Talanta 80 (2010) 1269–1276 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta The influence of indoxyl sulfate and ammonium on the autofluorescence of human urine Sandeep Menon Perinchery a , Unnikrishnan Kuzhiumparambil b , Subramanyam Vemulpad c , Ewa M. Goldys a,d,∗ a Department of Physics and Engineering, Macquarie University, Sydney 2109, NSW, Australia b Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney 2109, NSW, Australia c Faculty of Science, Macquarie University, Sydney 2109, NSW, Australia d ARC/NHMRC Network “Fluorescence Applications in Biotechnology and Life Sciences”, Macquarie University, Sydney 2109, NSW, Australia article info Article history: Received 25 August 2009 Received in revised form 11 September 2009 Accepted 13 September 2009 Available online 18 September 2009 Keywords: Autofluorescence Human urine Indoxyl sulfate Inner filter effect Quenching abstract Despite biological variability the spectral characteristics of undiluted human urine show relatively low autofluorescence at short UV (250–300 nm) excitation. However with dilution the fluorescence intensity remarkably increases. This paper examines the mechanisms behind this effect, by using excitation–emission matrices. Corrections for the inner filter effect were made for improved under- standing of the spectral patterns. We focused on three major fluorophores (tryptophan, indoxyl sulfate and 5-hydroxyindole-3-acetate) that are excited at these wavelengths, and whose content in urine is strongly linked with various health conditions. Their fluorescence was studied both individually and in combinations. We also examined the effect of ammonium on the fluorescence of these major fluo- rophores individually and in combinations. Through these studies we have identified the leading effects that reduce the UV fluorescence, namely higher concentration of indoxyl sulfate producing the inner filter effect and concentration quenching and quenching of fluorophores by ammonium. This result will assist in broader utilisation of UV fluorescence in medical diagnostics. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Human urine is a complex biological fluid containing a range of chemical compounds produced by the body, some of which are fluorescent [1–3]. The concentration of fluorophores in urine is influenced by many factors including body metabolism, dietary intake, age and disease [1,2]. Thus the measurement of autofluo- rescence of urine is, in principle, able to provide an indication of a number of health conditions, including urinary tract infection, proteinuria, renal disorder, nephrotic syndrome, hepatopathy, and neuronal ceroid lipofuscinosis [3–6]. However, despite the simplic- ity and speed of the fluorescence measurement, urine fluorescence is underutilised in medical diagnostics. One of the key impedi- ments is the observed high inter- and intra-individual variability of the fluorescence properties. Moreover, the broad spectral fea- tures of individual fluorophores tend to overlap, making the data interpretation quite complex and the identification of chemical constituents extremely difficult. Upon closer inspection, the spec- ∗ Corresponding author at: Department of Physics and Engineering, Faculty of Science, Macquarie University, Sydney 2109, NSW, Australia. Tel.: +61 2 98508902; fax: +61 2 98508115. E-mail address: goldys@ics.mq.edu.au (E.M. Goldys). tral characteristics of urine fluorescence are somewhat less variable at short UV excitation wavelengths (250–300 nm), corresponding to the region where the key, most abundant indole fluorophores, tryptophan and its metabolites can be excited (Supplemental material, Table 1) [7]. These fluorophores are directly linked to cellular metabolism and, indirectly, to various health conditions. However, they are practically difficult to observe in undiluted healthy human urine that shows very weak fluorescence with short UV excitation [5]. Thus the majority of earlier studies have focused on analyzing diluted urine [7]. The dilution greatly enhances the fluorescence of tryptophan and its metabolites, but it tends to compromise the fluorescence from less abundant components observable in other spectral regions, possibly reducing the diag- nostic potential of such measurement. Moreover, it would be preferable to be able to use undiluted urine for diagnostics due to simpler procedures. Our work is aimed at a better understanding of urine fluores- cence by identifying and quantifying the factors responsible for low intensity of fluorescence when excited at 250–300 nm. The princi- ples governing the observed fluorescence intensity of fluorophores are well established; they include the value of fluorophore quan- tum yield, the possibility of fluorescence quenching occurring at higher concentrations, and the reduced intensity of exciting light (or emitted light or both) known as the inner filter effect [8,9]. 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.09.020