Análisis e interpretación de los estados financieros

There is a general consensus of opinion that ATP levels within cells remain constant, hence its use as an index of biomass (Stanley, 1989c). This appears to be an over­ simplification. ATP plays a central role not only in the energy status of the cell but also as a regulator of enzyme activity. It is not surprising therefore that the overall internal cellular level of ATP remains fairly constant for a given set of environmental conditions and it is this factor that makes ATP a potentially useful index for microbial biomass. Studies have shown that ATP levels change during cell division and during the growth cycle of a culture. The former situation is not encountered in routine work because samples consist of a large number of microbial cells dividing in an asynchronous fashion and an "average" is always assayed. Changes in nutritional status can also effect ATP levels and a culture deprived of oxygen (eg during centrifugation or filtration), will suffer a rather rapid depletion of cellular ATP. This may be rapidly relieved (minutes) by resuplying oxygen or in some circumstances glucose. (Stanley, 1989b)

It therefore appears that ATP assaying is only an effective measure o f biomass as long as the content remains unchanged. Factors that effect the ATP levels per unit cell: (Stanley, 1986)

Age of cells or stage of growth Stage of cell division

Concentration of cells

Phage, virus and microbial infection

Action of agents that could change the cell type eg tumour promoting agents

Antibiotics

Disinfectants, pesticides and herbicides Heavy metals

Metabolic inhibitors, congeners of metabolites and toxins Radiation eg UV, microwave, X and gamma rays

Stanley (1986) also lists a number of environmental changes that are known to affect the levels of intracellular ATP per cell:

Change of growth rate

Change of nutrient(s) or their concentration. Change of gaseous environment eg DOT Change of temperature

Change of pH Change of pressure

Change of light flux (photosynthetic organisms)

In most situations it is not necessary to convert ATP back to colony forming units (CPU's). The value reported can be in ATP units or instrument readings. In such situations there is nothing special about having results expressed as CPU's. An average ATP content for a bacterial cell is lO'^^ g ATP cell‘d, yeast and mammalian cells have an average content of lO"^^ (Stanley, 1989a)

Fluctuations In Cellular ATP Content

Work carried out by Jirku (1989) examined the changes in adenylate pool size, energy charge values, and the whole sum of nucleotide concentrations o f free and covalently immobilised Saccharomyces cerevisiae. Free and immobilised cells were

Chapter 3 - Analytical development Introduction

Starved by shaking in citrate-phosphate buffer. During the 6 days of starvation the ATP, ADP, and AMP levels of free cells remained constant. More prolonged starvation produced a gradual decrease of ATP and an increase of ADP and AMP levels. In the immobilised cells the ATP, ADP and AMP levels remained constant for 11 days after which the levels abruptly changed. The EC values showed that the biosynthetic capacity and viability of the immobilised cells remained. The ATP/ADP ratio decreased, showing an increase in catabolic activity. The results indicated that immobilisation leads to a prolonged stabilisation of cell energy status of starving y east's. The more efficient maintenance of cell energetics is probably due to a specific physiological state of cells immobilised by this procedure.

Analysis of the ATP content of the fungus Trichoderma reesei was carried out on batch-grown cultures by Gaunt et a l. (1985). The ratio of ATP/ unit weight was found to remain constant during exponential growth.

It is apparent that some variation in intracellular ATP occurs. In work described by van Schie et al (1991), cells of A. calcoaceticus were grown in acetate-limited culture and then starved for 2-5 hours in order to lower the steady state concentration of intracellular ATP from 16 to 2 nmol ATP mg’* dry wt. Addition o f glucose resulted in a rapid increase of intracellular ATP to the original maximal level. This approach to ATP analysis could be of use in quantifying cells that have varying intracellular ATP concentrations by raising the ATP in all the cells in the sample to a maximum level. To determine viability of a cell sample after processing, raise to maximum ATP levels both control and processed cell samples. The difference between the two peak values is the proportional loss of viability.

3.1.3.4 Assay Techniques

There are a number of different methods of testing for ATP:

• Radiolabelling

• Fluorescence labelling • Chemiluminescence • Bioluminescence

Radiolabelling

Classic methods for the determination of metabolites have usually depended on the use of radioisotopes to label target analytes and their subsequent detection by microautoradiography. Such methods have been shown to be rapid and have great sensitivity. A method of ATP determination is described by Gonzalez and Garcia- Sancho (1980), using a radioenzymic assay based on the phosphorylation of radioactively labelled adenine sugar in the presence of the appropriate kinase. The labelled sugar-phosphate is then separated from the unreacted sugar using an ion exchange resin. This method has been shown to be extremely sensitive with detection in the range 10"^^ to 10"^^ mol ATP. The authors claim that accuracy could be increased using a radioactive sugar of higher specific activity.

Fluorescence labelling

A number of other methods are currently available for ATP determination. There are fluorimetric methods based on the coupling to the reduction of the pyridine nucleotides. Photoluminescence measuring procedures in the presence of the firefly and bacterial luciferin-luciferase system have also been well reported.

Chemi- and Bioluminescence

The trend away from isotopic labelling of proteins and other molecules, particularly for kits, is increasing and considerable interest is currently being shown in bioluminescence and fluorescence labelling. In addition to the health risks associated with the day to day use of radioisotopes there are the staff training and radioactive waste-disposal problems as well as the isotope half-lives which limit the useful shelf life of diagnostic kits. (Robinson, 1991) Chemiluminescence (CL) is the emission o f radiation, usually visible or near infra-red, caused by the decay of a chemical reaction product from an exited state to ground state. Bio luminescence (BL) is the CL produced by a wide range of organisms. Although known for several hundred

Chapter 3 - Analytical developm ent Introduction

years, only in the past two decades has there been analytical interest in luminescent reactions. Chemiluminescence coupled in assays with enzymatic or immunological methods allow the reactions to be adapted to detect and quantify a variety of analytes with great precision.

The most successful application of bioluminescence has been its use for ATP assay using the firefly luciferin-luciferase system. Under optimal conditions light intensity is proportional to ATP concentrations in the region of 5x1 O ' t o 10'^ molar. However contrary to the situation encountered in spectrophotometry and other photometric methods, the light emitted by bio- and chemiluminescent reactions depends not on the concentration of the substrates but also on their reaction rate. This further implies a more stringent control of the assay conditions, a situation further complicated by the fact that bioluminescence enzymes usually show a limited stability and a high sensitivity to various sample components, and that the emitted luminescence may more or less rapidly in the course of time. (Schram & van Witzenberg, 1989)

Using this reaction, two types of luminescence curve can be obtained. With high concentrations of the luminescent reactants, all the ATP is utilised within a few seconds and a high rapid peak occurs, so called "flash kinetics". With low concentrations of luciferin and luciferase, a continuous signal is produced which eventually diminishes as the ATP is utilised. The flash method offers greater sensitivity over the continuous output system. However, the continuous signal is more convenient and facilitates internal standardisation, and its introduction by Lundin in 1976 was considered to be somewhat of a milestone in ATP methodology (Jago, 1989).

3.1.3.5 Comparison Between Firefly ATP Assay And Conventional Culture