Enzymatic methods, measurement

Sensitive and biochemical methods for SBA are now currently available. The problem of its usefulness in diagnosis is still unsolved and a large-scale prospective studies on SBA related to disease are needed. A quality control study has been performed 11 laboratories located in c fferent parts of Italy have analyzed SBA by different techniques. The results obtained suggest that at the present time the analysis of serum bile acid requires further standardization, due to different analytical information obtained using various techniques. One of the more sensitive method for SBA seems to be the immunological procedure and several kits are commercially available, but for the above reasons none of them measures the same acid. On the other hand, the enzymatic method measures total bile acid with low sensitivity. 

As is well known the difficulty of analysis of a sample increases as its complexity increases. Analysis usually commences with a rather nonspecific clean-up step and requires that the separation step that follows be highly selective and depends on a detection step that is as specific as possible. As the selectivity of detection increases there is also an increase in the reliability of the identification and it is possible to reduce the demands made on the selectivity of the preceding separation method. This is the case for radiometric and enzymatic methods and also explains the popularity of fluorescence measurements. The latter obtain their selectivity from the freedom to choose excitation and measurement wavelengths. 

Schneider, A. J. Willis, H. J. Sources of variation in a standardized and semi-micro procedure for serum glutamic oxalacetic transaminase. Clin. Chem. (1958), 4, 392-408. Atwood, J. G. DiCesare, J. L. Making enzymatic methods optimum for measuring compounds with a kinetic analyzer. Clin. Chem. (1975), n, 1263-1269. 

There are some methods that are specific to HCHO. For example, the Hantzsch reaction of HCHO, collected with a diffusion scrubber, with ammonium acetate, acetic acid, and acetylacetone to form diacetyldihydrolutidine, which is measured using its fluorescence at 470 nm, has been applied to air measurements (Dasgupta et al., 1988, 1990 Kleindienst et al., 1988a,b Lawson et al., 1990 Khare et al., 1997). Reaction with 1,3-cyclohexanedione and ammonium acetate to form a dihydropyridine derivative that is measured by fluorescence has been used in conjunction with a diffusion scrubber (Fan and Dasgupta, 1994). Enzymatic methods have been used in which formaldehyde dehydrogenase catalyzes the oxidation of HCHO to HCOOH in the presence of -nicotinamide adenine dinucleotide, NAD+, which is reduced to NADH. The latter is measured by fluorescence at 450 nm (Lazrus et al., 1988 Ho and Richards, 1990). 

A number of intercomparison studies of the various methods of measurement of HCHO have been carried out. As might be expected given the specificity of spectroscopic methods, the results of FTIR, DOAS, and TDLS are generally in good agreement, within 15% of their mean value in one study in a polluted atmosphere (Lawson et al., 1990). During the same study, the diacetyldihydrolutidine derivative method and the DNPH method were lower by 15-25% than the spectroscopic mean, whereas the enzymatic method was higher by 25%. 

In another intercomparison using TDLS, several DNPH methods, the 1,3-cyclohexanedione diffusion scrubber, and the enzymatic method were compared using both spiked samples and ambient air. The TDLS was used as a standard for comparison. For ambient air measurements, results obtained with the 1,3-cyclohexanedione diffusion scrubber were about 30% higher than those obtained with TDLS, whereas results for the enzymatic method were about 35% lower. The DNPH cartridge measurements were quite variable, which may... 

For simultaneous blood measurement of the four metabolites (lactate, pyruvate, ACAC and ), blood that has been deproteinised with perchloric acid is used as a sample. Spectrophotometric enzymatic methods according to reactions given in Fig. 1.4.3 were developed for automated analysers to minimise sample volume and improve precision [1,10,11,17]. 

The same deproteinised blood as used for lactate, pyruvate, , and ACAC assays described above is used for enzymatic methods employing spectrophotometric measurement (Fig. 1.4.3) [10, 17]. The enzymes involved are LDH for pyruvate, lactate oxidase for lactate and HBDH for ACAC and . 

For faster results, thin layer chromatography has been used (66), but we are not confident that lower levels of malic acid (0.06% ) (cf. Ref. 11), sometimes found in California wine before malo-lactic fermentation, are easily detected by this means. Malic acid can be more precisely measured by using the quantitative enzymatic method (67). Only the l isomer, the natural form present in grapes and wine, is detected by this method. 

As shown in Table Dl.3.4, there is good agreement between the cholesterol contents of poultry meat and cheese determined by GC and those measured by the enzymatic method. 

Separation techniques have remained indispensable tools of the analytical chemist despite the great progress in selective measurement techniques like binding assays, enzymatic methods, or the use of tandem mass spectrometers. This is probably so... 

Apart from this, AT-methylhydantoin amidohydrolase is also useful for diagnostic purposes. JV-Methylhydantoin amidohydrolase and the corresponding enzyme in microbial creatinine transformation [i.e. N-carbamoylsarcosine amidohydrolase (EC 3.5.1.59)] are useful for enzymatically measuring creatinine in serum and urine, which is a marker of renal dysfunction [12]. This enzymatic method has proved to be simple and precise, and shows excellent sensitivity and specificity [38],... 

The objective of the experiments is to compare the affinity of wild-type antibody and wild-type immunogen (chicken lysozyme) with that of wild-type antibody and mutant antigen or with that of mutant antibody and wild-type immunogen. All the assays involve incubation of a constant concentration of one reactant with varying amounts of the complementary reactant, along with estimating the concentration of either bound or free reactant by an immunochemical or enzymatic method. In each case, the assumption is made that the measurement step does not disturb the equilibrium between antigen and antibody, and it is important that this assumption be validated experimentally. We summarize below several alternative methods. 

The suprahypothalamic neurotransmitter level can be assessed by a determination of catecholamines in circumscribed brain areas, the technique requires preparation of frozen tissue and isolation of specific nuclei by the micropunch technique. The catecholamines and indolamines can be measured by a radio-enzymatic methods and by a high-pressure liquid chromatography (HPLC) with electrochemical detection. These mechanistic investigations are mostly initiated due to questions arising from the receptor interaction profile of the drug candidate, they may be required to prove that such receptor interactions truly change the functional state of neurotransmitters (functional expression). Mostly, however, the peripheral effects of such neurotransmitter mechanisms (for instance prolactin secretion) are sufficiently distinct. 

Fig. 23. Relationship between glucose concentrations of human blood plasma samples measured by a glucose-sensitive FET sensor and by a conventional enzymatic method (F = 1.007X 14, r = 0.988, n = 101). (Re-...

Enzymatic methods have also been employed for measurement of acetaldehyde levels. The affinity of these enzymes for other aldehydes has not been completely determined (13, 38-40), therefore, these methods also suffer from lack of specificity. 

Enzyme-catalyzed reactions are used for the determination not only of substrates but also of the enzymes as well as activators and inhibitors of the enzymatic reaction. Substrate concentrations can be measured also by nonkinetic methods by allowing a reaction to proceed to completion before making measurements. Enzymatic methods of analysis have been reviewed by Guilbault. He listed over 150 enzymes with their sources many of these are now available in purified form with high specific activity. Urease was the first to be obtained in pure crystalline form. ... 

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