The cellular proportion of free and protein-bound NADH complexes is increasingly recognized as a metabolic indicator and biomarker. Because free and bound forms exhibit different fluorescence spectra, we consider whether autofluorescence shape sufficiently correlates with mitochondrial metabolism to be useful for monitoring in cellular suspensions. Several computational approaches for rapidly quantifying spectrum shape are used to detect Saccharomyces cereviseae response to oxygenation, and to the addition of mitochondrial functional modifiers and metabolic substrates. Observed changes appear consistent with previous studies probing free/protein-bound proportions, making this a potentially useful approach for the real-time monitoring of metabolism. (© 2015 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim).

Cellular NADH conformation is increasingly recognized as an endogenous optical biomarker and metabolic indicator. Recently, we reported a real-time approach for tracking metabolism on the basis of the quantification of UV-excited autofluorescence spectrum shape. Here, we use nanosecond-gated spectral acquisition, combined with spectrum-shape quantification, to monitor the long excited-state lifetime autofluorescence (usually associated with protein-bound NADH conformations) separately from the autofluorescence signal as a whole. We observe that the autofluorescence response induced by two NADH-oxidation inhibitors—cyanide and ethanol—are similar in Saccharomyces cerevisiae when monitored using time-integrated detection but easily distinguished using time-gated detection. Results are consistent with the observation of multiple NADH conformations as assessed using spectral phasor analysis. Further, because well-known oxidation inhibitors are used, changes in spectrum shape can be associated with NADH conformations involved in the different metabolic pathways, giving bioanalytic utility to the spectral responses.

The two-component nature of the autofluorescence response is indicative of whether a single or several pathways are affected.

Phasor analysis on fluorescence signals is a sensitive approach for analyzing multicomponent systems. Initially developed for time-resolved measurements, a spectral version has been used for the rapid identification of regions during the spectral imaging of biological systems. Here we show that quantitative information regarding conformation can be obtained from phasor analysis of fluorescence spectrum shape. Methanol denaturation of NADH and NADH binding to various dehydrogenase proteins are used as model reactions. Thermodynamic constants are calculated and compared with previous studies based on more direct measures of conformation. Next, the quantitative monitoring of UV-excited autofluorescence spectrum shape during chemically-induced metabolic transitions is presented and discussed in terms of NADH-utilizing pathways. Results show how phasor analysis is useful in assessing two-state behavior, and in interpreting autofluorescence as emission from an ensemble of cellular NADH forms.

1. The main metabolites of lacidipine were isolated from bile and plasma of rats and dogs following an oral dose of the 14C-labelled drug (10 mg/kg for rats: 2 and 1 mg/kg for dogs). They were identified by comparison of chromatographic and spectral data with authentic reference compounds synthesized ad hoc. 2. Five metabolites (I-V) were isolated and identified in dog bile by gradient h.p.l.c. with u.v. detection and h.p.l.c.-thermospray mass spectrometry. In all metabolites the heterocyclic ring has been oxidized to pyridine. Further biotransformation reactions involved hydroxylation of the methyl substituents and hydrolysis of the ethyl and t-butyl ester groups to produce carboxylic acids and a lactone. Some of these metabolites also occurred as glucuronide conjugates. 3. A metabolite retaining the intact dihydropyridine ring, the des-ethyl analogue of lacidipine (VI), was isolated from rat plasma where it accounted for 60% of the total circulating radioactivity up to 24 h after administration. To characterize this metabolite, h.p.l.c. with photodiode array u.v. detection also was employed. This compound was detected in dog plasma, but there was no evidence of its presence in dog bile samples. 4. Profiles of circulating metabolites were qualitatively similar in rats and dogs. Identified metabolites accounted for the large majority of total radioactivity in all the analysed samples.