(1)
Departement of Pathology, University of Michigan Health System, Ann Arbor, MI 48109-5054, USA
(2)
Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 2nd Street SW, Rochester, MN 55905, USA
Abstract
Urinary excretion of human serum albumin (HSA), a 6.65 kDa monomeric protein, is a sensitive marker of renal damage associated with many diseases including diabetes mellitus. Albumin is synthesized by the liver and functions as a transport protein for fat-soluble hormones and drugs and for maintaining plasma colloid osmotic pressure and pH. Albumin is not filtered at the glomerulus and its presence in the urine at concentration above 30 mg/day is suggestive of glomerular damage. Early diagnosis of microalbuminuria (30–300 mg/24 h urine albumin excretion or 30–300 mg/g creatinine in random collections) has prognostic value for monitoring disease progression and early clinical management of diabetic nephropathy in prediabetic patients. Current methods for quantitation of urine albumin are based on immunoassays or size exclusion high-performance liquid chromatography coupled with UV detection (SEC-HPLC-UV). Studies have demonstrated discordance between the existing methods. It has been suggested that while immunoassays underestimate albumin in urine, SEC-HPLC-UV method overestimates albumin as it cannot separate co-eluting interferences. This chapter describes a liquid chromatography tandem mass spectrometry LC-MS/MS candidate reference method for albumin quantitation.
Key words
LC-MS/MS Albumin Microalbuminuria Microalbumin 1 Introduction
Albumin , a 6.65 kDa monomeric protein, is the most abundant plasma protein in humans. Human liver synthesizes approximately 15 g (~200 mg/kg) albumin per day. Primary functions of albumin include maintenance of plasma oncotic pressure and blood pH, transport of fat soluble hormones, unconjugated bilirubin and drugs [1]. Albumin synthesis is regulated by nutritional status, colloid osmotic pressure, cytokines, and hormones. Colloid osmotic pressure is a form of osmotic pressure exerted by proteins , primarily albumin, which is responsible for about 80 % of the total colloid osmotic pressure exerted by blood plasma on the interstitial fluid. Physiological states that cause a reduction in plasma albumin including proteinuria and malnutrition lead to reduction of plasma oncotic pressure and increased capillary filtration and result in edema.
Presence of protein in the urine (proteinuria) is a marker of degree of renal damage in kidney disease [2, 3]. Proteinuria has been classified as glomerular, tubular, overflow, or post-renal based on the site of the filtration defect. In adults with normal kidney function, albumin is not filtered via glomerular filtration. Therefore, albuminuria is also termed as glomerular proteinuria and quantitation of urine albumin is clinically useful for assessment of glomerular permeability. The presence of 30–300 mg/24 h urine albumin excretion or 30–300 mg/g creatinine in random collections is termed as microalbuminuria and greater than 300 mg in a 24 h urine collection is termed as albuminuria. It is recommended to confirm the presence of microalbuminuria by repeating the 24 h urine measurement over a 2- to 3-month period. Microalbuminuria is an important prognostic marker of diabetic nephropathy and is an adverse predictor of glycemic control in prediabetic patients. Untreated diabetic nephropathy can lead to renal failure in 5–7 years. On the other hand, timely diagnosis and treatment of microalbuminuria can help slow the progression of diabetic nephropathy [3].
FDA-cleared immunoassays and size-exclusion high-performance liquid chromatography coupled with UV detection (SEC-HPLC -UV)-based assay (Accumin™, AusAm Biotechnologies) are used for albumin measurements [4, 5]. A study by Sviridov et al. suggested that SEC-HPLC-UV cannot separate transferrin, α1 acid glycoprotein, and α1 antitrypsin from albumin leading to overestimation compared to the immunoassay [6]. Due to discordance between the two methods, liquid chromatography tandem mass spectrometry (LC-MS/MS )-based methods have been developed [7, 8]. This chapter describes a candidate reference LC-MS/MS method for human albumin in urine.
2 Materials
2.1 Samples
Urine is an acceptable sample type for this method. Samples have to be maintained frozen prior to analysis and up to three freeze–thaw cycles are acceptable.
2.2 Reagents and Buffers
1.
Phosphate Buffered Saline (PBS) (Roche). Store at room temperature. Expiration: until date printed on box. Dilute appropriately with water (stable at room temperature for 3 months).
2.
Mobile Phase A (water, 0.1 % formic acid): Stable at room temperature for 1 month.
3.
Mobile Phase B (acetonitrile, 0.1 % formic acid): Stable at room temperature for 1 month.
4.
Clinical Laboratory Reagent Water (CLRW).
5.
Human serum albumin (HSA) (Sigma-Aldrich) fo r preparation of calibrators.
6.
Bovine serum albumin (BSA) (Sigma-Aldrich) for preparation of internal standard.
2.3 Calibrators, Internal Standard, and Quality Control Samples
1.
Dissolve 2 g HSA in 1 L PBS, Label as Intermediate 2 g/L Std. Store at −80 °C for 2 years. A dilution scheme for preparation of calibrators has been shown in Table 1.
Table 1
Dilution scheme for preparing HSA calibrator samples
Calibrator concentration mg/L | Intermediate 2 g/L Std (mL) | PBS (mL) |
|---|---|---|
0 | 0 | 250 |
12 | 1.5 | 248.5 |
24 | 3 | 247
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