Integrated Electroosmotic Perfusion of Tissue with Online Microfluidic Analysis to Track the Metabolism of Cystamine, Pantethine, and Coenzyme A

Abstract

We have developed an approach that integrates electroosmotic perfusion of tissue with a substrate-containing solution and online microfluidic analysis of products, in this case thiols. Using this approach we have tracked the metabolism of cystamine, pantethine and CoA in the extracellular space of organotypic hippocampal slice cultures (OHSCs). Currently, little is known about coenzyme A (CoA) biodegradation and even less is known about the regulation and kinetic characteristics for this sequential multi-enzyme reaction. We found that the steady state percentage yields of cysteamine from cystamine and pantethine during the transit through OHSCs were 91% ± 4% (SEM) and 0.01%–0.03%, respectively. The large difference in the yields of cysteamine can be used to explain the drugs’ different toxicities and clinical effectiveness against cystinosis. The kinetic parameters of the enzyme reaction catalyzed by the ectoenzyme pantetheinase are KM,C/α = 4.4 ± 1.1 mM and Vmax,C = 29 ± 3 nM/s, where α is the percentage yield of pantethine to pantetheine through disulfide exchange. We estimate that the percentage yield of pantethine to pantetheine through disulfide exchange is approximately 0.5%. Based on the formation rate of cysteamine in the OHSCs, we obtained the overall apparent Michaelis constant and maximum reaction rate for sequential, extracellular CoA degradation in an in situ environment, which are K′M = 16 ± 4 μM, V′max = 7.1 ± 0.5 nM/s. Kinetic parameters obtained in situ, although difficult to measure, are better representations of the biochemical flux in the living organism than those from isolated enzymes in vitro.

Chemical etching of bovine serum albumin-protected Au25 nanoclusters for label-free and separation-free detection of cysteamine

“…Endogenous cysteamine (CSH) is the terminal metabolite in coenzyme A (CoA) degradation, being formed by the enzymatic cleavage of panthetheine, and also a vital source for taurine (Ueki and Stipanuk, 2009;Wu et al, 2013). CSH has many important clinical applications.…”

“…CSH has many important clinical applications. For instance, CSH has been considered as a potential neuroprotective agent and exhibits significant beneficial properties in treating neurodegenerative diseases including Huntington's and Parkinson's diseases (Gibrat and Cicchetti, 2011;Mao et al, 2006;Wu et al, 2013). Also, CSH is the main drug used to treat children with nephropathic cystinosis caused by an excessive intralysosomal cystine accumulation due to a defect of its transport system in the lysosomal membrane (Kessler et al, 2008).…”

Proposed Therapies for Pantothenate-Kinase-Associated Neurodegeneration

“…(2) The HoPan-treated mouse model 12 was used to demonstrate that acetyl-phosphopantetheine reversed the effect of HoPan on liver CoA levels in mice. 9 Because pantothenate alone potently counteracts HoPan-mediated reduction of CoA levels 12 and acetyl-phosphopantetheine is degraded to pantothenate by digestion, 13-15 it is not proven that the therapy bypasses PANK. The HoPan-treated mouse model is also not a representative PKAN model because HoPan treatment can be used to reduce CoA in the liver and kidney, but HoPan does not reduce brain CoA.…”

Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures

“…To study this and other problems involving extracellular processing of thiols, we built an “all electric” microfluidic system for EO sampling coupled to a fluorogenic reaction, electrophoretic separation, and laser-induced-fluorescence detection [106–108]. As illustrated in Fig.…”