MONMOUTH JUNCTION, NJ.– Authors RA Hauser-Davis and team cites Cleanascite™ as an ideal lipid clarification reagent during sample preparation of fish bile containing matrix metalloproteinases (MMPs). The paper titled, “First-time report of metalloproteinases in fish bile and their potential as bioindicators regarding environmental contamination” is published in the journal Aquatic Toxicology.2012 Apr; 110-111:99-106. Scientists identified matrix metalloproteinases in the bile of mullets (Mugil liza) and tilapias (Tilapia rendalli) which required clarification and purification studies prior to performing SDS-PAGE and zymography analysis. Lipid removal was performed using the delipidizer Cleanascite™, which is a non-ionic adsorbent, used to precipitate lipid fat droplets, cell debris and mucinous impurities. It is ideal for clarifying ascites, serum, cell & tissue culture, bile and organ homogenates. 

MMPs are protease enzymes which require a metal (zinc or cobalt) for catalytic activity. MMPs function in wound repair, tissue development and embryogenesis by destroying extracellular matrix of normal physiological processes. Examples of MMPs are gelatinases, collagenases, stromelysins and similar proteinases. Published papers by authors Shapiro have shown the connection between overexpression of a specific MMP, either by tumor cells or the surrounding stroma, as being pro-tumorigenic. Moreover MMPs are recruited by tumor cells for reconstructing normal tissue and promote neovascularization, tumor spread and growth. Synthetic MMP inhibitors are being developed for slowing tumor growth and for anti-neoplastic therapy.

Gallbladder bile from 2 fish species, mullet (Mugil liza) and tilapias (Tilapia rendalli), contained substantial matrix metalloproteinases (MMPs). Extensive purification studies were conducted in order to obtain workable samples for SDS-PAGE and zymography analysis. Cleanascite™ is a solid-phase, non-ionic adsorbent supplied as a suspension in saline, ready for use. Simply add, centrifuge and/or filter. The clarified supernatant is ready for subsequent downstream processing or analysis.Cleanascite™ does not bind to DNA, RNA, enzymes, proteins and leaves glycoproteins, antibodies, nucleic acids, hemoglobin, proteoglycans, nucleic acids, serum components(such as hormones, nutrients, globulins, clotting factors, transport proteins) alone. Delipidizer treatments are known to extends the life of membrane and chromatographic columns and allows for downstream processing of large-scale therapeutic proteins, enzymes and monclonal antibodies. In this study scientists also conducted numerous inhibitor studies in which MMPS were inhibited. Differences in proteinase expression were observed in fish from a contaminated and reference site. Metalloproteinases in fish bile are routinely analyzed by mass spectrometry approaches such as inductively coupled plasma MS (ICP-MS), electrospray MS (ESI-MS), and matrix-assisted laser desorption ionization MS (MALDI-MS) alongside prior 2D-PAGE protein separation to rapidly and precisely identify gene products, metabolites, or proteins.

Fish is ideal for assessing water-borne or sediment-deposited toxins, provides knowledge of environmental contamination due to toxicants and therefore comparing fish proteomes to analyze proteome expression under complex field situations could provide knowledge of gene products, metabolites, or proteins for further studies. (Albertsson et al. 2007). Metalloproteins in fish could be used as biomarkers of environmental exposure for fish and have utility as biomarkers for environmental contamination (López-Barea and Gómez-Ariza 2006 ).

Quantifying total metal levels in fish organs, and directly relating them to environmental contamination provides information about toxic exposure levels (Moldovan et al. 2004).Total metal levels in fish are influenced by the amount of metals present in the form of metal-bound proteins and/or on-site contamination.

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Cleanascite™ References: Bile

Hauser-Davis RA, Lima AA, Ziolli RL, Campos RC.First-time report of metalloproteinases in fish bile and their potential as bioindicators regarding environmental contamination. Aquatic Toxicology.2012;110-111:99-106

Farina A, Dumonceau JM, Frossard JL. Proteomic Analysis of Human Bile from Malignant Biliary Stenosis Induced by Pancreatic Cancer Journal of Proteome Research.2009; 8(1):159-69

Guerrier L, Claverol S, Finzi L et al. Contribution of solid-phase hexapeptide ligand libraries to the repertoire of human bile proteins. Journal of Chromatography.2007;1176(1-2):192-205

Chen Bo, Zheng Jian-wei, Wang Jian-ming, et al. Establishment and preliminary analysis of a 2-D human biliary map Chinese Journal of Hepatobiliary Surgery.2007

Chen B, Dong JQ, Chen YJ et al Two-dimensional electrophoresis for comparative proteomic analysis of human bile.
Hepatobiliary & pancreatic diseases international.2007 Aug;6(4):402-6

Guerrier L, Claverol S, Finzi L et al Contribution of solid-phase hexapeptide ligand libraries to the repertoire of human bile proteins.Journal of Chromatography A.2007;1176(1-2):192-205

Kristiansen TZ, Bunkenborg J, Gronborg M et al A Proteomic Analysis of Human Bile Molecular and Cellular Proteomics.2004;3:715-728

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Suggested References

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Bode W, Maskos K (2003) Structural basis of the matrix metalloproteinases and their physiological inhibitors, the tissue inhibitors of metalloproteinases. Biol Chem 384:863–872

Dowling VA, Sheehan D (2006) Proteomics as a route to identification of toxicity targets in environmental toxicology. Proteomics 6:5597–5604

Gonzalez-Fernandez M, Garcia-Barrera T, Jurado J, Prieto-Alamo MJ, Pueyo C, Lopez-Barea J,Gomez-Ariza JL (2008) Integrated application of transcriptomics, proteomics, and metallomics in environmental studies. Pure Appl Chem 80:2609–2626

Jellum E, Thorsrud AK, Karasek FW (1983) Two-dimensional electrophoresis for determining toxicity of environmental substances. Anal Chem 55:2340–2344

López-Barea J, Gómez-Ariza JL (2006) Environmental proteomics and metallomics. Proteomics 6:S51–S62

Tay TL, Lin QS, Seow TK, Tan KH, Hew CL, Gong ZY (2006) Proteomic analysis of protein profi les during early development of the zebrafi sh, Danio rerio. Proteomics 6:3176–3188

Shapiro SD. Diverse roles of macrophage matrix metalloproteinases in tissue destruction and tumor growth. Thromb Haemost. 1999 Aug;82(2):846-9

Wang JS, Wei YH, Wang DZ, Chan LL, Dai JY (2008) Proteomic study of the effects of complex environmental stresses in the livers of goldfi sh (Carassius auratus) that inhabit Gaobeidian Lake in Beijing, China. Ecotoxicology 17:213–220