Blood Proteins, Analysis

The toxicological chemistry aspects of blood and the cardiovascular system are very important. Systemic poisons and their metabolites are carried to receptors in the body through this system. It carries phase I and phase II reaction products to the kidney and bladder for elimination. Toxicants can bind to blood proteins. Analysis of blood for toxicants and their metabolites is a common way of determining exposure to toxic substances and generally the most accurate means for evaluating systemic exposure. 

The antiradical capacity of proteins is thought to be an important component of the total antioxidant capacity of blood plasma. Analysis of the ACW of blood plasma showed, that under normal conditions, its main components are the UA and ASC. The rest of the total antiradical capacity (ACR), which can be... 

An internal standard should always be used for every analysis carried out. This is an amino acid that is known to be absent from the sample under investigation. For instance in blood plasma analysis either of the non-physio-logical amino acids, nor-leucine or a-amino-/3-guanidinobutyric acid, may be used. This should be added in a known amount to the sample prior to any sample pre-treatment (for example, removal of protein). 

Cheever, K.L., Richards, D.E. Plotnick, H.B. (1980) Metabohsm of ortho-, meta-, and para-toluidine in the adnlt male rat. Toxicol, appl. Pharmacol, 57, 361-369 Cheever, K.L., DeBord D.G, Sweaiengin, T.F. Booth-Jones, A.D. (1992) ortho-Toluidine blood protein addnets HPLC analysis with fluoreseenee deteetion after a single dose in the adult male rat. Fundam. appl Toxicol, 18, 522-531 Chemfinder (2000) ortiho-Toluidine Hydrochloride [http //www.ehemfinder.com/result.asp] Chemieal Information Serviees (1999) Directory of World Chemical Producers (Version 99.1.0) [CD-ROM], Dallas, TX... 

Bergen HR, Lacey JM, O Brien JF, et al. Online single-step analysis of blood proteins The transferrin story. Anal. Biochem. (2001) 296 122-129. 

Sulfur mustard forms adducts with the blood proteins hemoglobin and albumin. Adducts with histidine residues are the most abundant after exposure of hemoglobin in vitro to sulfur mustard. Analysis of adducted histidine by GC/MS is hampered by poor thermal stability of volatile derivatives. A sensitive method was developed using LC/ESI/MS/MS after... 

B.R. Capacio, J.R. Smith, M.T. DeLion, D.R. Anderson, J.S. Graham, D.E. Platoff and W.D. Korte, Monitoring sulfur mustard exposure by gas chromatography-mass spectrometry analysis of thiodiglycol cleaved from blood proteins, J. Anal. Toxicol., 28, 306-310 (2004). 

Robinson, J. C., B. S. Blumberg, and J. E. Pierce Studies on inherited variants of blood proteins. I. Two dimensional electrophoretic analysis of chromatographic fractions of serum proteins. J. Lab. Clin. Med. 60, 468 (1962). 

De-proteinisation of diluted whole blood, and acid extraction of lead from proteins with 2M HNOs is the preparation chosen for the National Bureau of Standards reference method for blood-lead analysis [28]. Centrifugation of blood samples treated in this way yields clear supernatant fractions that contain all of the lead present, and which are easily dispensed into electrothermal atomisers using auto-sampling techniques. This method gave results that compared very well with those obtained using anodic stripping voltammetry, r = 0.975, for concentrations of 10—900 pg l-1 of lead in blood [15]. 

Lawrence, R.J., Smith, J.R., Boyd, B.L. (2008). Improvements in the methodology of monitoring sulfur mustard exposiue by gas chromatography-mass spectrometry analysis of cleaved and derivatized blood protein adducts. J. Anal. Toxicol. 32(1) 31-6. 

Isotope dilution in combination with the substoichiometric principle is applied in various ways. The most important examples are radioimmunoassay for protein analysis and DNA analysis. In radioimmunoassay, radionuclides are used as tracers and immunochemical reactions for isolation. Radioimmunoassay was first described in 1959 by Yalow and Berson, and since then has found very broad application in clinical medicine, in particular for the measurement of serum proteins, hormones, enzymes, viruses, bacterial antigens, drugs and other substances in blood, other body fluids and tissues. Only one drop of blood is needed, and the analysis can be per-fonned automatically. Today more than 10 immunoassays are made annually in the United States. The most important advantages of the method are the high sensitivity and the high specificity. In favourable cases quantities down to 10 g can... 

The bioanalyst can be required to analyse most biofluids although the most common are urine and the aqueous phase of blood, i.e. plasma or serum. Other samples may be cell and tissue extracts, synovial fluid, cerebrospinal fluid (CSF) and saliva. In the case of urine and CSF with their very low protein content it might be possible to directly inject the sample into an HPLC column. With most silica-based packing materials, direct injection of blood proteins will rapidly lead to column deterioration. HPLC columns are expensive and their efficiency is easily lost so correct preparation of samples will not only improve column life but also improve the results. At its simplest it is only necessary to remove particulate matter from samples to prevent clogging of the column and frits. Modern HPLC packings are very susceptible to contamination by proteins, fats and other macromolecules from biological samples and it is necessary to remove these (except of course for protein analysis). 

Since no means has yet been devised for sampling blood plasma in situ (i.e., cell-free from the blood vessel), the clean separation of plasma or serum from blood is frequently the most expensive part of the total protein analysis. 

Analysis of Blood Samples. Urinary metabolites undergo relatively rapid elimination from the body, whereas blood components offer biomarkers that have the potential to be used for verification of sulfur mustard exposure long after the exposure incident. Three different approaches have been used for blood biomarker analysis. The intact macromolecule such as protein or DNA with the sulfur mustard adducts still attached can be analyzed. To date, this approach has only been demonstrated for hemoglobin using in vitro experiments. For proteins, an alternate approach is to enzymatically digest them to produce a smaller peptide with the sulfur mustard adduct still attached. Methods of this type have been developed for both hemoglobin and albumin. A third approach has been to cleave the sulfur mustard adduct from the macromolecule and analyze in a fashion similar to that used for free metabolites found in the urine. The later two approaches have both been successfully used to verify human exposure of sulfur mustard. 

Korte WD, Walker EM, Smith JR, et al. The determination of sulfur mustard exposure by analysis of blood protein adducts. Wehrmed. Mschr, 2005 49 327. 

Immunoassay is an application of the substoichiometric principle ( 9.3.4) developed by Yalow (Nobel laureate in 1977) for protein analysis. In the United States tens of millions radioimmunoassays are made annually in hospitals to measure hormones, enzymes, viruses, serum proteins, drugs, and so forth. Only a drop of the patient s blood is needed, reflecting the versatility and smisitivity of this technique, which can be performed automatically. Commercial RAST-kits (Radio Allergy Sorbent Tests) are used for rapid diagnosis of allergic reactions. 

Protein Glucose Ketones Bilirubin Occult blood Microscopic analysis... 

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