Tissue preparation measurement

Excitable tissue preparations were obtained fresh daily from live animals using the technique described by Dodd et al. (12). Protein was measured on each synapto-some preparation using the Coomassie Brilliant Blue dye technique described by Bradford (13) results were expressed as "toxin bound per mg synaptosome protein". 

Interpretation of these early experiments with crude tissue preparations was greatly complicated by the presence of very active phosphatases (ATPases) which rapidly hydrolyzed any ATP which might have been formed. Ochoa suggested that the amount of Pj apparently esterified should be corrected for the measurable rate of ATP hydrolysis by the preparation. This gave P/O ratios approaching 3. Belitzer and Tsibakowa and Ochoa realized that phosphorylation must occur not only when the substrate is dehydrogenated. .. but also during... 

Preliminary testing on whole animal (bioassay) and isolated tissue preparations (voltage-clamped squid giant axons) showed effects similar to those measured for STX and neo-STX. Again the presence of anionic cryptic forms of the toxins was indicated. 

A next-level assay is usually an isolated heart/cardiac tissue preparation. The canine Purkinje fiber assay (GLP) measures several action potential parameters, like resting membrane potential, upstroke velocity, action potential duration and shape, but also if a drug acts reverse-use dependently [72]. Based on changes of the action potential shape it is possible to conclude which ion channels are modulated (e.g., L-type calcium channel block would abolish the plateau phase). The papillary muscle assay (e.g., guinea pigs) determines similar parameters [73]. 

Although sensitive analytical methods are available for measuring the presence of aluminum in biological tissues and fluids, it is not known whether data collected using these techniques have been used to correlate the levels of aluminum in biological materials to exposure and effect levels. The problem of contamination during tissue preparation (Makjanic et al. 1998) makes this task more challenging. 

Pietra et al. (1990) studied the effects of some antidepressants by the in vitro inhibition of carbachol-induced contractions of rat detrusor strip preparations. The detrusor muscle tissue (bladder dome) was cut in a semicircular direction and further dissected into strip preparations measuring approximately 2 x 20 mm. 

Assay of Cu/Zn-SOD and Mn-SOD or Fe-SOD separately may be achieved as follows total SOD activity is measured as described above and the assay is then repeated following the addition of 2 mM cyanide which completely blocks the activity of the Cu/Zn-SOD. The activity of the Cu/Zn enzyme and the Mn enzyme (in mammalian tissue preparations) may then be obtained by difference. 

In experimental animals and with isolated tissue preparations and organ cultures, the test can be refined by measuring the production of G02 from [ C]histidine in the presence and absence of added methionine. If the impairment of histidine metabolism is the result of primary folate deficiency, the addition of methionine wUl have no effect. By contrast, if the problem is trapping of folate as methyl-tetrahydrofolate, the addition of methionine will restore normal histidine oxidation as a result of restoring the inhibition of methylene-tetrahydrofolate reductase by S-adenosylmethionine and restoring the activity of 10-formyl-tetrahydrofolate dehydrogenase, thus permitting more normal folate metabolism (Section 10.3.4.1). 

One approach that has been used quite widely to quantitate neurotransmitter release employs radiolabeled (tritiated) neurotransmitter analogs (e.g.. Reference 67). First, tissue is incubated in a buffer solution that contains tritiated neurotransmitter. During this time, the radiolabeled transmitter is taken up into cells by endogenous plasma-membrane transporters and packaged into vesicles by vesicular transporters. The tissue preparation then is rinsed in buffer to remove extracellular radiolabeled transmitter leaving only that which was taken up into cells. This stored transmitter is then released over time by exocytosis. To quantitate its release, the tissue is continuously perfused with buffer, and time-dependent aliquots are collected. Radioactivity is measured in the aliquots with a scintillation counter and is used as an index of endogenous neurotransmitter release. Rather than estimate absolute neurotransmitter release, this method is typically used to compare the relative release between two or more conditions. 

The tissue preparations were diluted (1 12) in heat-inactivated sera (1 hour at 55°C), and incubated in 0.02 M Veronal buffer (pH 9.8) containing 0.018 M disodium phenyl phosphate for 2 hours at 37°C. Phenol was measured via a diazo coupling procedure. Conditions for heat-inactivating the tissue enzymes were 16 minutes at 55°C. By subtracting from the total activity the intestinal component, which is measured by n-phenylalanine sensitivity, one obtains the sum of the activities of liver and bone. The ratio of the two was computed from the heat inactivation minus that attributed to intestine, employing 91.2% heat inactivation to represent 100% bone and 51.4% heat inactivation indicating all liver. In this way one arrives at values for bone, liver, and intestinal alkaline phosphatase. 

As part of a laboratory s quality control program, all steps of the test should be described separately, and parameters of each step established and monitored in order to ensure consistency of performance and reproducibility of results. Daily records of control results are maintained, and corrective actions are undertaken and documented when results are unacceptable. In practice the surgical pathology histology laboratory typically falls short of the ideal standards in key areas, especially with regard to tissue preparation. In this section, we will discuss quality control issues as they pertain to the validation of antibodies and the use of controls. We will also address measures that may be adopted to minimize variability deriving from inconsistent tissue preparation, including fixation. 

Phosphorus NMR is also used for vascularly perfused preparations, such as heart, skeletal muscle, bladder, or uterus. In these cases, the contributions to the spectra by the perfusate may be important. Phe-nylphosphonate or similar derivatives are often used for such experiments. PPA has a direct C-P bond, unlike phosphates that have O-P bonds. As a result, PPA resonates downfield (to the left) of the naturally occurring phosphorus compounds. In mechanical and NMR experiments, the effects of PPA on the isolated, perfused bladder were measured (Fisher and Dillon, 1987a). PPA in concentrations up to 20 mM did not produce a significant reduction in force generation. Its NMR peak position was shown to be pH sensitive with a pK of 7.09, making it ideal for measurements of extracellular pH. It did not produce any alteration in the natural phosphorus spectrum, and it was able to be washed into and out of the perfused tissue without measurable residue, indicating that it did not cross cell membranes. In addition to being a pH indicator, it is therefore also useful as a marker for extracellular space. 

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