Assays in Body Fluids

The multiple actions of individual cytokines mean that cell lines rarely respond only to one cytokine. This is well illustrated by the efforts to develop specific bioassays for lL-1. Although the original mouse thymocyte proliferation assay was thought to be IL-1 specific, it is now clear that many cytokines influence the assay. IL-6 and TNF can replace IL-1 (G7, U4), and IL-2 and IL-4 can sygerize with 

Measurement Biologically active molecules Numbers of available epitopes (protein or peptide) 

Specificity May be high using specific antibodies High but may be inappropriate 

Interference Native inhibitors, anti-cytokine antibodies Serum proteins, anti-cytokine and heterophile antibodies, rheumatoid factors 

Precision Poor (10-100% coefficient of variation) Should be good (5-10% coefficient of variation) 

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Recently, creativity in chromogenic macrocycle synthesis has expanded. New spherand species have been synthesized that act as highly preorganized chromogenic-specific indicators for Li+ and Na (Cram et al., 1985), and an azophenol dye has been prepared with perfect selectivity for Li (Kaneda et al., 1985). Many of these chromogenic macrocycles and more complicated species such as the hemispherands and cryptohemispherands have found commercial use for Na" and K assays in body fluids (see 41) (Helgeson et al., 1989 Czech et al., 1990). 

Assay in body fluids and tissue (i) Sulphadimidine in bovine kidney, liver, muscle and fat tissue. Partition Bondapak C18 /Porasil B 2.5% isopropyl alcohol in phosphate buffer (pH 7.7)... 

A very important consequence of enzyme specificity is that it is often possible to detect a particular enzyme and even measure its catalytic activity in the presence of many other enzymes and molecules. (Of course, in the cell the enzyme must be able to carry out its function in the presence of many other enzymes and substrates.) A simple enzyme can often be assayed in a cell or unpurified extract of a cell by adding a substrate and measuring the amount of product released. Several conditions must carefully be controlled, to take account of the enzyme kinetics and the presence of regulatory substances and cofactors, but under the right conditions, enzyme assay in body fluids or unpurified extracts is a valuable clinical and research tool. 

Assay of Enzymes In body fluids, enzyme levels aie measured to help in diagnosis and for monitoiing treatment of disease. Some enzymes or isoenzymes are predominant only in a particular tissue. When such tissues are damaged because of a disease, these enzymes or isoenzymes are Hberated and there is an increase in the level of the enzyme in the semm. Enzyme levels are deterrnined by the kinetic methods described, ie, the assays are set up so that the enzyme concentration is rate-limiting. The continuous flow analyzers, introduced in the early 1960s, solved the problem of the high workload of clinical laboratories. In this method, reaction velocity is measured rapidly the change in absorbance may be very small, but within the capabiUty of advanced kinetic analyzers. 

The microbial assay is based on the growth of l ctobacillus casei in the natural (72) or modified form. The lactic acid formed is titrated or, preferably, the turbidity measured photometrically. In a more sensitive assay, l euconostoc mesenteroides is employed as the assay organism (73). It is 50 times more sensitive than T. casei for assaying riboflavin and its analogues (0.1 ng/mL vs 20 ng/mL for T. casei). A very useful method for measuring total riboflavin in body fluids and tissues is based on the riboflavin requirement of the proto2oan cHate Tetrahjmenapyriformis which is sensitive and specific for riboflavin. 

GC/MS ASSAYS FOR ABUSED DRUGS IN BODY FLUIDS. Rodger L. Foltz, Ph.D. Allison F. Fentiman, Jr., Ph.D. and Ruth B. Foltz. 

Levels of a number of metabolites as well as a number of enzymes in body fluids are indicative of disease conditions. Many of the enzymatic reactions mentioned above have been used in solution clinical assays as well as in test strips.446,497-508 512-515 Assays for hydrogen peroxide and the enzyme peroxidase using NADH and a tetrazolium salt have been de-scribed.509,5io Assays of exogenous substances (e.g., drugs or their metabolites) also utilize this chemistry. The determination of alcohol using alcohol dehydrogenase is an example.511 As mentioned above, the assay of enzyme levels can also be achieved using tetrazolium salts.516-520... 

HPLC has had considerable success in separating compounds as diverse as steroids, carbohydrates, vitamins, dyestuffs, pesticides and polymers. It is used routinely for the assay of pharmaceutical products, the monitoring of drugs and metabolites in body fluids and for other biomedical, biochemical and forensic applications, such as the detection of drugs of abuse. The determination of additives in foodstuffs and beverages including sugars,... 

The recognition of their structure permits the determination of vitamins by the tools of analytical chemistry, but while such methods are widely used in industrial production, the minute quantities in body fluids and tissues limit the purely chemical approach to a few members of this group present in relatively high concentration, e.g., vitamin C (K5). Microchemical methods are in use for the determination of thiamine, riboflavin, and some of the fat-soluble vitamins, based on the most sensitive colorimetric and, in particular, fluorometric techniques. Vitamin D, on the other hand, is determined by animal assay. 

It may be determined by various chemical methods, but its analysis in body fluids lends itself to a microbiological assay method. 

The establishment of quantitative methods for the determination of vitamins in body fluids and tissues by microbiological assay techniques should stimulate the search for the significance of vitamins in disease, not only in nutritional deficiency, but in the much wider field of all metabolic disturbances. Functional vitamin deficiencies are produced by malabsorption, by inhibitors of the vitamin function through products of the body, and particularly through drugs and other toxic substances. Vitamin deficiencies may be relative deficiencies whenever an individual s metabolism is deranged so as to require enhanced quantities of a given vitamin to cure or to counteract certain symptoms as, e.g., in Darier s disease (keratosis follicularis) (P2a). 

Items of general medical interest and an assay finder to help researcher find methods or labs to measure a wide variety of hormones, metals, enzymes and drugs in body fluids... 

A much more serious genetic disease, first described by Foiling in 1934, is phenylketonuria. Here the disturbance in phenylalanine metabolism is due to an autosomal recessive deficiency in liver phenylalanine hydroxylase (Jervis, 1954) which normally converts significant amounts of phenylalanine to tyrosine. Phenylalanine can therefore only be metabolized to phenylpyruvate and other derivatives, a route which is inadequate to dispose of all the phenylalanine in the diet. The amino acid and phenylpyruvate therefore accummulate. The condition is characterized by serious mental retardation, for reasons which are unknown. By the early 1950s it was found that if the condition is diagnosed at birth and amounts of phenylalanine in the diet immediately and permamently reduced, mental retardation can be minimized. The defect is shown only in liver and is not detectable in amniotic fluid cells nor in fibroblasts. A very sensitive bacterial assay has therefore been developed for routine screening of phenylalanine levels in body fluids in newborn babies. 

Schmerr and Jenny established a CE-based immunoassay for the detection of prion protein (24). In this competitive assay, peptides derived from the prion protein and labeled with fluorescein were used. This allowed them to distinguish scrapie-infected brain preparations from noninfected. For identification, the ratio between the peaks resulting from the free and the com-plexed peptide with a specific antibody was used. The results were in agreement with other data on the brain preparations achieved by Western blot analysis. The CE-based assay provides the advantage of direct detection of the scrapie protein in blood and tissue preparations with high sensitivity. Furthermore, due to the small sample amount needed for analysis, the CE-based assay is applicable to the putative diagnostics of prion protein in body fluids. 

Another approach to detect CsA in body fluids is a CE-based competitive immunoassay using labeled CsA and an anti-CsA antibody (25). In comparison to the direct assay, an immobilization of an antibody is not... 

Bioassay may also be of low-precision design (multiple samples on large plates, i.e., >3 manual turbidimetric assays, small-plate assays). These types of assay are useful for trace analyses (cleaning validation), and are often used for the analysis of samples in body fluids as they are capable of dealing with the large numbers of samples that may be generated in these studies. 

Several alternative methods for the determination of sialic acid in body fluids and tissues have been described. Most of these methods make use of the classic periodate-TBA assay in combination with purification using HPLC [13]. Another method makes use of fluorometric HPLC of sialic acids after derivatization with a fluorogenic compound [9]. The most promising new method for the determination of free sialic acid in urine (and probably also other body fluids and tissues) is the HPLC-tandem mass spectrometry method [19]. This method is rapid, accurate, and sensitive, and is more robust than earlier methods. The only disadvantage is the expensive equipment that is required, which makes it only economical for specialized metabolic laboratories. Since this equipment is used for many different metabolic assays, the investment is certainly warranted, and nowadays almost essential for any metabolic laboratory. 

Porphyrins and porphyrin precursors are assayed most often in a 24-h urine collected without additive. Alternatively, untimed urine samples may be used and excretion standardized to creatinine. The latter is especially recommended for children and in emergency situations. Alternative specimens for porphyrins are plasma, erythrocytes, and feces, depending on the medical indication. During collection and until arrival at the laboratory, specimens should be kept cold, preferably at about 4°C, and protected from light. Specimens in the laboratory are best kept frozen, as the metabolites in body fluids are stable at -20°C for at least 3 months. Some exceptions have been noted below. 

In addition, a number of other assays have been developed for screening antibiotic residues in body fluids of animals at slaughter. They include an agar plate assay for detecting tilmicosin in bovine serum (88), modified CFT and BR-test assays for screening penicillin G residues in plasma of healthy steers (89), a modified CFT for screening plasma and urine samples from healthy market... 

One of the more important areas of use of ultraviolet instruments is the identification and determination of biologically active substances. Many components in body fluids can be determined either directly or through colorimetric methods. Drugs and narcotics can be measured both in the body as well as in formulations. Vitamin assay is another related activity. Nearly all metals and nonmetals can be determined through their ultraviolet absorption or by colorimetric methods. In recent years, ultraviolet instruments have been used extensively for the determination of air and water pollutants, such as aldehydes, phenolics, and ozone ... 

Some work has demonstrated that pulse voltammetry at the carbon paste electrode can be applied to the assay of drugs in body fluids [197,198]. Methods based on the electrooxidation of theophylline [197] and acetaminophen [198] were described at concentrations of approximately 5-10 fxg/mL plasma. 

HPLC is the technique of choice for determining the purity of moxalactam disodium in raw materials, formulated products, and in body fluids. Moxalactam is determined in a system containing 0.05 to 0.1 M ammonium acetate with about 6 percent methanol present. An ES Industries Chromegabond C18 column or other alternative column with similar retention characteristics is used to determine the purity of the moxalactam sample. The substance may be monitored at 254 nm or, when available, a variable wavelength detector can be operated at 271 nm for assay. The sample is dissolved in water or in 0.1 M ammonium acetate solution. Under conditions of this method, the assay should be completed within 4 hours of sample dissolution. 

The following methods have been used for the assay of sulindac in body fluids and tissues. 

W. Roth and K. Beschke, Fully-automated assay by liquid chromatography for routine drug monitoring in body fluids—method development with biological samples, /. Pharm. Biomed. Anal. 2 (1984), 289-296. 

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