Total luminescence spectroscopy of fluorescence changes during aging in Caenorhabditis elegans

Total luminescence spectroscopy was employed to characterize and quantitate age-related changes in fluorescence in the nematode Caenorhabditis elegans, an established model for aging research. The excitation wavelength was varied between 250 and 590 nm in 10-nm increments. At each excitation wavelength, the emission wavelength was varied between 300 and 600 nm. Contour plots of corrected spectra were made. All fluorescence increased severalfold with age, except for that ascribed to tryptophan of soluble protein fractions. This general increase included fluorescence due to flavins, which is not expected to increase with age but has previously been observed to do so in this species. Blue emission peaks that approximated Schiff base product fluorescence were detected in whole aqueous homogenates, chloroform/methanol extracts, and detergent-cleaned cuticle preparations. Age-related increases in emission intensities of these peaks were demonstrated in aqueous homogenates and isolated cuticles. Cuticle preparations, known to be rich in collagenous protein, exhibited a fluorescence peak that approximated the recently described pyridinoline cross-link of vertebrate collagen. This peak, as well as the entire cuticle emission spectrum between 300 and 500 nm, increased dramatically with age. A fluorescence peak tentatively identified as cuticle tyrosine, characteristic of collagenous protein, also increased in older worms. The effectiveness of the spectroscopic technique in distinguishing individual fluorescence peaks in complex mixtures was demonstrated, and the potential of the nematode cuticle for age-related collagen studies was identified.