This research is funded as part of EPSRC grants EP/K016938/1 and EP/P019943/1 (ZW, HRF) and of an EPSRC/AstraZeneca CASE conversion award (BFJ). The University of Southampton is thanked for additional support. The EPSRC is further thanked for a core capability grant EP/K039466/1.

1. Partitioning
<ol>
	<li>Add 4,4,4-trifluorobutan-1-ol (compound X, ca. 6.0 mg) and 2,2,2-trifluoroethanol (reference compound, ca. 3.0 mg) to a 10 mL pear-shaped flask, dissolve in n-octanol (HPLC grade, ca. 2 mL), and add water (HPLC grade, ca. 2 mL). 
	Note: This experiment is run in triplicate. Compound solubility in water and n-octanol must be checked. The amount of the compound used for partition must be carefully considered to avoid oversaturation of the compound in any layer. The mass ratio between compound X and reference compound ref must also be considered to avoid that the integral ratios of a given NMR sample are outside a 10/1 to 1/10 range. For example, if there is a difference of &#60;2 logP units between compound X and ref, optimal mass ratio can assure that integration ratios in water and 1-octanol NMR samples are within a 10/1 to 1/10 range. In contrast, if an integration ratio of 50/1 in one layer is obtained, there will be more likely relatively larger errors in the integration for the peak with lower concentration. The equation below can be used to predict optimal compound mass ratio: 
	mX / mref = {(cPX / Pref)-0.5 * (MX/ Mref) * [(1 + cPX) / (1 + Pref)]} / (NX / Nref) 
	m, mass; M, molecular mass; N, number of F atoms; P, partition coefficients; cP, calculated partition coefficients.</li>
	<li>Place the flasks inside a temperature-controlled receptacle above a stirplate, and connect to a recirculating chiller. Stir the biphasic mixture at 25 &#176;C for 2 h, with stirring speed set at 600 rpm.</li>
	<li>Equilibrate the mixture at 25 &#176;C overnight (ca. 16 h), to allow for complete phase separation. 
	Note: In some cases, the formation of a foam between the n-octanol and water boundary can be observed. In this case, the mixture was transferred into a 4 mL glass vial and centrifuged till the disappearance of the foam. The biphasic mixture was then left to equilibrate again at 25 &#176;C overnight.</li>
</ol>
2. NMR Sample Preparation
<ol>
	<li>Fix the flask to a retort stand with a clamp.</li>
	<li>Take an aliquot of ca. 0.70-0.85 mL from both water and n-octanol layers, by using 1 mL disposable plastic syringes with long needles.
	<ol>
		<li>For taking the water aliquot, draw ca. 0.02 mL of air into the syringe before putting the needle into the mixture. While moving the needle through the upper n-octanol layer into water layer, gently push out the air to prevent n-octanol solution from entering the needle.</li>
		<li>Remove the long needle from the mixture. Discard a small amount of water sample, leaving ca. 0.6 mL of sample left in the syringe. Carefully wipe the needle with dry tissue, and inject ca. 0.5 mL of water sample into a clean NMR tube. Quickly close the NMR tube with a cap.</li>
		<li>For the n-octanol sample, remove the long needle from the n-octanol layer. Discard a small amount of n-octanol sample, leaving ca. 0.6 mL of sample left in the syringe. Carefully wipe the needle with dry tissue and inject ca. 0.5 mL of n-octanol sample into a clean NMR tube. Quickly close the NMR tube with a cap.</li>
	</ol>
	</li>
	<li>Visually inspect both n-octanol and water samples for any contamination (e.g., small droplets of n-octanol in water sample or small droplets of water in n-octanol sample). 
	Note: If there is any contamination, the aliquot sample needs to be re-prepared from the biphasic mixture. As the measurement is done in triplicate, six NMR tubes are obtained.</li>
	<li>To each NMR tube, add 0.1 mL of a deuterated NMR solvent that is miscible with both n-octanol and water (e.g., acetone-d6) to enable signal lock during NMR acquisition.</li>
	<li>For compounds with low boiling points (e.g., &#60;120 &#176;C), seal the NMR tubes using a blow torch, and, after cooling, invert the tube to check for any leaks. Carefully invert the sealed or non-sealed NMR tubes 20 times to obtain a homogenous solution for 19F NMR experiments.</li>
</ol>
3. NMR Experiments
<ol>
	<li>Run, using standard NMR parameter settings (NS 64, D1 1 s, SW 300 ppm, O1P -100 ppm), 19F{1H} NMR experiments to identify chemical shifts of 4,4,4-trifluorobutan-1-ol (compound X) and 2,2,2-trifluoroethanol (reference compound) in both n-octanol and water NMR samples.</li>
	<li>Measure the spin-lattice relaxation time (T1) for diagnostic fluorine nuclei by using an inversion-recovery sequence22. Gauge the level of appropriate pulse delay time (D1, set as &#8805; 5*T1) from the obtained T1 values for accurate quantitative NMR integration. 
	Note: This is very time-consuming, but a D1 of 60 s for the water phase sample, and of 30 s for the octanol phase sample, are conservative settings which will safely fulfill the D1 &#8805; 5*T1 criterium.</li>
	<li>Run 19F{1H} NMR experiments again with adjusted parameter settings as follows: a) use D1 &#8805; 5*T1; b) center the frequency offset point (O1P) between the two diagnostic fluorine signals so both nuclei can be equally excited; c) Set the spectral width (SW) as 300 ppm, but reduce if a better SNR ratio if needed; d) Set the number of transients (NS) as 64 but increase if higher SNR is required. 
	Note: Non-decoupled 19F NMR experiments can be also used for NMR data acquisition. However, proton-decoupled 19F NMR experiments are preferred here as it simplifies the fluorine signals by removing proton-fluorine couplings which also increases signal-to-noise ratio. We use inverse-gated decoupling to obtain a decoupled spectrum without nOe (nuclear Overhauser effect) enhancements23. For quantitative integration, a signal-to-noise ratio (&#8805;300) is desired.24</li>
</ol>
4. Data Processing
<ol>
	<li>Process the obtained data using ACD/NMR Processor Academic Edition or other custom NMR processing software.
	<ol>
		<li>Open the NMR data file, then open the pdata folder, followed by folder 1. Delete the 1r file.</li>
		<li>Return to the NMR data file and drag the fid file into the ACD/NMR Processor window.</li>
		<li>Click the WFunctions button, select Exponential, set LB value as 2, and click the OK button.</li>
		<li>Click the Zero Filling button, increase the Points Count to 4 times of its Original Points Count by clicking a small button next to the number, and click the OK button.</li>
		<li>Click the Fourier Tr. button.</li>
		<li>Click the Phase button, then click the Mouse Ph. button, click and hold the left mouse button, move the mouse forward or backward till the major peak of the spectrum is properly phased.
		<ol>
			<li>Click and hold the right mouse button, move the mouse forward or backward until the other peak(s) of the spectrum is properly phased. Then unclick the Mouse Ph. button, zoom into the spectral area with the fluorine peaks, click Fine Tuning, perform the phase correction if needed as described earlier till all peaks are correctly phased, and then click the Tick button.</li>
		</ol>
		</li>
		<li>Click the Baseline button, then the Options button. Select Spectrum Averaging for Automatic Models, adjust the number of points for Box Half Width if needed (in particular for spectrum with low S/R ratio), click OK | Auto, and then click the Tick button.</li>
		<li>Click Integration, integrate the diagnostic fluorine peaks, and click the Tick button. 
		Note: If the integral curve is not parallel to the baseline, click the Bias Corr. button, and adjust the tilt and slope until the curve is parallel to the baseline.</li>
	</ol>
	</li>
	<li>Obtain the integration ratios from n-octanol and water NMR samples and use in the logP calculation equation (Figure 1, eq. 4) to obtain the logP value of of 4,4,4-trifluorobutan-1-ol (compound X).</li>
</ol>

<ol>
	<li>Arnott, J. A., Planey, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+lipophilicity+in+drug+discovery+and+design.">The influence of lipophilicity in drug discovery and design.</a> Expert Opinion on Drug Discovery. 7 (10), 863-875 (2012).</li><li>Lipinski, C. A., Lombardo, F., Dominy, B. W., Feeney, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+computational+approaches+to+estimate+solubility+and+permeability+in+drug+discovery+and+development+settings.">Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings.</a> Advanced Drug Delivery Reviews. 23 (1), 3-25 (1997).</li><li>Leeson, P. D., Springthorpe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+drug-like+concepts+on+decision-making+in+medicinal+chemistry.">The influence of drug-like concepts on decision-making in medicinal chemistry.</a> Nature Reviews Drug Discovery. 6, 881 (2007).</li><li>Perola, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Analysis+of+the+Binding+Efficiencies+of+Drugs+and+Their+Leads+in+Successful+Drug+Discovery+Programs.">An Analysis of the Binding Efficiencies of Drugs and Their Leads in Successful Drug Discovery Programs.</a> Journal of Medicinal Chemistry. 53 (7), 2986-2997 (2010).</li><li>Tarcsay, A., Nyiri, K., Keseru, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Lipophilic+Efficiency+on+Compound+Quality.">Impact of Lipophilic Efficiency on Compound Quality.</a> Journal of Medicinal Chemistry. 55 (3), 1252-1260 (2012).</li><li>Tarcsay, &. #. 1. 9. 3. ;., Keser&#369;, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+Molecular+Properties+to+Drug+Promiscuity.">Contributions of Molecular Properties to Drug Promiscuity.</a> Journal of Medicinal Chemistry. 56 (5), 1789-1795 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> OECD Guidelines for Testing of Chemicals. , (1992).</li><li>Tsang, S. C., Yu, C. H., Gao, X., Tam, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+nanomagnetic+absorbent+for+partition+coefficient+measurement.">Preparation of nanomagnetic absorbent for partition coefficient measurement.</a> International Journal of Pharmaceutics. 327 (1), 139-144 (2006).</li><li>Andersson, J. T., Schr&#228;der, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+Measuring+1-Octanol&#8722;Water+Partition+Coefficients.">A Method for Measuring 1-Octanol&#8722;Water Partition Coefficients.</a> Analytical Chemistry. 71 (16), 3610-3614 (1999).</li><li>Danielsson, L. -. G., Yu-Hui, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanized+determination+of+n-octanol/water+partition+constants+using+liquid-liquid+segmented+flow+extraction.">Mechanized determination of n-octanol/water partition constants using liquid-liquid segmented flow extraction.</a> Journal of Pharmaceutical and Biomedical Analysis. 12 (12), 1475-1481 (1994).</li><li>Scherrer, R. A., Donovan, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+Potentiometric+Titrations+in+KCl/Water-Saturated+Octanol:+Method+for+Quantifying+Factors+Influencing+Ion-Pair+Partitioning.">Automated Potentiometric Titrations in KCl/Water-Saturated Octanol: Method for Quantifying Factors Influencing Ion-Pair Partitioning.</a> Analytical Chemistry. 81 (7), 2768-2778 (2009).</li><li>Poole, S. K., Poole, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+methods+for+estimating+octanol-water+partition+coefficients.">Separation methods for estimating octanol-water partition coefficients.</a> Journal of Chromatography B. 797 (1), 3-19 (2003).</li><li>Ishihama, Y., Oda, Y., Uchikawa, K., Asakawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Solute+Hydrophobicity+by+Microemulsion+Electrokinetic+Chromatography.">Evaluation of Solute Hydrophobicity by Microemulsion Electrokinetic Chromatography.</a> Analytical Chemistry. 67 (9), 1588-1595 (1995).</li><li>Jorabchi, K., Smith, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Droplet+Separations+and+Surface+Partition+Coefficient+Measurements+Using+Laser+Ablation+Mass+Spectrometry.">Single Droplet Separations and Surface Partition Coefficient Measurements Using Laser Ablation Mass Spectrometry.</a> Analytical Chemistry. 81 (23), 9682-9688 (2009).</li><li>Kaliszan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+structure-retention+relationships.">Quantitative structure-retention relationships.</a> Analytical Chemistry. 64 (11), 619A-631A (1992).</li><li>Mo, H., Balko, K. M., Colby, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+deuterium-free+NMR+method+for+the+rapid+determination+of+1-octanol/water+partition+coefficients+of+pharmaceutical+agents.">A practical deuterium-free NMR method for the rapid determination of 1-octanol/water partition coefficients of pharmaceutical agents.</a> Bioorganic &amp; Medicinal Chemistry Letters. 20 (22), 6712-6715 (2010).</li><li>St&#233;en, E. J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simple+proton+nuclear+magnetic+resonance-based+procedure+to+estimate+the+approximate+distribution+coefficient+at+physiological+pH+(logD7.4):+Evaluation+and+comparison+to+existing+practices.">Development of a simple proton nuclear magnetic resonance-based procedure to estimate the approximate distribution coefficient at physiological pH (logD7.4): Evaluation and comparison to existing practices.</a> Bioorganic &amp; Medicinal Chemistry Letters. 27 (2), 319-322 (2017).</li><li>Soulsby, D., Chica, J. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+partition+coefficients+using+1H+NMR+spectroscopy+and+time+domain+complete+reduction+to+amplitude-frequency+table+(CRAFT)+analysis.">Determination of partition coefficients using 1H NMR spectroscopy and time domain complete reduction to amplitude-frequency table (CRAFT) analysis.</a> Magnetic Resonance in Chemistry. 55 (8), 724-729 (2017).</li><li>Tetko, I. V., Poda, G. I., Ostermann, C., Mannhold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+In+Silico+log+Predictions:+One+Can't+Embrace+the+Unembraceable.">Accurate In Silico log Predictions: One Can't Embrace the Unembraceable.</a> QSAR &amp; Combinatorial Science. 28 (8), 845-849 (2009).</li><li>Waring, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipophilicity+in+drug+discovery.">Lipophilicity in drug discovery.</a> Expert Opinion on Drug Discovery. 5 (3), 235-248 (2010).</li><li>Linclau, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+the+Influence+of+(Deoxy)fluorination+on+the+Lipophilicity+of+Non-UV-Active+Fluorinated+Alkanols+and+Carbohydrates+by+a+New+log+P+Determination+Method.">Investigating the Influence of (Deoxy)fluorination on the Lipophilicity of Non-UV-Active Fluorinated Alkanols and Carbohydrates by a New log P Determination Method.</a> Angewandte Chemie International Edition. 55 (2), 674-678 (2016).</li><li>Derome, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Modern NMR Techniques for Chemistry Research. , (1997).</li><li>Claridge, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> High-Resolution NMR Techniques in Organic Chemistry. , (1999).</li><li>Zhang, F. -. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+sitagliptin+using+the+19F-NMR+method:+a+universal+technique+for+fluorinated+compound+detection.">Quantitative analysis of sitagliptin using the 19F-NMR method: a universal technique for fluorinated compound detection.</a> Analyst. 140 (1), 280-286 (2015).</li><li>Muller, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+is+a+trifluoromethyl+group+more+lipophilic+than+a+methyl+group?+partition+coefficients+and+selected+chemical+shifts+of+aliphatic+alcohols+and+trifluoroalcohols.">When is a trifluoromethyl group more lipophilic than a methyl group? partition coefficients and selected chemical shifts of aliphatic alcohols and trifluoroalcohols.</a> Journal of Pharmaceutical Sciences. 75 (10), 987-991 (1986).</li><li>Hansch, C., Leo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Substituent constants for correlation analysis in chemistry and biology. , (1979).</li><li>Dillingham, E. O., Mast, R. W., Bass, G. E., Autian, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+Methyl-+and+Halogen-Substituted+Alcohols+in+Tissue+Culture+Relative+to+Structure-Activity+Models+and+Acute+Toxicity+in+Mice.">Toxicity of Methyl- and Halogen-Substituted Alcohols in Tissue Culture Relative to Structure-Activity Models and Acute Toxicity in Mice.</a> Journal of Pharmaceutical Sciences. 62 (1), 22-30 (1973).</li><li>Leo, A., Hansch, C., Elkins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partition+coefficients+and+their+uses.">Partition coefficients and their uses.</a> Chemical Reviews. 71 (6), 525-616 (1971).</li><li>Fujita, T., Iwasa, J., Hansch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Substituent+Constant,+&#960;,+Derived+from+Partition+Coefficients.">A New Substituent Constant, &#960;, Derived from Partition Coefficients.</a> Journal of the American Chemical Society. 86 (23), 5175-5180 (1964).</li><li>Zhong-Xing, J., Xin, L., Eun-Kee, J., Bruce, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetry-Guided+Design+and+Fluorous+Synthesis+of+a+Stable+and+Rapidly+Excreted+Imaging+Tracer+for+19F+MRI.">Symmetry-Guided Design and Fluorous Synthesis of a Stable and Rapidly Excreted Imaging Tracer for 19F MRI.</a> Angewandte Chemie International Edition. 48 (26), 4755-4758 (2009).</li></ol>

The authors have nothing to disclose.

The protocol described in the paper is a straightforward method for logP measurement of fluorinated compounds. This method is applicable to fluorinated compounds with a logP value from -3 to 3. For more hydrophilic (logP &#60; -3) or lipophilic compounds (logP &#62; 3), this method can still be used but will require much longer NMR experiment time as extended number of transients are needed to obtain a good signal-to-noise ratio. Hence, this is a limitation of the method. There is no requirement for the frequency of NMR spectrometer, as long as the conditions (NMR parameter settings and sufficient SNR) for quantitative integration are met. As for any shake flask method, it is critical to avoid oversaturation and contamination during the layer sampling.
Compared with previous shake-flask method and its variations, there are several advantages in our method with respect to existing methods. 1) Measurements of solute mass, volume of partition solvents and aliquots for NMR sample are not necessary. 2) The compound for measurement can be impure provided that the fluorine chemical shifts of the impurities are different from that of the measured compound. 3) Because of the intrinsic compensation effect when working with the ratio of a ratio, systematic errors are eliminated. 4) This method is applicable to non-UV-active fluorinated compounds. 5) This method is easy to use with open-access NMR facilities as no special NMR settings are needed (such as solvent suppression, applying a small excitation angle, etc.).
Currently, we are using this method to measure the lipophilicities of fluorinated carbohydrates, fluorohydrins and fluorinated amides, in order to investigate the influence of fluorination on lipophilicity and to identify fluorinated moieties with lipophilicity-lowering effect. Method development for logP measurement of more lipophilic compounds (logP &#62;3) and for fluorinated amines is ongoing in our group.
It can be pointed out that 19F NMR can also be used for critical micelle concentration (CMC) determination30.

Lipophilicity is a key physicochemical parameter of drug molecules which influences the properties of drug candidates in many aspects, including drug solubility, bioavailability, and toxicity1. Lipophilicity is measured as the logarithm (logP) of the ratio of compound concentrations after partitioning between n-octanol and water. Optimal lipophilicity ranges have been proposed based on statistical data of orally administered drugs, of which the Lipinski&#39;s &#34;rule of 5&#39;&#39; is the most famous example2,3. Indeed, controlling lipophilicity has shown to be essential for improving the prospect of drug candidates. Increasing drug binding affinity by elevated lipophilicity has been identified as one of the main problems in drug discovery projects during the past few decades, leading to increased attrition rates3. Therefore, it has been suggested that successful drug development is associated with keeping the molecular lipophilicity of the drug candidates within optimal boundaries during the affinity optimization process3,4. In that regard, new concepts (such as lipophilic efficiency indices) have been introduced5,6.
It is thus of great importance to accurately measure lipophilicity during the drug development process. Besides, the availability of straightforward methods for lipophilicity measurement is in demand as fundamental research aims to identify solutions for logP modulation. Currently, numerous established methods are accessible for lipophilicity determination1. The standard &#39;shake-flask (SF)&#39; method7and its variations are commonly employed to measure logP values directly, which in most cases depend on UV-Vis spectroscopy for quantification. The main disadvantage of this classic SF method is its labor-intensive nature. In addition, the formation of emulsions may occur, especially for highly lipophilic compounds8,9.Several methods were developed to circumvent such issues, such as by using flow injection analysis, dialysis tubing, etc.9,10.However, none of those methods are straightforward or easily applicable in non-specialized laboratories.
There are also many indirect methods available for use, such as potentiometric titration11, electrophoretic methods12,13, RP-HPLC-based chromatographic methods, mass-spectrometry-based methods14, etc. These are indirect methods, as the logP values are obtained by calibration curves. Among these methods, the RP-HPLC method has been widely used because it is user-friendly and time-saving. Nevertheless, its accuracy depends on the training set used to establish the calibration curve, and the estimated lipophilicity depends on the partition system used13,15.
There are a number of 1H NMR-based methods reported in the literature for lipophilicity determination. Mo et al. developed a method for logP measurement using 1H NMR without deuterated solvents. Water and octanol, as partition solvents, were used as references for the quantification of solute concentration in each phase16. Herth and co-workers also reported an approach, by which the partition experiment occurred directly in an NMR tube, where the NMR data of the bottom D2O aqueous layer were collected before and after the extraction with 1-octanol, to obtain the distribution coefficient17. In addition, Soulsby et al. exploited 1H NMR as an analysis tool, determining the amplitude of signals by using complete reduction to amplitude-frequency table software. The ratio of the amplitudes in both layers led to the measured partition coefficient18. These methods are relatively simple to use but often require the calibration of selective pulses and power levels or the use of shaped gradient pulses to ensure appropriate solvent suppression and signal selectivity.
Calculated logP (clogP) values for compounds can also be obtained. Several calculation methods and commercially available software are available. Such clogP values are commonly used in the pharmaceutical industry when evaluating large numbers of drug molecules. However, large errors from clogP values are not uncommon19,20.
The requirements of UV-activity for concentration analysis and the establishment of calibration curves for logP calculation impede research progress in this field. In particular, this is the case for non-UV-active aliphatic compounds. Fluorinated aliphatic moieties have become increasingly attractive for drug design in recent years, and their influence on overall lipophilicity of the compound is a research topic in our group21. In addition, 19F is a highly sensitive NMR-active nucleus, making 19F NMR a useful tool for analyzing fluorinated compounds. It also has a larger chemical shift range compared to that of 1H. Therefore, it is worthwhile to develop a straightforward method for logP determination of non-UV-active fluorinated compounds by 19F NMR spectroscopy. Hence, the overall goal of this method is to achieve convenient lipophilicity determination of fluorinated compounds.
The key principle of our 19F NMR-based method is to add a fluorinated reference compound in the partition experiment (Figure 1)21. Compound X and reference compound (ref) are partitioned between water and n-octanol. After equilibrating, an aliquot from each phase is taken into an NMR tube, and 19F NMR experiments are run on both NMR samples. The intensity of the fluorine peaks is proportional to compound concentration (C) and the number of fluorine atoms (n) of the compounds. Between compound X and ref, integral ratios can be obtained for both phases. The ratio in n-octanol layer is defined as &#961;oct, and &#961;aq for water layer (eq. 1). The ratio of &#961; values equals the ratio of partition coefficients (P) of compound X and ref (eq. 2). This leads to the final equation (eq. 4) for logP measurement of compound X. Therefore, in order to determine the logP value of an unknown compound X, only integration ratios (&#961;oct and &#961;aq) in both layers are needed to be measured by 19F NMR.

<table><tbody><tr><td>NMR (400 MHz) with Bruker 5 mm SEF probe</td><td>Bruker</td><td>n/a</td><td>AVIIIHD400</td></tr><tr><td>NMR (400 MHz) with Bruker 5 mm SMART probe</td><td>Bruker</td><td>n/a</td><td></td></tr><tr><td>DrySyn Snowstorm reactor</td><td>Asynt</td><td>ADS13-S</td><td></td></tr><tr><td>recirculating chiller</td><td>Asynt</td><td>n/a</td><td>model:Grant-LTC2</td></tr><tr><td>magnetic stirplate</td><td>Asynt</td><td>ADS-HP-NT</td><td></td></tr><tr><td>ACD/NMR processor software</td><td>ACD/Labs</td><td>n/a</td><td>ACD/NMR processor academic edition or ACD/Spectrus processor 2015</td></tr></tbody></table>

a new straightforward method for lipophilicity (logp) measurement using 19f nmr spectroscopy

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction is to modulate the lipophilicity of the compound. In our group, we are interested in the study of the impact of fluorination on lipophilicity of aliphatic fluorohydrins and fluorinated carbohydrates. These are not UV-active, resulting in a challenging lipophilicity determination. Here, we present a straightforward method for the measurement of lipophilicity of fluorinated compounds by 19F NMR spectroscopy. This method requires no UV-activity. Accurate solute mass, solvent and aliquot volume are also not required to be measured. Using this method, we measured the lipophilicities of a large number of fluorinated alkanols and carbohydrates.

Two sets of data as control experiments are shown in Figure 221. Using 2,2,2-trifluoroethanol as reference compound, logP values were obtained for 2-fluoroethanol and 3,3,3,2,2-pentafluoropropanol as -0.75 and +1.20, respectively (Figure 2A). Subsequently, the lipophilicity of 2-fluoroethanol was determined again but with 3,3,3,2,2-pentafluoropropanol as the reference (using its previous experimentally measured logP value +1.20). The measured logP value was -0.76, which only had a difference of 0.01 logP units when compared with the value measured using 2,2,2-trifluoroethanol as reference.
Likewise, for cis-2,3-difluoro-1,4-butanediol, the difference in measured logP values by using 2-fluoroethanol and its trans isomer is also very small (0.01 logP units, Figure 2B). This demonstrated that the selection of reference compound does not have impact on the logP measurement. In addition, a rather small standard deviation (&#60;0.01) indicated good reproducibility of our method.
Using our method, a series of compounds with known logP values was measured as shown in Table 1. The difference between literature data and the values measured using our method is shown in the last column of the Table. Overall, the experimentally obtained logP values (at 25 &#176;C) have good to excellent accordance with the literature values, which further validated our method.
Additional selected examples21 were shown in Figure 3. All these non-UV-active aliphatic compounds (from fluorinated carbohydrates to fluorohydrins) can be easily measured with our method.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58567/58567fig1.jpg" /> 
Figure 1: Principle of the logP determination method. This figure has been reproduced with permission from Wiley-VCH Verlag GmbH &#38; Co. KGaA.21. This shake-flask method is based on 19F NMR spectroscopy. A reference compound is used for partition experiment. Aliquots for both n-octanol and water phase were taken for NMR experiment. Integration ratios between reference compound and the compound to be measured are obtained for the determination of logP value. Detailed mathematical deduction of equations, which leads to the final equation for measurement, are also given. <a href="https://www.jove.com/files/ftp_upload/58567/58567fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58567/58567fig2.jpg" /> 
Figure 2: Examples for internal validation21. Two sets of control experiments, using two different reference compounds to measure logP value of one compound, were conducted. The logP difference between those experiments is negligible. Standard deviation (&#60;0.01) from experiments run in triplicate shows good reproducibility of the method. <a href="https://www.jove.com/files/ftp_upload/58567/58567fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58567/58567fig3.jpg" /> 
Figure 3: Further selected examples of logP measurement using our method. Applying this method, logP values for 8 fluorinated compounds (such as fluorinated carbohydrates, acyclic alkanols and conformationally restricted fluorohydrins) were obtained. <a href="https://www.jove.com/files/ftp_upload/58567/58567fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Comparison between literature data and the experimental logP values using our method21. logP values for 14 fluorinated compounds (with known logP data) were measured using this new method. The reference compounds used for each measurement were also tabulated. Comparision (logP) between literature values and logP results from our method demonstrated good accuracy of this method. a2,2,2-Trifluoroethanol (TFE), 2-Fluoroethanol (FE); bAveraged logP value from at least three experiments; cExperimentally measured logP value by our method (-0.75) was used as reference. <a href="https://www.jove.com/files/ftp_upload/58567/Table1.xlsx">Please click here to download this file.</a>

Watch this Scientific Journal Video about A New Straightforward Method for Lipophilicity logP Measurement using 19F NMR Spectroscopy at JoVE.com

A New Straightforward Method for Lipophilicity logP Measurement using 19F NMR Spectroscopy

A novel and straightforward variation of the shake-flask method was developed for accurate lipophilicity measurement of fluorinated compounds by 19F NMR spectroscopy.

A New Straightforward Method for Lipophilicity (logP) Measurement using 19F NMR Spectroscopy

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction ...

a-new-straightforward-method-for-lipophilicity-logp-measurement-using

Nanyang Technological University

Research

JoVE Journal

Chemistry

14.2K Views.  University of Southampton. A novel and straightforward variation of the shake-flask method was developed for accurate lipophilicity measurement of fluorinated compounds by 19F NMR spectroscopy.

Video: A New Straightforward Method for Lipophilicity logP Measurement using 19F NMR Spectroscopy

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

Biochemistry

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the average content of fluorinated drugs in their formulated drug product forms: tablets or capsules. This method is specific to fluorinated drugs because it detects only the content of fluorine, avoiding interference from the excipients that lack fluorine. The advantages of measuring the active content of fluorinated drugs using low-field 19F TD-NMR versus high-field 19F solid-state (SS) NMR are the simplicity of the method; the low cost; and the non-destructive nature of the technique, with all samples recoverable in intact forms (e.g., powders, tablets, and capsules), making this technique affordable for any laboratory.
We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content.
Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

A simple and non-destructive technique that measures the average content of drug substances in formulated drug products containing fluorine using low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) is presented here. The technique can be applied to the development and manufacturing of drugs in the pharmaceutical industry.

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Environment

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Medicine

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.

We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

Bioengineering

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral budding, among other vital cellular processes. For efficient biological activity, cell membranes should be rigid enough to maintain the integrity of the cell and its compartments yet fluid enough to allow membrane components, such as proteins and functional domains, to diffuse and interact. This delicate balance of elastic and fluid membrane properties, and their impact on biological function, necessitate a better understanding of collective membrane dynamics over mesoscopic length and time scales of key biological processes, e.g., membrane deformations and protein binding events. Among the techniques that can effectively probe this dynamic range is neutron spin echo (NSE) spectroscopy. Combined with deuterium labeling, NSE can be used to directly access bending and thickness fluctuations as well as mesoscopic dynamics of select membrane features. This paper provides a brief description of the NSE technique and outlines the procedures for performing NSE experiments on liposomal membranes, including details of sample preparation and deuteration schemes, along with instructions for data collection and reduction. The paper also introduces data analysis methods used to extract key membrane parameters, such as the bending rigidity modulus, area compressibility modulus, and in-plane viscosity. To illustrate the biological importance of NSE studies, select examples of membrane phenomena probed by NSE are discussed, namely, the effect of additives on membrane bending rigidity, the impact of domain formation on membrane fluctuations, and the dynamic signature of membrane-protein interactions.

This paper describes the protocols for sample preparation, data reduction, and data analysis in neutron spin echo (NSE) studies of lipid membranes. Judicious deuterium labeling of lipids enables access to different membrane dynamics on mesoscopic length and time scales, over which vital biological processes occur.

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral ...

Biology

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are sequentially cleaved from the glycerol backbone, resulting in the release of free fatty acids and glycerol. Due to the low expression of glycerol kinase in white adipocytes, glycerol re-uptake rates are negligible, while fatty acid re-uptake is dictated by the fatty acid binding capacity of media components such as albumin. Both glycerol and fatty acid release into media can be quantified by colorimetric assays to determine the lipolytic rate. By measuring these factors at multiple time points, one can determine the linear rate of lipolysis with high confidence. Here, we provide a detailed protocol for the measurement of lipolysis in in vitro differentiated adipocytes and ex vivo adipose tissue from mice. This protocol may also be optimized for other preadipocyte cell lines or adipose tissue from other organisms; considerations and optimization parameters are discussed. This protocol is designed to be useful in determining and comparing the rate of adipocyte lipolysis between mouse models and treatments.

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Triglyceride lipolysis in adipocytes is an important metabolic process resulting in the liberation of free fatty acids and glycerol. Here, we provide a detailed protocol to measure basal and stimulated lipolysis in adipocytes and ex vivo adipose tissue from mice.

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are ...

A New Straightforward Method for Lipophilicity (logP) Measurement using ¹⁹F NMR Spectroscopy

Podsumowanie

Przeglądaj więcej filmów

Fatty Acid ¹³C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

Application and Methodology of the Non-destructive ¹⁹F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

In vivo ¹⁹F MRI for Cell Tracking

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro