Analysis of aqueous humour in uveitis by high performance liquid chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Abstract

Aqueous humour from patients with Fuchs' heterochromic cyclitis (FHC) and other types of uveitis was analysed by high performance liquid chromatography (HPLC) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Using HPLC, the number of peaks and their respective elution times were similar for the FHC, uveitis and control groups. SDS-PAGE and silver staining showed immunoglobulin G migrating as two to four distinct bands in all non-reduced samples. In the reduced state, 1-5 unidentified low MW bands (10-19 kD) were seen. Two bands at 19 and 10 kD appeared to be specific for the uveitis group, and a band at 11 kD was present in 76% of uveitis and 19% of FHC patients. None of these three bands were detected in any of the control group. As yet the identity of these low MW proteins and their possible significance in the pathogenesis of intraocular inflammation are unknown.

The Analysis of Aqueous Humor Constituents Using Capillary Zone Electrophoresis

“…Using techniques such as SDS-PAGE electrophoresis (Ando et al, 1993 ;Ku$ chle et al, 1994 ;Murray et al, 1992) or isoelectric focusing (Yu and Okamura, 1987) the resolution and number of individual proteins identified may exceed that demonstrated using the capillary electrophoresis technique described here, although accurate quantitation is difficult.…”

“…However, the resolution of HPLC techniques for separation of aqueous humor components have been very limited to date. Murray et al, (1992) were able to resolve aqueous humor into 5 or 6 peaks, of which they were able to identify IgG, albumin, ascorbate and uric acid. No other proteins were resolved and they were unable to distinguish any differences between uveitis patients and controls with respect to peak number and elution time.…”

“…However the samples required an initial 20 fold concentration prior to analysis and although a number of protein peaks were identified the resolution was poorer than that achieved using capillary electrophoresis. HPLC techniques using UV detection (Murray et al, 1992) will, like capillary electrophoresis, provide qualitative and quantitative information and will detect any separated compound with absorption characteristics at the detection wave-F. 4.…”

“…Attempts to further our understanding of the aetiology of diseases of the eye, such as uveitis, glaucoma and cataract may in part at least depend upon analysis of the changes that occur in the composition of the aqueous humor in these conditions (Murray et al, 1992 ;Tripathi et al, 1992 ;Huang et al, 1997).…”

“…Quantitative immunoassays are generally specific for one constituent, hence a limited amount of data can be obtained from a single sample. Chromatographic techniques such as high pressure liquid chromatography (HPLC) can potentially provide qualitative and quantitative information on the composition of aqueous humor however techniques currently available have provided limited sensitivity (Murray et al, 1992).…”