Analytical measurements in small pulmonary vessels

Abstract

Micro-sensors and ultra small devices, the latter often discussed in the context of the field of nanotechnology, have attracted burgeoning interest in recent years. Michael Thompson in consultation with Flavio Coceani of Scuola Superiore di Studi Universitari e di Perfezionamento Sant’ Anna, Pisa, Italy, outlines the background and challenge represented by the design of sensors required for the quantitative determination of NO and CO in very small pulmonary vessels. The solution to this problem will require a highly interdisciplinary research effort involving micro-electronic or -optical fabrication, physiology, chemometrics and surface chemistry.

For some time there has been increasing research interest in the pulmonary circulation of vessels in the animal fetus or neonatal state. In particular, extensive work has been performed with respect to the chemical factors (vasoactive agents) that are responsible for the constriction or dilatation of specific blood vessels. As a direct consequence of all this work, the conclusion has been reached that quantitative measurement of molecules such as NO, O₂ and CO inside small vessels, in a specific location of dimensions of the order of a micrometer, is highly desirable. Although recognised as extremely challenging in terms of acquisition, such data would prove to be invaluable in the conduction of medical research in physiology. An additional, but exciting problem, is whether it is feasible to mount such a putative detection system in tandem with a device that is capable of physical measurement such as that of vessel internal pressure (relevant to the resistance to blood flow). In order to provide a backcloth as to the precise nature of the analytical challenge, some research examples over recent years with respect to aspects of physiology are traced in the following brief remarks.

In the middle 1990s an instrument was devised for the measurement of the isometric tension from isolated small arteries and veins obtained from the lungs of term fetal lambs.¹ The particular interest in studying these vessels, which have an internal diameter around 200 μm, was to examine the influence of known agents such as endothelin-I on the prostaglandin synthetic system, with respect to perinatal pulmonary hemodynamics. This agent had been thought to mediate a dilatation or constriction of vessels. In overall terms there was an interest as to the role played by oxygen tension and the molecule, indomethacin. In summary, among a number of observations, it was found that small arteries are endowed with a prostaglandin relaxing mechanism that becomes functional on raising the PO₂ from fetal to neonatal levels. In a further study, an investigation of the mechanism of hypoxic vasoconstriction (pulmonary vasculature constriction) in the same animal was performed.² At the time of this work there was debate as to whether the mechanism was connected to enhanced production of a constrictor or the reduced formation of a dilator in the vessel wall itself. In this connection it had previously been demonstrated that prostaglandin and NO-based relaxation processes are partially governed by oxygen tension. In summary, hypoxia contracted both vessels in vitro, with the contraction being greater in arteries. From these results and a number of studies performed with various agents it was concluded that hypoxia tone could be ascribed primarily to the intramural production of endothelin-I.

In contemporary times, questions have been posed regarding NO-activation.³ To look at this issue further preterm lamb was studied. This was instigated by the idea that arteries operate both as an analytical sensor and effector when it comes to responses to oxygen tension. (This notion will be of considerable interest to the bioanalytical scientist). Experimentally, the role of inhibitors to NO synthesis and the effects of various agents, which influence pulmonary relaxation, were examined. The overall conclusion was reached that preterm pulmonary arteries possess viable effector mechanisms for contraction and relaxation, although the capability for these processes to be activated by PO₂ changes is much less than previously encountered.

Very recently, attention has been switched to addressing pulmonary issues regarding mouse physiology, as a model for the human configuration. As an example, one of the interests with respect to human physiology is the structure, ductus arteriosus, which is a muscular shunt in the fetus, which connects the pulmonary artery with the aorta.⁴ This allows blood to bypass the unexpanded lungs. At birth this structure closes as the infant begins normal lung function. The effectors, which cause the ductus to retain patency and closure, are the subjects of some debate. Again, as for the cases described above the mechanisms are thought to involve prostaglandins and NO. Using mice strains with targeted deletion of genes allows the study of particular aspects of physiology, in this case the system for ductus closure. In vitro and in vivo methods for the investigation of genetically modified mice were employed with respect to the activity of the entity (receptor) responsible for the closure of the ductus. It was concluded that the receptor involved does mediate the response to oxygen, but that the ductus still closes postnatally, likely a process connected the withdrawal of relaxing influences. There is a view that this type of research is highly relevant with respect to the medical welfare of infants.

Turning to the nature of the problem to be posed to the analytical scientist, it is manifestly obvious that a comprehensive understanding of the interaction of complex biological systems with active molecules such as NO, and much more recently CO, in blood requires not only quantification of levels of these species in the structure but also measurement of concentration with time in a specific location. Historically, a wide variety of methods for the detection of NO, in particular, have appeared in the literature. These include intermittent and continuous measurement based on chemiluminescence, electron spin resonance, spectrophotometry, chromatography, electrochemical, optrode and semiconductor technologies. With regard to the requirement for continuous detection in biological applications, Kikuchi et al.⁵ used a fiber-optic configuration for the chemiluminescence measurement of NO release from a kidney on-line. An electrochemical method composed of a selective Nafion membrane (Ni-porphyrin complex) and differential pulse voltammetry was used to examine NO in blood and plasma.⁶ However, realistic in vivo measurements have not figured prominently in the literature. In 1996, Tschudi et al.⁷ used a bundle of carbon fibers for the detection of NO in aorta and mesenteric arteries of rats. With regard to measurement in humans an amperometric sensor was used to measure NO concentration in a vein in the hand.⁸ Very recently the Schuhmann group has been employing porphyrin-based electrochemical devices to monitor NO from biological samples such as the retina of rats.⁹

Clearly, in order to progress to the measurement of NO (and CO) in resistance vessels of genetically-modified mice, quantum advances with respect to detection strategy are needed. This application requires quantification of active molecules present in the fluid cross-section-wise across the vessel. The arteries and veins of mice have very small internal diameters and, accordingly, to generate site-specific chemical information the detecting device must be of a true ‘nanotechnology’ dimension. As indicated above, it is recognised that the analytical literature contains possible ingredients for such a sensor. However, the fabrication of a sensor capable of assisting the physiologist here must measure concentration faithfully even in the face of likely protein surface adsorption. Furthermore, there is also the daunting task of including a system that can be interrogated so that chemical data can be collected for future evaluation. Finally, it is worth noting that there is interest in the process of performing research on ultra small devices fabricated for the purpose of the measurement of physical parameters such as pressure. It would be highly desirable to generate detectors that operate in a tandem fashion, that is, combine physical and chemical measurement (for both NO and CO) in the same structure. There is clearly a dearth of such structures in the microelectronic fabrication literature.

Michael Thompson is the Scientific Editor of The Analyst I-Section

References

1. Y. Wang and F. Coceani, Circulation Res., 1992, 71, 320.
2. Y. Wang, Y. Coe, O. Toyoda and F. Coceani, J. Physiol., 1995, 482, 421.
3. Y.-A. Liu, J. G. W. Theis and F. Coceani, Biol. Neonate, 2000, 77, 253.
4. F. Coceani, Y.-A. Liu, E. Seidlitz, L. Kelsey, T. Kuwaki, C. Ackerley and M. Yanagisawa, Am. J. Physiol., 1999, 277, 1521H.
5. K. Kikuchi, T. Nagano, H. Hayakawa, Y. Hirata and M. Hirabe, Anal. Chem., 1993, 65, 1794.
6. T. Malinski, Z. Taha, S. Grunfeld, A. Burewicz, P. Tomboulian and F. Kiechle, Anal. Chim. Acta, 1993, 279, 135.
7. M. R. Tschudi, S. Mesaros, T. F. Luscher and T. Malinski, Hypertension, 1999, 27, 32.
8. P. Vallance, S. Patton, K. Bhagat, R. MacAllister, M. Radomski, S. Moncada and T. Mallinski, The Lancet, 1995, 346, 153.
9. P. Heiduschka, M. Groppe, N. Diab, S. Thanos and W. Schuhmann, Abstracts of the 53rd Annual Congress of the International Electrochemical Society, 2002, p. 61.

---