Human matrices

Abstract We have reviewed the human exposure to selected emerging organic contaminants, such new brominated flame retardants, organophosphate flame retardants, phthalate substitutes, triclosan, synthetic musks, bisphenol-A, perchlorate, and polycyclic siloxanes. Levels of these emerging contaminants in matrices relevant for human exposure (air, dust, food, water, etc.) and in human matrices (blood, urine, or tissues) have been reviewed, together with some of the relevant health effects reported recently. 

Table 2 Overview of HBCDs, TBBPA, and NBFRs levels in various human matrices...

As a compound proceeds into first-in-human clinical trials, the assay method reaches an apex in terms of performance. These methods require maximal sensitivity to be able to support dose escalation studies. The selectivity of the method is well established versus matrix components, concomitant medications, and metabolites. The assay is revalidated in the human matrices (plasma, serum, and urine) and will again meet well-established guidelines for inter- and intraday precision and accuracy. If significant changes are made to the method, a comparison of methods study will be conducted to understand the relative accuracy of the methods. After this benchmark, assay requirements, especially limit of quantitation, are usually less demanding and use of the assay becomes more routine, as it is applied again and again to additional clinical studies for pharmacokinetic support. 

The errors associated with the steps identified in Section 12.13.5.1 have been discussed and the approaches used to establish and combat them will now be summarized. For the extraction step, QA considerations mean the extraction efficiency needs to be validated and this can be done either by spike recovery experiments or by using a representative CRM. The main criticism of spike recovery experiments is that the spike is not bound in the sample matrix in the same way as the naturally occurring analyte being measured. However, if a low recovery is apparent using the method chosen, it would indicate an inadequate protocol or one that requires further developmental work. The range of CRM available with values for some of the more important chemical species is increasing, and includes various sediments, fish and shellfish tissue, and human matrices such as hair and urine. However, real samples are rarely identical to the matrix CRM available, so care should be taken when validating methods to be used for real samples. 

Namciu SJ, Blochhnger KB, Eoumier RE (1998) Human matrix attachment regions insulate transgene expression from chromosomal position effects in Drosophila melanogaster. Mol Cell Biol 18 2382-2391... 

Chapter 2 describes several repositories from completed or current projects in Europe and the United States. The human-matrix repositories vary widely and include commercial banks, national or international project banks, and much smaller biorepositories associated with scientific laboratories or universities. The latter may have 100,000 samples or fewer larger biorepositories, such as CDC or the National Cancer Institute, have many millions of samples (Gunter 1997 Goodman et al. 2006). Each sample needs to have a secure chain of custody, processing, location, and temperature-stability records compiled in an accessible location. Nanobarcoding and partial to complete automation of a biorepository help to address these issues (Holland et al 2005). 

Briknarova, K., Gehrmann, M., Banyai, L., Tordai, H., Patthy, L., and Llinas, M. (2001). Gelatin-binding region of human matrix metalloproteinase-2 Solution structure, dynamics, and function of the COL-23 two-domain construct. J. Biol. Chem. 276, 27613-27621. 

P. Baridc, S. Raha, J. A. Calvo, S. Balaz, Similarity of Binding Sites of Human Matrix Metalloproteinases, /. Biol. 

Hackeng, T. M., Rosing, J., Spronk, H. M. H., and Vermeer, C. (2001) Total chemical synthesis of human matrix Gla protein. Protein Science 10, 864-870. 

Range from bacterial digestive proteins to human matrix metalloproteinases and ecto sheddses. 

Table 3. Alignment of the sequences of the catalytic domain of selected human matrix metallo proteases (Matrixins). The sequences have been colored according to the secondary structure, with the zinc-chelating residues shown in white and the catalytic glutamic acid shown in red. Yellow residues identify amino acids discussed in the text.

Mutations in the gene encoding the human matrix Gla protein cause Keutel syndrome. Nat Genet 21 142-144... 

Stability Complete sample analysis ASAP due to lack of stability data Determine short and long-term stability for critical reagents and QCs in matrix Determine short and long-term stability for critical reagents and QCs in human matrix... 

F7. Fujimoto, N., Mouri, N., Iwata, K., Ohuchi, E., Okada, Y., and Hayakawa, T., A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) using monoclonal antibodies. Clin. Chim. Acta 221, 91-103 (1993). 

Netzel-Amett, S., Mallya, S.K., Nagase, H., Birkedal-Hansen, H., Van Wart, H.E. Continuously recording fluorescent assays optimized for five human matrix metalloproteinases. Analytical Biochemistry 195, 86-92 (1991)... 

Matrix Effect and Recovery For LC-MS/ MS-based methods, the signal suppression or enhancement of the analyte due to the presence of the matrix interferences (matrix effects) in MS/MS detection should be evaluated by comparing the response (peak area) of the analyte and the IS from the extracted blank samples post-fortified with the analyte and the IS with the response of neat solutions with both the analyte and the IS at the same concentrations as above. Matrix effects should be evaluated in one pooled batch of animal matrix or in at least three different batches of human matrix, using three replicates at a minimum of three QC concentrations (e.g., low quality control [LQC], medium quality control [MQC], high quality control [HQC]) with IS at working concentration. The coefficient of variation (CV%) of the matrix effect variability should be <15% at each concentration level and between the three (LQC, MQC, and HQC) concentration levels. 

The recovery of an analyte and the associated IS in an LC-MS/MS assay is the percentage (%) of the LC-MS/ MS response (peak area) obtained from the extracted QC samples (LQC, MQC, and HQC) prepared from one pooled batch of animal matrix or from at least three different batches of human matrix in at least three replicates for each concentration level compared with those... 

Human matrix metalloproteinases may be processed from their proenzyme forms to their active forms by two new and unique mechanisms (Maeda et al. 1998) Firstly, by bacterial proteases such as Pseudomonas elastase and Vibrio cholerae protease, which cleave off the N-terminal autoinhibitory domain (so-called cysteine switch) from pro-matrix metalloproteinases. The second mechanism depends on free radical generation by activated polymorphonuclear leucocytes. In this case, peroxyni-trite (ONOO") or nitrogen dioxide radical ("NOj) are the key reagents. 

Joseph J, Ranganathan S, Mehta JL. Low density lipoproteins modulate collagen metabolism in fibroblasts. J Cardiovasc Pharmacol Ther 2003 8 161-166. Lemaitre V, O Byme TK, Borczuk AC, et al. ApoE knockout mice expressing human matrix metalloproteinase-1 in macrophages have less advanced atherosclerosis. J Clin Invest 2001 107 1227-1234. 

Traditionally, least-squares methods have been used to refine protein crystal structures. In this method, a set of simultaneous equations is set up whose solutions correspond to a minimum of the R factor with respect to each of the atomic coordinates. Least-squares refinement requires an N x N matrix to be inverted, where N is the number of parameters. It is usually necessary to examine an evolving model visually every few cycles of the refinement to check that the structure looks reasonable. During visual examination it may be necessary to alter a model to give a better fit to the electron density and prevent the refinement falling into an incorrect local minimum. X-ray refinement is time consuming, requires substantial human involvement and is a skill which usually takes several years to acquire. 

Applicability/Limitations. In-situ treatment can be used when it is uneconomical to haul or when infeasible or uneconomical to dig or pump the contaminated waste matrix for treatment in a reactor. This approach should be used whenever excavation or removal causes an increased threat to human health. It can reduce the cost of a remediation program. Because chemicals are applied to the contaminated waste matrix, specifically soil and groundwater, a potential exists for reaction with the soil. Permeability problems can occur as the result of precipitate formation. This can result in inadequate mixing of the contaminant with the treatment chemical. Gas generation may also occur. 

A critical assembly is a split bed on which fissionable material used to mock up up a separated reactor core that is stacked half on each half. One half is on roller guides so that the two halves may be quickly pulled apart if the neutron multiplication gets too high. Use the Preliminary Hazards Analysis method described in section 3,2.1 to identify the possible accidents that may occur and the qualitative probabilities and consequences. List the initiators in a matrix to systematically investigate the whole process. Don t forget human error. 

List the initiators in a matrix to systematically investigate the whole process. Don t forget human error. 

FIGURE 12.31 A model for chromosome structure, human chromosome 4. The 2-um DNA helix is wound twice around histone octamers to form 10-um uucleosomes, each of which contains 160 bp (80 per turn). These uucleosomes are then wound in solenoid fashion with six uucleosomes per turn to form a 30-nm filament. In this model, the 30-nm filament forms long DNA loops, each containing about 60,000 bp, which are attached at their base to the nuclear matrix. Eighteen of these loops are then wound radially around the circumference of a single turn to form a miniband unit of a chromosome. Approximately 10 of these minibands occur in each chromatid of human chromosome 4 at mitosis. 

COMPARTMENTALIZED PYRUVATE CARBOXYLASE DEPENDS ON METABOLITE CONVERSION AND TRANSPORT The second interesting feature of pyruvate carboxylase is that it is found only in the matrix of the mitochondria. By contrast, the next enzyme in the gluconeogenic pathway, PEP carboxykinase, may be localized in the cytosol or in the mitochondria or both. For example, rabbit liver PEP carboxykinase is predominantly mitochondrial, whereas the rat liver enzyme is strictly cytosolic. In human liver, PEP carboxykinase is found both in the cytosol and in the mitochondria. Pyruvate is transported into the mitochondrial matrix, where it can be converted to acetyl-CoA (for use in the TCA cycle) and then to citrate (for fatty acid synthesis see Figure 25.1). /Uternatively, it may be converted directly to 0/ A by pyruvate carboxylase and used in glu-... 

---