Changes in urine macromolecular composition during processing

Purpose: To determine the urinary crystallization inhibitory activity, urine is generally centrifuged and/or filtered. These preparative procedures may result in a total or partial removal of many macromolecular constituents implicated in crystallization. The main purpose of this study was to investigate the changes in urinary macromolecular composition following centrifugation and filtration. Materials and Methods: Twenty-four hour urine samples were collected from human volunteers. Each was divided into 4 aliquots; one was filtered, the other was centrifuged, another was centrifuged and filtered. The control sample was neither filtered nor centrifuged. Total protein and lipid contents of each sample were determined. Proteins were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis was performed using antibodies against osteopontin (OPN), prothrombin (PT)related proteins, inter-alpha-inhibitor (I alpha I) related proteins, Tamm-Horsfall protein (THP), and albumin (ALB). The effect of processing on incorporation of urinary proteins in crystal matrices was also examined. Calcium oxalate crystals were produced in processed and unprocessed urine samples by the addition of sodium oxalate. Crystals were harvested, de-mineralized and their proteins analyzed by SDS-PAGE and Western blotting. Results: Processing reduced the amounts of both proteins and lipids in the urine. Previously we identified phospholipids in the matrix of calcium oxalate crystals as well as the filtrate and retentate removed during filtration and centrifugation. Phospholipids have a high affinity for calcium-containing crystals. In the case of proteins, those with high molecular weights appeared to be clearly affected by filtration and centrifugation. Processing also appeared to influence the incorporation of proteins in the crystals. The matrix of crystals produced in processed urine contained less THP than those made in unprocessed urine, apparently a result of the loss of this higher molecular weight protein during processing. Incorporation of PT-related proteins, particularly fragment 1, was increased. Conclusions: We propose that selective inclusion of macromolecules is a result of an increase in available binding sites on crystal surfaces because of the removal of certain calcium binding substances such as phospholipids and proteins. Removal of larger macromolecules from the milieu may also provide a better access to the crystal surfaces.