

Outline

• Albumin molecules in urine

• Albumin measurement

• Total protein measurement

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

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

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

PT example: CAP urine albumin (USA)

pooled human urine supplemented with albumin, creatinine and other substances, liquid, within method comparison

0

5

10

15

20

25

30

0 20 40 60 80 100

Peer group mean, mg/L

Am

on

g la

bo

rato

ry C

V, %

Inst. 1 (N=11)

Inst. 2 (N=79)

Inst. 3 (N=69)

Inst. 4 (N=194)

Inst. 5 (N=59)

Inst. 6 (N=59)

Inst. 7 (N=44)

Inst. 8 (N=39)

Inst. 9 (N=207)

Inst. 10 (N=112)

Inst. 11 (N=86)

Inst. 12 (N=123)

Inst. 13 (N=82)

Inst. 14 (N=18)

Inst. 15 (N=66)

mg/L CV, % ±2 SD

11 3-26 8-15

26 5-10 21-31

87 3-8 74-100

PT example: Finland, Norway, Canada

pooled or single donation human urine (may be supplemented with albumin and creatinine), liquid, among lab/method comparison

Method N Mean CV ±2SD rangemg/L % mg/L

Finland/Norway 136 19 15.4 14-25

Norway (GPs) 1012 35 12.1 27-44

Canada 28 20 16.5 14-26

Current status of albumin measurement

• Within subject biological variability (CVi) 30-40%

• Limitation: the biological variability data was not uniformly acquired nor analyzed; CVi 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

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”

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)

Outline

• Albumin molecules in urine

• Albumin measurement

• Total protein measurement

Proteins in Urine

• Albumin

• Others• Immunoglobulins

• Bence-Jones

• Tamm-Horsfall

• Lysozyme

• Myoglobin

• Hemoglobin

• Bacterial origin

• Peptides

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)

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

Benzethonium chloride Pyrocatechol 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

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 acidSSA-SS - sulfosalicylic acid sodium sulfateTCA – trichloroacetic acidBC – benzethonium chlorideCBB – comassie brilliant bluePR-M – Pyrogallol red molybdenumTCA-B - Trichloroacetic acid precipitation biuret

Patients:(3) nephrotic syndrome(1) diabetic nephropathy(1) systemic lupus(1) acute glomerulonephritis(2) multiple meyoloma(4) cancer

Interfering substances with urine protein methods

A non-inclusive list:

• Creatinine

• Aminoglycosides

• Phenothiazines

• Amino acids

• Organic acids

• Peptides

• Other substances

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

Questions

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