| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| Other Sizes |
| Targets |
18:1 BMP (S,R) targets the endolysosomal cholesterol trafficking pathway. BMP binds directly to the lysosomal protein NPC2, with a Kd in the low micromolar range. This interaction facilitates the transfer of cholesterol from NPC2 to the lysosomal membrane, allowing its export to other cellular compartments. Loss of BMP (or its stereoisomer configuration) leads to cholesterol accumulation in lysosomes, similar to NPC disease. BMP also interacts with other proteins: it binds to and activates the acid sphingomyelinase (ASM) enzyme and is required for the degradation of sphingomyelin and gangliosides. BMP is also a ligand for the anti-phospholipid antibodies (aPL) in autoimmune diseases. In addition, BMP is a structural component of the lipid bilayer in late endosomes/lysosomes, promoting the formation of intraluminal vesicles. In research, the compound is used to study these interactions. It serves as a substrate for enzymes (e.g., group XV phospholipase A2, PLA2G15) and as a standard for lipidomics. The target is not a single receptor; BMP is a lipid that modulates protein function through direct binding.
|
|---|---|
| ln Vitro |
In vitro, 18:1 BMP (S,R) is used in protein-binding assays and as a standard for mass spectrometry. In a binding assay with recombinant NPC2 (0.5 ug), addition of BMP (1-50 uM) leads to a dose-dependent increase in NPC2 thermal stability (DSF, deltaTm up to 4degC), indicating direct interaction. In a fluorescent cholesterol transfer assay, NPC2 (100 nM) and NBD-cholesterol (0.5 uM) are incubated with liposomes containing 10 mol% BMP (S,R) (0.5 mM total lipid). The transfer of NBD-cholesterol to liposomes is measured by fluorescence increase (Ex 470 nm, Em 530 nm) upon dilution of the self-quenched probe. BMP accelerates cholesterol transfer by 3-5-fold. In enzyme assays, BMP (1-10 mol% in liposomes) activates recombinant human acid sphingomyelinase (ASM, 0.1 U) by 2-3-fold, as measured by the conversion of [3H]-sphingomyelin to [3H]-phosphorylcholine. In cell-based assays, addition of 18:1 BMP (S,R) (10-100 uM, complexed with cyclodextrin or liposomes) to NPC1-deficient fibroblasts (NPC patient cells) partially restores cholesterol export from lysosomes, as visualized by filipin staining (reduced perinuclear fluorescence). In viability assays, BMP (up to 100 uM) is non-toxic to fibroblasts (MTT viability >90%). In lipidomics, BMP is quantified by LC-MS/MS as a biomarker for NPC disease; levels of BMP (especially 18:1) are elevated in NPC patient fibroblasts. In enzymology studies, BMP is a substrate for PLA2G15 (lysosomal phospholipase A2), which cleaves one fatty acyl chain to produce lyso-BMP and free fatty acid. The enzyme activity is measured by LC-MS detection of the product.
|
| ln Vivo |
In vivo, 18:1 BMP (S,R) is not administered therapeutically. It is an endogenous lipid present in all cells, particularly in late endosomes/lysosomes. In NPC1-deficient mice (Npc1-/- mice) and NPC patients, BMP levels are elevated by 5-10× in liver, spleen, and brain due to lysosomal storage. The compound is used as a biomarker to monitor disease progression and response to therapy. In preclinical studies, treatment of Npc1-/- mice with HPbetaCD (4000 mg/kg, SC, weekly) reduces BMP levels in the liver by 50-60% after 8 weeks, correlating with reduced cholesterol storage. For biodistribution studies, deuterated 18:1 BMP (d5-18:1) can be injected intravenously into mice (1 mg/kg). The lipid is rapidly taken up by the liver (within 15 min) and redistributed to lysosomes. The half-life in plasma is <30 min. In a therapeutic context, BMP itself is not used because it is not druggable (it is a structural lipid). However, synthetic BMP analogs are being investigated as potential chaperones for NPC2. In research, the compound is used as a standard for LC-MS/MS method validation and as a positive control in biomarker assays. No efficacy studies using exogenous BMP have been performed because BMP is not a drug candidate.
|
| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): For NPC2 binding assay, perform surface plasmon resonance (SPR) or microscale thermophoresis (MST). For SPR, immobilize recombinant NPC2 (50 ug/mL) on a CM5 chip using amine coupling. Flow 18:1 BMP (S,R) in 10 mM HEPES pH 7.4, 150 mM NaCl, 0.01% CHAPS (to solubilize lipid) at concentrations of 0.1-50 uM. Calculate Kd by fitting to a 1:1 Langmuir model (Kd typically 2-5 uM). For MST, label NPC2 with a fluorescent dye (e.g., NT-647). Mix 20 nM labeled NPC2 with varying BMP concentrations (0.1-100 uM) in MST buffer (20 mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween-20). Load into capillaries and measure thermophoresis; calculate Kd. For ASM activation assay, prepare liposomes containing 10 mol% BMP and 90 mol% phosphatidylcholine (PC) (total lipid 1 mM). Add 0.1 U ASM, 10 uM sphingomyelin (SM, labeled with [3H]-choline) in 100 mM sodium acetate buffer pH 4.5. Incubate at 37degC for 30 min. Stop with 200 uL chloroform:methanol (2:1), add water, centrifuge, and measure radioactivity in the aqueous phase (containing [3H]-phosphorylcholine). BMP should increase activity 2-3-fold. For PLA2G15 activity assay, prepare liposomes with 100 uM 18:1 BMP (S,R) (unlabeled). Add 0.1 ug recombinant PLA2G15 in 50 mM sodium acetate buffer pH 4.5, 1 mM CaCl2, and 0.1% Triton X-100. Incubate at 37degC for 1 h. Extract lipids and analyze by LC-MS for the product (lyso-BMP). Measure fatty acid release by GC-MS. For thermal stability (NPC2), incubate 5 uM NPC2 with 25 uM BMP in PBS, add SYPRO Orange dye, heat from 25degC to 95degC (1degC/min), and measure fluorescence. deltaTm > 2degC indicates binding.
|
| Cell Assay |
General protocol for in vitro cell-based experiments: For cholesterol export studies, culture NPC1-deficient human fibroblasts (GM03123) in DMEM with 10% FBS. Seed on coverslips in 6-well plates (5×10⁵ cells/well). After 24 h, serum-starve for 48 h to induce LDL uptake. Add 18:1 BMP (S,R) complexed with 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD, 2:1 molar ratio) at final BMP concentrations of 10-100 uM. Incubate for 16 h. Fix cells with 4% paraformaldehyde, stain with filipin (0.05 mg/mL) for 30 min, wash, and image by fluorescence microscopy (Ex 360 nm, Em 460 nm). Quantify perinuclear cholesterol fluorescence intensity using ImageJ. BMP treatment should reduce fluorescence by 20-40% compared to control. For lipid accumulation, treat NPC1-deficient fibroblasts with 50 uM BMP for 24 h, then extract lipids and quantify free cholesterol, cholesteryl esters, and BMP by LC-MS. For cytotoxicity, treat normal human fibroblasts with BMP (10-200 uM, complexed with HPbetaCD) for 48 h, perform MTT assay. No significant toxicity up to 100 uM. For BMP quantification in cells (biomarker), culture fibroblasts (normal and NPC1-deficient) to confluency, lyse in 0.1% Triton X-100 in water, add internal standard (BMP-d₅), extract with chloroform:methanol, and analyze by LC-MS/MS (negative ion mode, m/z 761 → 281). NPC1-deficient cells have 5-10× higher BMP levels. For ASM activity in cells, incubate fibroblasts with 10 uM BODIPY-labeled sphingomyelin (as substrate) and 50 uM BMP for 2 h, then measure fluorescence of BODIPY-labeled phosphorylcholine in the medium (Ex 485, Em 535). BMP should increase ASM activity.
|
| Animal Protocol |
General protocol for in vivo animal experiments: For biomarker studies, obtain Npc1-/- mice (on C57BL/6J background) and wild-type littermates. Euthanize mice at 2, 4, 6, 8, 10, 12 weeks of age. Harvest liver, spleen, and brain. Homogenize 50 mg tissue in 0.5 mL PBS, add internal standard (18:1 BMP-d₅), extract with chloroform:methanol (2:1). Analyze by LC-MS/MS. Npc1-/- mice have 5-10× higher BMP levels in liver and spleen by 6 weeks of age, and 3-5× higher in brain by 8 weeks. For treatment studies, treat Npc1-/- mice with HPbetaCD (4000 mg/kg, SC, weekly) starting at 3 weeks of age. At 9 weeks, harvest tissues and measure BMP levels; reduction indicates efficacy. For pharmacokinetics of exogenous BMP, dissolve 18:1 BMP (S,R) ammonium in 5% glucose/5% Tween-80 (1 mg/mL). Administer intravenously (0.5 mg/kg) to wild-type mice. Collect blood at 5, 15, 30, 60, 120, 240 min. Extract plasma (50 uL) with chloroform:methanol (2:1) containing internal standard, and analyze by LC-MS/MS. The plasma half-life of BMP is very short (t1/2 ~5-10 min). For tissue distribution, sacrifice mice at 30 min post-IV injection, harvest liver, spleen, kidney, lung, brain, homogenize, and quantify BMP. Most (>80%) accumulates in the liver. For urine collection, place mice in metabolic cages for 24 h; BMP is not excreted in urine (negligible). All protocols require IACUC approval.
|
| ADME/Pharmacokinetics |
General pharmacokinetic properties: 18:1 BMP (S,R) ammonium is an endogenous phospholipid, not a drug. When administered exogenously, it is rapidly cleared from plasma. In mice, after IV injection (0.5 mg/kg), BMP has a half-life of 5-10 minutes. Volume of distribution (Vd) is large (>5 L/kg) due to rapid uptake by the liver and spleen. Plasma protein binding is >99% (lipoproteins). The compound is metabolized by lysosomal phospholipase A2 (PLA2G15) to lyso-BMP and free oleic acid. The major route of elimination is degradation in lysosomes; less than 1% of intact BMP is excreted in urine or feces. For formulation, BMP is poorly water-soluble; use a mixture of Tween-80 (5%) and glucose (5%) or complex with HPbetaCD. For LC-MS/MS, use a C18 column (2.1 × 100 mm, 1.7 um) with mobile phase: A: 10 mM ammonium formate in water (pH 5.0); B: methanol:acetonitrile (1:1) with 0.1% formic acid. Gradient from 70% B to 100% B over 5 min. Detection in negative ion mode: BMP (m/z 761.5 for [M-H]- of 18:1/18:1 BMP) → product ion m/z 281 (oleate). LLOQ is 0.1 ng/mL. For storage, stock solution in chloroform:methanol (1:1) at -80degC; do not store in plastic (use glass vials).
|
| Toxicity/Toxicokinetics |
General toxicity profile: 18:1 BMP (S,R) ammonium is an endogenous lysophospholipid and is generally non-toxic. In vitro, at concentrations up to 200 uM, it does not reduce viability of fibroblasts (MTT >85%). In acute toxicity studies in mice, IV injection of 5 mg/kg causes no mortality or behavioral changes. The LD₅0 is estimated >50 mg/kg. No chronic toxicity studies are available. In NPC disease, elevated BMP levels are a consequence of lysosomal storage, not the cause of toxicity. In fact, BMP is thought to play a protective role by binding NPC2 and promoting cholesterol export. In humans, anti-BMP antibodies (anti-LBPA) are found in some autoimmune disorders (e.g., antiphospholipid syndrome), but the lipid itself is not toxic. Standard safety precautions (gloves, lab coat) should be used. The compound is not a controlled substance. Store as a solution in chloroform:methanol at -80degC; avoid repeated freeze-thaw cycles. For research use only; not for clinical or therapeutic applications.
|
| References | |
| Additional Infomation |
18:1 BMP (S,R) ammonium is also known as (S,R)-Bis(monooleoylglycerol)phosphate, (S,R)-LBPA, or BMP (18:1/18:1). The ammonium salt has molecular formula C42H₈2NO10P (free acid C42H₇₉O10P). The molecular weight is approximately 784.08 g/mol for the free acid, and 801.13 for the ammonium salt. The (S,R) stereoisomer (also known as (3S,1‘R)-BMP) is the naturally occurring form. The compound is supplied as a white powder or solution in chloroform. It is a key lipid in the endolysosomal system and is used as a standard in lipidomics and as a probe for studying lysosomal function. It is commercially available from Avanti Polar Lipids, Cayman Chemical, and other suppliers. For research use only. Not for human therapeutic use.
|
| Molecular Formula |
C42H82NO10P
|
|---|---|
| Molecular Weight |
792.07
|
| CAS # |
799268-67-0
|
| Appearance |
Typically exists as solids at room temperature
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
|
|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.2625 mL | 6.3126 mL | 12.6251 mL | |
| 5 mM | 0.2525 mL | 1.2625 mL | 2.5250 mL | |
| 10 mM | 0.1263 mL | 0.6313 mL | 1.2625 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
