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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 multienzyme 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/? = 4.4 ± 1.1 mM and Vmax,C = 29 ± 3 nM/s, where ? 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 ?M, 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.

SUBMITTER: Wu J

PROVIDER: S-EPMC3899583 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Integrated electroosmotic perfusion of tissue with online microfluidic analysis to track the metabolism of cystamine, pantethine, and coenzyme A.

Wu Juanfang J Sandberg Mats M Weber Stephen G SG

Analytical chemistry 20131122 24

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 sequent ...[more]

PMID: 24215585

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Project description:Pancreatic islets of Langerhans release glucagon to maintain blood glucose levels, and release of this peptide is dysregulated in diabetes mellitus. Although the importance of proper secretion of this peptide has been shown, no measurement of its release at the single islet level has been reported. In previous work, a non-competitive assay for glucagon was developed with a 6 pM limit of detection, low enough to measure from a single islet. To incorporate this method in an online assay, a microfluidic system with several distinct features was developed. To maintain appropriate flow rates in the presence of the high concentration of salt that was required for the assay, a piezo-actuated pressure transducer with in-line flow sensors was used to drive sample flow through 80 × 50 μm (width × depth) channels, while electroosmotic flow was used to gate the sample away from 15 × 5 μm separation channel. Flow rates tested with this system were 50 - 200 nL min-1 with relative standard deviations (RSDs) ranging from 1 - 4 %. Use of the pressure-driven flow was found to increase the amount of clogs in the system, so a method to incorporate in-line filters into the channels was developed. A total of 4 low resistance, in-line microfabricated filters were evaluated, with all designs prolonging the operation time of the microfluidic device to more than 4 hours without clogs observed. Use of this system enabled highly reproducible injections (3-6% RSD). During initial incorporation of the noncompetitive assay for glucagon, it was determined that Joule heating was problematic and temperature measurements revealed the separation channel increased to more than 50°C during operation. A 3D-printed manifold was used to hold a Peltier cooler in place on the microfluidic device which produced a 2.6-fold improvement in the amount of the noncovalent glucagon complex that was detected compared to without cooling. These features are expected to be useful for not only long-term monitoring of the glucagon release from islets of Langerhans, but has the potential to be applied to a number of other microfluidic separation-based assays as well.

Dataset Information

Integrated electroosmotic perfusion of tissue with online microfluidic analysis to track the metabolism of cystamine, pantethine, and coenzyme A.

Publications

Integrated electroosmotic perfusion of tissue with online microfluidic analysis to track the metabolism of cystamine, pantethine, and coenzyme A.

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