Phthalaldehyde microprotein method: Usefulness and potential errors

Abstract

Two major technical advances introduced by Brenner et al [l, 2] have been responsible for recent breakthroughs in understanding the process of glomerular ultrafiltration at the single nephron level. These are the development of a method for directly measuring glomerular capillary pressure [1] and the microadaptation of the colorimetric protein method of Lowry et al [2, 3] to estimate changes in colloid osmotic pressure between the afferent and efferent ends of the glomerular vasculature [4]. The good correlation between colloid osmotic pressure as calculated from the arterial protein concentrations and direct measurement of the colloid osmotic pressure has established the usefulness of the protein method to accurately measure intravascular oncotic forces [5]. Because the colorimetric Lowry microprotein method is both time consuming and necessitates the use of a frequently unstable, relatively high intensity colorimetric scale for sample reading, attempts have been made to use other microprotein techniques as substitutes for the Lowry method [6–9]. Viets et al [7] have compared the usefulness of two fluorescent microprotein methods in micropuncture studies. Of these, the phthalaldehyde fluorescent microprotein method was preferable and appeared to be equally as useful as the Lowry method, with certain minor limitations. Our laboratory has spent the last 2 years evaluating the applicability of the phthalaldehyde technique to microprotein analysis in micropuncture studies. Although we have found the method to have the advantage of being less time consuming than the Lowry technique, we have noted certain limitations and potential errors that could artificially lower the estimate of protein content in plasma samples. Spuriously low protein concentrations could result in under-estimations of afferent and efferent arteriolar oncotic pressures and overestimates of single nephron filtration fractions. This report is designed to describe the limitations of phthalaldehyde fluorescent protein technique and point out its potential errors in measuring protein concentration while comparing its usefulness to that of the colorimetric micro-Lowry method