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Oxidized Low Density Lipoprotein Overview
It has been thought for many years that oxidized-LDL may play an important part in the atherogenic process in vivo (7,8,9,10). Ox-LDL generated by a variety of means, including copper mediated oxidation, has been used in hundreds of published studies. The biological effects of oxidized LDLs on macrophages, smooth muscle cells, and endothelial cells have been extensively studied in many different laboratories around the world. The chemical composition of the Ox-LDL has been demonstrated to vary with the degree of oxidation. Oxidized lipids, such as oxidized phospholipids and oxysterols, and oxidized protein adducts increase with increasing time and degree of oxidation of LDL (4,11,14). Each of these classes of chemical components can have a multitude of different effects on each of the cell types in the artery wall. Most investigators interested in the biological effects of oxidized LDL such as gene regulation to tend to use regularly oxidized LDL (as measured by time of oxidation/TBARs). Their needs are met by our product BT-910 which is routinely available. Investigators interested in cytotoxic effects may find our BT-910X, which has a higher TBARs level more suitable. Investigators interested in certain biological effects of Ox-LDL may find a very lightly oxidized LDL (as measured by time of oxidation) more suitable. BTI will provide product BT-910 Low TBAR (oxidized for 2 hrs) for these investigators on request. Investigators will need to determine empirically which form and concentration of Ox-LDL works best for their combination of cell system and the cell pathway/marker being investigated.
Ascribing specific biological effects of Ox-LDL to defined chemical components of Ox-LDL has been accomplished in a number of laboratories. Up-regulation of IL-1 alpha in rabbit arterial derived foam cells was associated with phospholipid oxidation product 13-HODE if presented in a LDL vehicle and also with the oxidized protein components of copper oxidized LDL (5). Oxidized phospholipid adducts have been demonstrated to be associated with the lysine groups of ApoB (11) and induce biological effects in vascular cells (5). Both 9-HODE and cholesteryl ester 9-HODE produced during LDL oxidation can induce IL-1 beta from human monocyte derived macrophages (3,13)). Oxidized phospholipid HOOA-PC increases production of both IL-8 and MCP-1 from endothelial cells (12). The unsaturated aldehydes 2,4 decadienal, and 2-octenal formed as advanced breakdown products of lipid peroxidation also induce IL-1 beta in human monocyte derived macrophages (13). Oxysterols 7-ketocholesterol and 7-hydroxycholesterol which are formed during LDL oxidation have been shown to be responsible for Ox-LDL cytotoxicity on porcine aortic smooth muscle cells (2). In addition, regulation of the nuclear signaling pathway via PPAR gamma ligands and activators has been demonstrated using an oxidatively fragmented alkyl phospholipid (azPC), and 9-HODE, 13-HODE from Ox-LDL (1,6). These are but a few examples of the fascinating biological effects associated with oxidized LDL. Undoubtedly there will be more biological effects ascribed to oxidized LDL and its components as additional pathways in the cells comprising the vascular wall are studied.
References:
1. Davies SS, Pontsler AV, Marathe, GK, Harrison, KA, Murphy RC, Hinshaw JC, Prestwich GD, St. Hilaire A ,Prescott SM, Zimmerman G, and McIntyre TM. Oxidized alkyl phospholipids are specific high affinity peroxisome proliferator-activated receptor gamma ligands and activators. J Biol Chem 2001; 276:16015-16023.
2. Hughes H, Mathews B, Lenz ML, and Guyton JR. Cytotoxicity of oxidized LDL to porcine aortic smooth muscle cells is associated with oxysterols 7-ketocholesterol and 7-hydroxycholesterol. Atheroscler Thromb Vasc Biol 1994; 14: 1177-1185.
3. Ku G, Thomas, CE, Akeson, AL, and Jackson, RL. Induction of interleukin-1 beta from human peripheral blood -derived macrophages by 9-hydroxyoctadecadienoic acid. J Biol. Chem. 1992; 267: 14183-14188.
4. Lenz ML, Hughes H, Mitchell JR, Via DP, Guyton JR, Taylor AA, Gotto, AM Jr, and Smith CV. Lipid hydroperoxy and hydroxyl derivatives in copper-catalyzed oxidation of low density lipoprotein. J lipid Research 1990; 1043-1050.
5. Lipton BA, Parsanathy, S., Ord VA, Clinton, SK, Libby P, and MR Rosenfeld. Components of the protein fraction of oxidized LDL stimulate interleukin-1 alpha production by rabbit arterial macrophage derived foam cells. J lipid Res 1996; 36:2232-2242.
6. Nagy L, Tontonoz P, Alvarez JG, Chen H, and Evans, RM. Oxidized LDL regulates macrophage gene expression through ligand activation of PPARgamma. Cell 1998; 17:229-240.
7. Parthsarathy S, Steinberg D, and Witzum JL. Role of oxidized low density lipoproteins in the pathogenesis of atherosclerosis. Annu. Rev. Med 1992; 43:219-225.
8. Parthasarathy S, Litvinov D, Selvarajan K, and Garelnabi M. Lipid Peroxidation and decomposition-conflicting roles in plaque vulnerability and stability. Biochem. Biophys. Acta 2008: 1781:221-223.
9. Ravandi A, Babaei S, Monge JC, Hoppe G, Hoff H, Kamido H, and Kukiss A. Phospholipids and oxophospholipids in atherosclerotic plaques at different stages of plaque development. Lipids 2004; 39:97-109.
10. Steinberg D, Parthasarathy S, Carew TE, Witzum JL. Beyond cholesterol: modification of low-density lipoproteins that increase its atherogenicity. N Engl J Med 1989; 320:915-924.
11. Steinbrecher, UP. Oxidation of human low density lipoprotein results in derivatization of lysine residues of Apoliprotein B by lipid peroxide decomposition products. J Biol Chem 1987; 262:3603-3608.
12. Subbabagounder G, Deng Y, Borromeo C, Dooley AN, Berliner JA, and Solomon RG. Hydroxy alkenal phospholipids regulate inflammatory functions of endothelial cells. Vascul Pharmacol 2002; 38:201-209.
13. Thomas CE, Jackson RL, Ohweiler DF, and Ku, G. Multiple lipid oxidation products in low density lipoproteins induce interleukin-1 beta release from human blood mononuclear cells. J Lipid Res 1994; 35: 417-427.
14. Wang T, Yu W-G, Powell, WS. Formation of monohydroxy derivatives of arachidonic acid, linoleic acid, and oleic acid during oxidation of low density lipoprotein by copper ions and endothelial cells. J Lipid Res. 1992; 33: 525-537.
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BTI Carbamylated LDL, c-LDL
Studies in humans have shown a link between elevated c-LDL levels and increased risk of Atherosclerosis. LDL and other proteins are modified by urea-derived cyanate normally present in human blood. Patients with kidney disease have elevated levels of c-LDL and have an increased risk of atherosclerosis. Carbamylated LDL induces endothelial cell death and makes way for the proliferation of smooth muscle cells.
BTI Carbamylated Human LDL is made with Potassium Cyanate. Each lot is analyzed for the degree of carbamylation (via diacetylmonoxime assay and the e-carbamyl-Lysine present versus native LDL in nanomoles/mg protein) and the c-LDL migration versus the native LDL on agarose gel electrophoresis. The product is membrane filtered, aseptically packaged and endotoxin tested. Both [I125]cLDL and DiI-cLDL can be prepared if needed.
Carbamylated LDL, c-LDL
Catalog No. BT-924
Quantity: 1mg
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Lipoprotein Deficient Serum
BTI Lipoprotein Deficient Serum, LPDS, is prepared by ultracentrifugation, all of the lipoproteins are removed and it is extensively dialyzed. The resultant LPDS is membrane filtered and packaged aseptically. Each lot is evaluated in Human Skin Fibroblast or other cells in culture with our LDL and [I125] LDL (or DiI-LDL and DiO-LDL) to determine the degree of up regulation of LDL receptors. Typical lots give a 3 to 4 fold increase in [I125] LDL binding. We do not dilute the LPDS, typical protein concentration for the bovine LPDS is 75 -120 mg/ml. and for the human LPDS is ~50mg/ml.
Bovine LPDS Catalog No: BT-907 * Quantity: On Request
Human LPDS Catalog No: BT-931* Quantity: On Request
Reference
1. Grove, R.I. et al. J. Lipid. Res. 32: 1889-1897 (1991).
DiI-HDL
Catalog No: BT-915
On Request
DiO-LDL, Cell Marker
Catalog No: BT-916
Quantity: 200ug 5x200ug
Human Lipoprotein [a], Lp [a]
Catalog No: BT-917
Quantity: 100ug
anti-Human Apo [a] (LDL Adsorbed)
Catalog No: BT-918
Quantity: 1ml
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DiI-Ac-LDL & LDL Reagents
- Labels Endothelial Cells & Macrophages
- DiI Labeled Cells can be Trypsinized
- Does Not Label Matrix Associated Proteins
- DiI Labeled Cells Remain Viable
- Directly Applicable to Cell Sorting & Fluorescence Microscopy
Introduction
DiI-Ac-LDL, Acetylated Low Density Lipoprotein, labeled with 1,1'-dioctadecyl – 3,3,3',3'-tetramethyl-indocarbocyanine perchlorate, labels both vascular endothelial cells and macrophages. It can be used to identify and/or isolate these cells from mixed cell populations. When cells are labeled with DiI-Ac-LDL, the lipoprotein is degraded by lysosomal enzymes and the DiI (fluorescent probe) accumulates in the intracellular membranes. Labeling cells with DiI-Ac-LDL has no effect on cell viability. Pure cultures of vascular endothelial cells can be isolated from complex primary cultures using fluorescent activated cell sorting based on their increased metabolism of the DiI-Ac-LDL. Contaminating cell types (fibroblasts, smooth muscle, pericytes, epithelial cells) are not labeled. Macrophages can be differentiated from mixed cell populations (including endothelial cells) because they are more brightly labeled.
Labeling endothelial cells with DiI-Ac-LDL has many advantages over labeling other endothelial cell associated antigens. The labeling procedure is one step, and once the cells are labeled, the fluorescent probe (DiI) is not removed by Trypsin. Both low density and confluent cultures of vascular endothelial cells are effectively labeled. No other cell type (other than macrophages) is labeled to the same level as vascular endothelial cells. Each lot of DiI-Ac-LDL is evaluated for the specific labeling of bovine aortic endothelial cells and murine macrophages to assure consistent results. A complete labeling protocol is included with each shipment. We also offer an "FITC-like" label DiO-Ac-LDL, which is useful for fixed wavelength FACS Cell sorters.
Procedural Outline
1. Dilute DiI-Ac-LDL to 10ug/ml in complete growth media.
2. Add to cells and incubate for 4 hours at 37ºC.
3. Remove media.
4. Wash with probe-free media.
5. Visualize via Fluorescence Microscopy and/or trypsinize (or EDTA) for cell sorting.
References
1. Goldstein, J.L., Y.K., Ho. S.K. Basu, and M.S. Brown. 1979. Binding site on macrophages that mediates uptake degradation of actylated low density lipoprotein, producing massive cholesterol deposition. Proc. Nat. Acad Sci. USA 76: 335-337.
2. Folgelman, A.M., I. Schechter, J. Seager, M. Hokum, J.S. Child and P.A. Edwards. 1980. Malondialdehyde alteration of low density lipoproteins leads to cholesterylester accumulation in human monocyte-macrophages. Proc. Nat. Acad. Sci 77: 2214-2218.
3. Stein, O. and Y. Stein 1980. Bovine aortic endothelial cells display macrophage-like properties towards acetylated [I125]-labeled low density lipoprotein. Biochem. Biophys. Acta 620: 631-635.
4. Pitas, R.E., T.L. Innerarity, J.N. Weinstein, and R. W. Mahley. 1981. Acetoacetylated lipoproteins used to distinguish fibroblasts from macrophages in vitro by fluoresence microscopy. Arteriosclerosis. 1: 177-185.
5. Voyta, J.C., D.P. Via, C.E. Butterfield and B.R. Zetter. 1984. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J. Cell Biology. 99: 2034-2040.
6. Giulian, D. and D. G. Young. 1986. Brain peptides and glial growth. II. Identification of cells that secrete glia-promoting factors. J. Cell Biology. 102: 812-820.
7. Netland, P.A., B.R. Zetter, D.P. Via and J. C. Voyta. 1985. Insitu labeling of vascular endothelium with fluorescent acetylated low density lipoprotein. Histochemical Journal. 17: 1309-1320.
8. Pitas, R.E., J. Boyles, R. W. Mahley and D.M. Bissell. 1985. Uptake of chemically modified low density lipoproteins in-vivo is mediated by specific endothelial cells. J. Cell Biology. 100: 103-117.
9. Giulian, D., and T. J. Baker 1986. Characterization of ameboid microglia isolated from developing mamalian brain. J. Neuroscience. 6: 2163-2178.
10. Bjorling, D.E., R. Saban, M.W. Tengowski, S.M. Gruel and V.K. Rao. 1992. Removal of Venous Endothelium with Air. J. Pharm. & Tox. Methods. 28: 149-157.
11. Lysco, P.G., J. Weinstock, Chris. T. Webb, M.E. Brawner and N.A. Elshourbagy. 1999. Identification of a Small-Molecule, Nonpeptide Macrophage Scavenger Receptor Antagonist. J. of Pharm. and Experimental Therapeutics. 289 No.3: 1277-1285.
12. Murphy, H.S., R.L.Warner, N. Bakopoulos, M.K. Dame, J. Varani and P.A. Ward. 1999. Endothelial Cell Determinants of Susceptibility to Neutrophil-Mediated Killing. Shock 12:111-117.
13. Nugent, M. A. et al. 2000. Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia. PNAS. 97 No.12: 6722-6727.
Catalog Information
DiI-Ac-LDL
Catalog No: BT-902
Quantity: 200ug
(Including labeling protocol)
Refrigerate at 4ºC. Do Not Freeze.
DiO-Ac-LDL
Catalog No: BT-925
Quantity: 200ug
(Including labeling protocol)
Refrigerate at 4ºC. Do Not Freeze.
FOR RESEARCH USE ONLY. NOT FOR USE IN HUMANS OR AS AN IN-VITRO DIAGNOSTIC.
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Human LDL & Ac-LDL
BTI Human Low Density Lipoprotein, LDL, is isolated from blood bank produced human plasma. It is purified via ultracentrifugation to homogeneity determined by agarose gel electrophoresis. Each lot of BTI LDL is evaluated for receptor binding to human skin fibroblasts in conjunction with our [I125] LDL or DiI-LDL.
Acetylated Human Low Density Lipoprotein, Ac-LDL, is also available. The BTI Ac-LDL is purified to homogeneity as demonstrated on agarose gel electrophoresis. Each lot of BTI Ac-LDL is evaluated for receptor binding to murine peritoneal macrophages in conjunction with our [I125] Ac-LDL or DiI-Ac-LDL.
LDL, Human
Catalog No: BT-903
Quantity: 5mg
Ac-LDL, Human
Catalog No: BT-906
Quantity: 2mg
[I125] Human Ac-LDL
Human Ac-LDL (99% Pure, AGE) is radioiodinated by the Iodine Monochloride Method. The resulting [I125] Ac-LDL is purified via column chromatography. Each lot is evaulated for binding to murine peritoneal macrophages.
[I125] Human Ac-LDL
Specific Activity: 100-300 cpm/ng Protein
Catalog No: BT-912R
Quantity: 300uCi
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[I125] Human LDL
Human LDL (99% Pure, AGE) is radioiodinated by the Iodine Monochloride Method. The resulting [I125] LDL is purified via column chromatography. Each lot of BTI [I125] LDL is evaluated for binding to human skin fibroblasts.
[I125] Human LDL
Specific Activity: 100-300 cpm/ng Protein
Catalog No: BT-913R
Quantity: 300uCi
DiI-LDL, Cell Marker
DiI-LDL, Low Density Lipoprotein, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine
perchlorate is used to visualize LDL binding sites in cultured cells or to screen for cell mutants defective in the
expression of LDL receptors. It may also be useful in evaluating receptor levels by fluorescent activated cell sorting,
and examining tissue levels of receptor in vivo. DiI-LDL is an excellent marker of cellular endocytosis.
A complete protocol is included with each shipment.
DiI-LDL, Cell Marker
Catalog No: BT-904
Quantity: 200ug
DiO-LDL(FITC-like)
Catalog No: BT-916
Quantity: On Request
References
1. Pitas R.E., et al. Arteriosclerosis 1: 177 (1983).
2. Pitas R.E., et al. Arteriosclerosis 3: 1 (1983).
3. Barak L.S., Webb WW. J. Cell Biology 90: 595 (1981).
4. Barak L.S., Webb WW. J. Cell Biology 95: 846 (1982).
5. Kingsley D.M., Kreiger M. PNAS, USA, 81: 454 (1984).
6. Voyta J.C., et al. J. Cell Biology 99: 2034 (1984).
7. Kreiger, M., et al. J. Receptor Res. 3: 361 (1983).
8. Pitas RE., et al. J. Cell Biology 100: 103 (1985).
9. Herman B., Albertini D.F., J. Cell Biology 98: 565 (1984).
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Other Proteins
Calmodulin
BTI Calmodulin is purified to >99.5% as demonstrated by silver stained, two dimensional polyacrylamide gel electrophoresis. The biological activity is 48,000 units/mg (one unit of Calmodulin gives a 50% activation of 0.14 units of bovine retina adenylate cyclase where one unit of adenylate cyclase is equivalent to the production of one nanomole of Cyclic AMP per minute per milligram of protein at 37ºC). This product is suitable for radioiodination and as a standard in biological and immunohistochemical experiments.
Catalog No: BT-372
Quantity: 1mg
References
1. Hamaguchi Y., Iwasa F., Biomedical Research. 1: 502-509 (1980).
2. Pardue R.L., et al, Cell. 23: 533-542 (1981).
3. Zavortink M., et al, Exp Cell Res. 149: 375-385 (1983).
4. Luby, Phelps K., et al, JCB. 101: 1245-1256 (1985).
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Tamm-Horsfall Glycoprotein
Tamm-Horsfall Glycoprotein, Uromodulin, is the most abundant protein present in normal human urine. Recent studies have implicated THG as a unique regulatory glycoprotein limiting the circulating activity of a number of potent cytokines. BTI THG is purified from normal human urine via precipitations with sodium chloride and then by Sepharose gel chromatography. The protein shows a single homogeneous band on 5-18% PAGE run under reducing and non-reducing conditions. BTI THG is packaged in aqueous solution with a stabilizer.
Catalog No: BT-381
Quantity: 100ug
anti-Tamm-Horsfall Glycoprotein
BTI anti-Human Tamm-Horsfall Glycoprotein is prepared in rabbits using chromatographically pure THG. No significant cross-reactivity with urinary or other related proteins was detected. This antibody is suitable for Elisa assays (1:150,000 against solid phase antigen) and localization studies on cells and tissues (1:100 to 1:250).
Catalog No: BT-590
Quantity: 0.5ml
References
1. I. Tamm and F.L. Horsfall, Proc. Soc. Exp. Biol. Med. 74: 108-114 (1950).
2. J. R. Hoyer, and M.W. Seiler, Kidney Int. 16: 279-289 (1979).
3. Janssens, P.M.J. et al. Clinical Chemistry 38: 216-222 (1992).