Analysis methods of Transport through biological membranes
The transport of substances through membranes is a fundamental phenomenon for a living. All living beings are separated from the environment by special barriers made up of membranes, which make possible a selective exchange of material with the external environment and ensure that the internal composition remains constant over time. Furthermore, all biological phenomena are in practice strictly connected to membrane processes. In fact, membranes play a role of primary importance both in the transformation of chemical energy into mechanical, osmotic, or electrical work. Here we will discuss the analysis methods of transport through biological membranes.
The flows of matter through the membranes represent a fundamental method of analysis. To calculate the flow, i.e., the quantity of a substance that is transported in the unit of time through the membrane surface, we must measure the variation of the concentration of the substance in the volume adjacent to the membrane as a function of time. If the volume varies, a correction must be applied, which takes into account the change in volume as a function of time. To carry out these measurements, membranes of a size large enough to separate two half-cells are placed, and the trend over time of the concentration variation in the external phases is determined by continuous sampling or by continuous and direct photometric or electrical analysis of the liquid contained in the cell. To avoid that, differences in concentration arise in the external phases, and these must be mixed continuously and vigorously by appropriate stirrers.
If the transport through cell membranes of cell suspensions, membrane vesicles or liposomes is measured (these are microscopic liquid-filled vesicles, consisting of a double lipid layer which are obtained by subjecting the lipid emulsions to ultrasound and which are used to characterize the type of transport in artificial lipid membranes). The concentration variation of the external phase can be measured with the help of selective electrodes, or the concentration trend can be followed at internal of the cells or vesicles separating them rapidly (at set times) from the external phase by filtration or centrifugation and then determining the total mass of the test substance on the filter or in the sediment. To measure the concentration, a small dose is often added to the marked phase substance with a radioactive isotope (the so-called 'tracer'). If the specific activity of the substance that interests us (i.e., the ratio between the concentrations of the marked and the unmarked component) is constant everywhere in the system, then the tracer flow can be considered as a measurable parameter of the net flow also of the component not radioactive. If the flow of the tracer is divided by the specific activity, the net flow is obtained. This method makes it possible to measure extremely small flows of substance, for which there are no sufficiently sensitive chemical determination methods.
If, however, the tracer is added to the liquid only from one side of the membrane, then the so-called unidirectional flows are measured, the interpretation of which is, however, complicated by the matching with the tracer flow. The specific activity gradient represents, in experiments of this type, an independent thermodynamic force next to the chemical gradient of the unmarked component. The permeability measured by means of the tracers is identical to the permeability of the unlabeled component only when the interactions with the tracer flow are negligible as in the diluted aqueous solutions. Interactions with a tracer flow have often been observed in the transport of molecules not only through biological membranes but also through artificial membranes.
The best way to determine water flow is to determine volumetric variations. The contribution of ions or non-electrolytes in the volumetric flows of biological systems is usually negligible due to their strong dilution.
It must be kept in mind, during the measurement and interpretation of ionic flows, that the condition of electro neutrality (the same number of positive and negative charges in each phase) must be maintained. It may happen that the flow of an ion is infinitely small or zero, despite the fact that there are high differences in electrochemical potential and high permeability at stake. It occurs when no other ion can cross the membrane or when electro neutrality cannot be maintained by the flow of ions from the electrodes.
Biological liquids are mainly made up of aqueous solutions of electrolytes (ions), and it is for this reason that in the transport through biological membranes, electrical phenomena appear as membrane potentials and transport of electric charges (electric current). For this reason, the field of analysis methods expands considerably.
The membrane potential can be used to measure the properties of the membrane, as it depends on the concentration gradients of electrolytes and non-electrolytes and on the metabolism. The so-called microelectrodes are normally used to measure the membrane potential of individual cells. These are glass capillaries, which are filled with a very concentrated solution of potassium chloride. If the tip of these electrodes haves the diameter of less than one-thousandth of an mm (about 0.2 μm), it can be inserted inside individual cells, without shorting the membrane.
1. Voltage pre-selection (voltage clamp)
With this technique, it comes to measure the current that is needed to keep the membrane potential constant at a certain value with the help of a circuit capable of counter-reacting. Due to the dependence of currents on potential and concentration, it has been possible to identify the ionic flows that take part in the phenomenon of nervous excitement (see electrophysiology and neuron and nerve impulse).
2. The measurement of the short-circuit current
It represents a particular case of the voltage pre-selection technique. With this technique, the current that is needed to fix the membrane potential to zero is measured. In many epithelia, this short-circuit current is a direct measure for the active transport of sodium.
3. Analyzing the membrane currents
(for example, the short-circuit current) with high sensitivity and with high resolution, as a rule, there are small oscillations of current due to the presence of ‛pores 'or‛ channels' that close and open spontaneously in the membrane. From the frequency spectra of the oscillation of the current, the number, and the average opening time of the channels can be detected in favorable cases.
4. If blunt electrodes are applied to a cell membrane
So that the electrode isolates a fraction of 1 square micrometer of the membrane from the surrounding environment (patch clamp), the current flow of individuals can be observed through the electrode channels.
5. If the microelectrodes are filled, instead of with a solution of KCl, with ion exchange resins selective for an ionic species (e.g., for Na + or for Cl -), and these electrodes are inserted in single cells. It is possible to determine the intracellular activity of the respective ionic species and calculate the ionic concentration of the cytoplasm knowing the electrical membrane potential.
Other analytical techniques
The study using electron microscopy techniques is essential above all for the analysis of the ultrastructure of individual cell membranes, as well as the constitution of compound membranes. Such as for example, the epithelial (in the latter case, the cryodeck technique is of great importance). However, electron microscopy has also been used for the analysis of intracellular ion concentrations (electron beam microprobe).
Biochemical methods are used to determine the composition of membranes and to isolate their individual components, as well as to enrich the structural analysis.