Analysis of dipalmitoyl phosphatidylcholine in amniotic fluid by high-performance liquid chromatography

A novel method for the determination of dipalmitoyl phosphatidylcholine (DPPC) in amniotic fluid by high-performance liquid chromatography (HPLC) is described. Aliquots of 50 μl of amniotic fluid were hydrolyzed with phospholipase C from Bacillus cereus and the resulting dipalmitoylglycerol analyzed by HPLC. Run-to-run and day-to-day precision for DPPC analysis were 4.2 and 6.1%, respectively, and analysis time was 10 min. Recoveries for DPPC ranged between 92 and 98%. In summarizing, this method provides a high precision and fast turnaround time means for the analysis of DPPC in amniotic fluid.     

Lipid bilayer structure determined by the simultaneous analysis of neutron and X-ray scattering data

Quantitative structures were obtained for the fully hydrated fluid phases of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) bilayers by simultaneously analyzing x-ray and neutron scattering data. The neutron data for DOPC included two solvent contrasts, 50% and 100% D₂O. For DPPC, additional contrast data were obtained with deuterated analogs DPPC_d62, DPPC_d13, and DPPC_d9. For the analysis, we developed a model that is based on volume probability distributions and their spatial conservation. The model's design was guided and tested by a DOPC molecular dynamics simulation. The model consistently captures the salient features found in both electron and neutron scattering density profiles. A key result of the analysis is the molecular surface area, A. For DPPC at 50°C A = 63.0 Å², whereas for DOPC at 30°C A = 67.4 Å², with estimated uncertainties of 1 Å². Although A for DPPC agrees with a recently reported value obtained solely from the analysis of x-ray scattering data, A for DOPC is almost 10% smaller. This improved method for determining lipid areas helps to reconcile long-standing differences in the values of lipid areas obtained from stand-alone x-ray and neutron scattering experiments and poses new challenges for molecular dynamics simulations.     

Order parameters and areas in fluid-phase oriented lipid membranes using wide angle X-ray scattering

We used wide angle x-ray scattering (WAXS) from stacks of oriented lipid bilayers to measure chain orientational order parameters and lipid areas in model membranes consisting of mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol in fluid phases. The addition of 40% cholesterol to either DOPC or DPPC changes the WAXS pattern due to an increase in acyl chain orientational order, which is one of the main properties distinguishing the cholesterol-rich liquid-ordered (Lo) phase from the liquid-disordered (Ld) phase. In contrast, powder x-ray data from multilamellar vesicles does not yield information about orientational order, and the scattering from the Lo and Ld phases looks similar. An analytical model to describe the relationship between the chain orientational distribution and WAXS data was used to obtain an average orientational order parameter, S_x-ray. When 40% cholesterol is added to either DOPC or DPPC, S_x-ray more than doubles, consistent with previous NMR order parameter measurements. By combining information about the average chain orientation with the chain-chain correlation spacing, we extended a commonly used method for calculating areas for gel-phase lipids to fluid-phase lipids and obtained agreement to within 5% of literature values.     

Hypoxic incubation leads to concerted changes of carbonic anhydrase activity and 2.3 DPG concentration of chick embryo red cells

Red cell carbonic anhydrase activity, 2.3 DPG concentration, and activities of three key enzymes controlling DPG metabolism (PK, PFK and DPGM) were measured in normoxic and hypoxic (incubation in 13.5% O₂) chick embroys. In normoxia 2.3 DPG concentration and carbonic anhydrase activity begin to increase by the third week of incubation. Hypoxia induces a rise of 2.3 DPG concentration and carbonic anhydrase activity by Day 8 of development, i.e., 1 week earlier. Since during normal development chick embryos become hypoxic in the third week of incubation, the results suggest that PO₂ has a controlling influence on the timing of differentiation events of definitive embryonic red cells.     

Mixing of oxidized and bilayer phospholipids

Composition and phase dependence of the mixing of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), with the oxidized phospholipid, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) were investigated by characterizing the aggregation states of DPPC/PGPC and DOPC/PGPC using a fluorescence quenching assay, dynamic light scattering, and time-resolved fluorescence quenching in the temperature range 5–60 °C. PGPC forms 3.5 nm radii micelles of aggregation number 33. In the gel phase, DPPC and PGPC fuse to form mixed vesicles for PGPC molar fraction, X_PGPC ≤ 0.3 and coexisting vesicles and micelles at higher X_PGPC. Data suggest that liquid phase DPPC at 50 °C forms mixed vesicles with segregated or hemi fused DPPC and PGPC for X_PGPC ≤ 0.3. At 60 °C, DPPC and PGPC do not mix, but form coexisting vesicles and micelles. DOPC and PGPC do not mix in any proportion in the liquid phase. Two dissimilar aggregates of the sizes of vesicles and PGPC micelles were observed for all X_PGPC for T ≥ 22 °C. DOPC–PGPC and DPPC–PGPC mixing is non-ideal for X_PGPC > 0.3 in both gel and fluid phases resulting in exclusion of PGPC from the bilayer. Formation of mixed vesicles is favored in the gel phase but not in the liquid phase for X_PGPC ≤ 0.3. For X_PGPC ≤ 0.3, aggregation states change progressively from mixed vesicles in the gel phase to component segregated mixed vesicles in the liquid phase close to the chain melting transition temperature to separated coexisting vesicles and micelles at higher temperatures.     

Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A2 Action at the Single Molecule Level

We monitored the action of phospholipase A₂ (PLA₂) on L- and D-dipalmitoyl-phosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity and diffusion behavior of single PLA₂ molecules in a heterogeneous lipid environment during active hydrolysis. The experiments showed that enzyme molecules adsorbed and interacted almost exclusively with the fluid region of the DPPC monolayers. Domains of gel state L-DPPC were degraded exclusively from the gel-fluid interface where the buildup of negatively charged hydrolysis products, fatty acid salts, led to changes in the mobility of PLA₂. The mobility of individual enzymes on the monolayers was characterized by single particle tracking. Diffusion coefficients of enzymes adsorbed to the fluid interface were between 3.2 μm²/s on the L-DPPC and 4.9 μm²/s on the D-DPPC monolayers. In regions enriched with hydrolysis products, the diffusion dropped to ≈0.2 μm²/s. In addition, slower normal and anomalous diffusion modes were seen at the L-DPPC gel domain boundaries where hydrolysis took place. The average residence times of the enzyme in the fluid regions of the monolayer and on the product domain were between ≈30 and 220 ms. At the gel domains it was below the experimental time resolution, i.e., enzymes were simply reflected from the gel domains back into solution.     

Characterization of complexes formed in fully hydrated dispersions of dipalmitoyl derivatives of phosphatidylcholine and diacylglycerol.

The phase diagram of fully hydrated binary mixtures of dipalmitoylphosphatidylcholine (DPPC) with 1,2-dipalmitoylglycerol (DPG) published recently by López-García et al. identifies regions where stoichiometric complexes of 1:1 and 1:2 DPPC:DPG, respectively, are formed. In this study, the structural parameters of the 1:1 complex in the presence of pure DPPC was characterized by synchrotron low angle and static x-ray diffraction methods. Structural changes upon transitions through phase boundaries were correlated with enthalpy changes observed by differential scanning calorimetry in mixtures of DPPC with 5, 7.5, 10, and 20 mol% DPG dispersed in excess water. Phase separation of a complex in gel phase could be detected by calorimetry in the mixture containing 5 mol% DPG but was not detectable by synchrotron low angle x-ray diffraction. Static x-ray measurements show evidence of phase separation, particularly in the reflections indexing chain packing. In the mixture containing 7.5 mol% DPG, two distinct lamellar repeat spacings could be seen in the temperature range from 25 to 34 degrees C. The lamellar spacing of about 6.6 nm was assigned to pure gel phase DPPC because the change in the spacing corresponds with thermal transition of the pure phospholipid, and a longer repeat spacing of about 7.2 nm was assigned to domains of the 1:1 complex of DPPC-DPG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ether vs. ester linkage on lipid bilayer structure and water permeability

The structure and water permeability of bilayers composed of the ether-linked lipid, dihexadecylphosphatidylcholine (DHPC), were studied and compared with the ester-linked lipid, dipalmitoylphosphaditdylcholine (DPPC). Wide angle X-ray scattering on oriented bilayers in the fluid phase indicate that the area per lipid A is slightly larger for DHPC than for DPPC. Low angle X-ray scattering yields A = 65.1 Å² for DHPC at 48 °C. LAXS data provide the bending modulus, K_C = 4.2 × 10⁻¹³ erg, and the Hamaker parameter H = 7.2 × 10⁻¹⁴ erg for the van der Waals attractive interaction between neighboring bilayers. For the low temperature phases with ordered hydrocarbon chains, we confirm the transition from a tilted L_β′ gel phase to an untilted, interdigitated L_βI phase as the sample hydrates at 20 °C. Our measurement of water permeability, P_f = 0.022 cm/s at 48 °C for fluid phase DHPC is slightly smaller than that of DPPC (P_f = 0.027 cm/s) at 50 °C, consistent with our triple slab theory of permeability.     

High performance thin layer chromatography as a method to authenticate Hoodia gordonii raw material and products

Hoodia gordonii which contains the perceived active molecule, P57, is a plant used in many weight loss products that are highly susceptible to adulteration due to increased public demand and limited availability. Rapid and simple methods for authentication and confirmation of the presence of P57 are desirable for the quality control of H. gordonii raw material and products. High performance thin layer chromatography (HPTLC) analysis of several H. gordonii raw material samples collected from different locations as well as weight loss products was carried out on silica gel plates and developed in a mobile phase of toluene:chloroform:ethanol (40:40:12.5 v/v/v). Liebermann–Burchard (LB) reagent was used as derivatising agent since it is specific for glycosides and triterpenes (such as P57) and the plates were viewed under UV light at 365 nm. This method produced good separation of the compounds in complex mixtures with well-defined bands including that of the P57 band (R_f 0.42), which was confirmed by liquid chromatography coupled to mass spectrometry (LC–MS) after preparative thin layer chromatography (TLC). All the HPTLC results obtained for the H. gordonii raw materials and products were confirmed with quantitative LC–MS analyses, which confirmed the qualitative reliability of the HPTLC method. The HPTLC method was used successfully to develop a chemical fingerprint for authentication and reliable confirmation of the presence of P57 in H. gordonii raw material and products.     

Clustering of spin-labeled cholesterol analog diluted in bilayers of saturated and unsaturated phospholipids

Clustering of spin-labeled cholesterol analog, 3β-doxyl-5α-cholestane (DChl), diluted in bilayers comprised of either saturated dipalmitoyl-glycero-phosphocholine (DPPC) or unsaturated dioleoyl-glycero-phosphocholine (DOPC) phospholipids was studied. DChl molar fraction X varied between 0.005 and 0.04. EPR spectroscopy applied at low temperatures (200 K) enabled exploring magnetic dipole-dipole (d-d) interaction between spin labels. For DOPC bilayers, EPR spectra were found to broaden remarkably with X increase. The broadening was simulated for the models of 2-dimentional (2-D) clusters with enhanced local concentration, X_loc, which was several times larger than X, and for 1-dimensional (1-D) DChl clusters. The distance of closest approach in these simulations attained the intermolecular lateral distance in the membrane (~0.7 nm). For DPPC bilayers, EPR spectra showed only small broadening, which in these simulations could not be reproduced even if X_loc was taken as small as X. However strong concentration dependence was found for electron spin echo (ESE) decays. Both the EPR and ESE data for DPPC bilayers were explained within the model assuming encapsulation of DChl molecules in lipid shells so preventing them to approach each other closer than a certain distance, R_min. The R_min value was found to vary between ~2.5 nm and 5 nm, for X varying between 0.04 and 0.005; X_loc in these simulations was several times larger than X. So the DChl clustering in DOPC bilayers is driven by attractive lipid-mediated forces, while in DPPC bilayers long-range nanoscale lipid-mediated repulsive/attractive forces take place for distances smaller and larger R_min, correspondingly.     

Incorporation of bovine thyroid peroxidase in liposomes

Bovine thyroid peroxidase (TPO), an enzyme requiring lipids for demonstrating catalytic activity, was incorporated in liposomes made of pure phospholipids. The enzyme did not show high differences in activity when bilayer thickness was changed, but dipalmitoyl phosphatidyl choline (DPPC) seemed to be more appropiate for activity. The perturbation caused on lipid fluidity by enzyme incorporation was studied by differential scanning calorimetry (DSC) and fluorescence polarization of the apolar probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The complexes of TPO with dimyristoyl phosphatidyl choline (DMPC), DPPC, and distearoyl phosphatidyl choline (DSPC) bilayers showed transition temperatures (T_c) which were lower than the characteristic ones shown by liposomes with the respective phospholipids alone. The microsomal fraction from which TPO was extracted was in the fluid state at 37°C, the temperature at which thyroid peroxidase works ‘in vivo’. Since the effect of the protein in lowering the transition temperature of the phospholipids was so low, the contribution of phospholipids containing unsaturated fatty acids has to be essential for obtaining a fluid bilayer at body temperature.     

Interactions of ciprofloxacin with DPPC and DPPG: Fluorescence anisotropy, ATR-FTIR and P NMR spectroscopies and conformational analysis

The interactions between a drug and lipids may be critical for the pharmacological activity. We previously showed that the ability of a fluoroquinolone antibiotic, ciprofloxacin, to induce disorder and modify the orientation of the acyl chains is related to its propensity to be expelled from a monolayer upon compression [1]. Here, we compared the binding of ciprofloxacin on DPPC and DPPG liposomes (or mixtures of phospholipids [DOPC:DPPC], and [DOPC:DPPG]) using quasi-elastic light scattering and steady-state fluorescence anisotropy. We also investigated ciprofloxacin effects on the transition temperature (T_m) of lipids and on the mobility of phosphate head groups using Attenuated Total Reflection Fourier Transform Infrared-Red Spectroscopy (ATR-FTIR) and ³¹P Nuclear Magnetic Resonance (NMR) respectively. In the presence of ciprofloxacin we observed a dose-dependent increase of the size of the DPPG liposomes whereas no effect was evidenced for DPPC liposomes. The binding constants K_app were in the order of 10⁵ M^− 1 and the affinity appeared dependent on the negative charge of liposomes: DPPG > DOPC:DPPG (1:1; M:M) > DPPC > DOPC:DPPC (1:1; M:M). As compared to the control samples, the chemical shift anisotropy (Δσ) values determined by ³¹P NMR showed an increase of 5 and 9 ppm for DPPC:CIP (1:1; M:M) and DPPG:CIP (1:1; M:M) respectively. ATR-FTIR experiments showed that ciprofloxacin had no effect on the T_m of DPPC but increased the order of the acyl chains both below and above this temperature. In contrast, with DPPG, ciprofloxacin induced a marked broadening effect on the transition with a decrease of the acyl chain order below its T_m and an increase above this temperature. Altogether with the results from the conformational analysis, these data demonstrated that the interactions of ciprofloxacin with lipids depend markedly on the nature of their phosphate head groups and that ciprofloxacin interacts preferentially with anionic lipid compounds, like phosphatidylglycerol, present at a high content in these membranes.     

Synthesis of Novel Phosphatidyldihydroxyacetone via Transphosphatidylation Reaction by Phospholipase D

Phospholipase D (PLD) from Streptomyces sp. catalyzed the transfer reaction of the dipalmitoylphosphatidyl residue of 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) to dihydroxyacetone (DHA) in a biphasic system, to afford 1,2-dipalmitoyl-3-sn-phosphatidyldihydroxyacetone (DPP-DHA). The structure of DPP-DHA was identified by NMR, FAB-mass, IR, and UV spectra. PLD also catalyzed the transphosphatidylation reaction of DPP-DHA to choline, and DPPC was reproduced. In the presence of phospholipase C (PLC) from Bacillus cereus, DPP-DHA was hydrolyzed to 1,2-dipalmitoylglycerol (DPG). DPP-DHA might be another source of DHA phosphate.     

Material Studies of Lipid Vesicles in the Lα and Lα-Gel Coexistence Regimes

In this work, we utilize micropipette aspiration and fluorescence imaging to examine the material properties of lipid vesicles made from mixtures of palmitoyloleoylphosphocholine (POPC) and dipalmitoylphosphatidylcholine (DPPC). At elevated temperatures/low DPPC fractions, these lipids are in a miscible liquid crystalline (L_α) state, whereas at lower temperatures/higher DPPC fractions they phase-separate into L_α and gel phases. We show that the elastic modulus, K, and critical tension, τ_c, of L_α vesicles are independent of DPPC fraction. However, as the sample temperature is increased from 15°C to 45°C, we measure decreases in both K and τ_c of 20% and 50%, respectively. The elasticity change is likely driven by a change in interfacial tension. We describe the reduction in critical tension using a simple model of thermally activated membrane pores. Vesicles with two-phase coexistence exhibit material properties that differ from L_α vesicles including critical tensions that are 20–40% lower. Fluorescence imaging of phase coexistent POPC/DPPC vesicles shows that the DPPC-rich domains exist in an extended network structure that exhibits characteristics of a solid. This gel network explains many of the unusual material properties of two-phase membranes.     

Nonelectrolyte substitution for water in phosphatidylcholine bilayers

Glycerol substitutes for water in multilamellar phosphatidylcholine liposomes in that the fluid spaces between bilayers, as well as their main transition temperatures, heat capacities, and ethalpies are very similar in water and in pure glycerol. One major difference is that the gel state phase of dipalmitoylphosphatidylcholine (DPPC) in glycerol consists of bilayers with fully interdigitated hydrocarbon chains. Interdigitated DPPC phases are also formed in ethylene glycol or in methanol (at low methanol content). In solutions of glycerol and water, the fluid spacing between bilayers is a function of mole fraction of glycerol X_g, reaching maximum values at $\text{X}{}_{\mathrm{<mtext>g</mtext>}}\text{≌ 0.1}$ for lipid in the liquid crystalline phase and at $\text{X}{}_{\mathrm{<mtext>g</mtext>}}\text{≌ 0.3}$ for the gel phase. These changes are explained in terms of a modification of the long-range Van der Waals attractive forces by glycerol.     

Modifying dipalmitoylphosphatidylcholine monolayers by n-hexadecanol and dipalmitoylglycerol

The monolayer structure of pure dipalmitoylphosphatidylcholine (DPPC) and equimolar mixtures of DPPC/n-hexadecanol (C(16)OH) and DPPC/dipalmitoylglycerol (DPG) are studied by the film balance technique and grazing incidence X-ray diffraction measurements. At 20 degrees C, the binary systems exhibit complete miscibility. In contrast to pure DPPC monolayers, a condensing effect is observed in the presence of both non-phospholipid additives; but the phase transition behavior differs. The tilt angle of the hydrocarbon chains in the DPPC/C(16)OH mixture is significantly smaller than in pure DPPC monolayers. The tilt of the chains is even further reduced in the mixed monolayer of DPPC/DPG. A comparison of the three systems reveals distinct structural features such as phase state, chain tilt, and molecular area over a wide range of surface pressures. Therefore, these monolayers provide a highly suitable model to investigate the influence of structural parameters on biological processes occurring at the membrane surface, e.g. enzymatic reactions and adsorption events.     

Ceramide-1-Phosphate, in Contrast to Ceramide, Is Not Segregated into Lateral Lipid Domains in Phosphatidylcholine Bilayers

Sphingolipids are key lipid regulators of cell viability: ceramide is one of the key molecules in inducing programmed cell death (apoptosis), whereas other sphingolipids, such as ceramide 1-phosphate, are mitogenic. The thermotropic and structural behavior of binary systems of N-hexadecanoyl-D-erythro-ceramide (C₁₆-ceramide) or N-hexadecanoyl-D-erythro-ceramide-1-phosphate (C₁₆-ceramide-1-phosphate; C₁₆-C1P) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied with DSC and deuterium nuclear magnetic resonance (²H-NMR). Partial-phase diagrams (up to a mole fraction of sphingolipids X = 0.40) for both mixtures were constructed based on DSC and ²H-NMR observations. For C₁₆-ceramide-containing bilayers DSC heating scans showed already at X_cer = 0.025 a complex structure of the main-phase transition peak suggestive of lateral-phase separation. The transition width increased significantly upon increasing X_cer, and the upper-phase boundary temperature of the mixture shifted to ∼65°C at X_cer = 0.40. The temperature range over which ²H-NMR spectra of C₁₆-ceramide/DPPC-d₆₂ mixtures displayed coexistence of gel and liquid crystalline domains increased from ∼10° for X_cer = 0.1 to ∼21° for X_cer = 0.4. For C16-C1P/DPPC mixtures, DSC and ²H-NMR observations indicated that two-phase coexistence was limited to significantly narrower temperature ranges for corresponding C1P concentrations. To complement these findings, C₁₆-ceramide/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and C16-C1P/POPC mixtures were also studied by ²H-NMR and fluorescence techniques. These observations indicate that DPPC and POPC bilayers are significantly less perturbed by C₁₆-C1P than by C₁₆-ceramide and that C₁₆-C1P is miscible within DPPC bilayers at least up to X_C1P = 0.30.     

Penetration Depth of Surfactant Peptide KL4 into Membranes Is Determined by Fatty Acid Saturation

KL₄ is a 21-residue functional peptide mimic of lung surfactant protein B, an essential protein for lowering surface tension in the alveoli. Its ability to modify lipid properties and restore lung compliance was investigated with circular dichroism, differential scanning calorimetry, and solid-state NMR spectroscopy. KL₄ binds fluid lamellar phase PC/PG lipid membranes and forms an amphipathic helix that alters lipid organization and acyl chain dynamics. The binding and helicity of KL₄ is dependent on the level of monounsaturation in the fatty acid chains. At physiologic temperatures, KL₄ is more peripheral and dynamic in fluid phase POPC/POPG MLVs but is deeply inserted into fluid phase DPPC/POPG vesicles, resulting in immobilization of the peptide. Substantial increases in the acyl chain order are observed in DPPC/POPG lipid vesicles with increasing levels of KL₄, and POPC/POPG lipid vesicles show small decreases in the acyl chain order parameters on addition of KL₄. Additionally, a clear effect of KL₄ on the orientation of the fluid phase PG headgroups is observed, with similar changes in both lipid environments. Near the phase transition temperature of the DPPC/POPG lipid mixtures, which is just below the physiologic temperature of lung surfactant, KL₄ causes phase separation with the DPPC remaining in a gel phase and the POPG partitioned between gel and fluid phases. The ability of KL₄ to differentially partition into lipid lamellae containing varying levels of monounsaturation and subsequent changes in curvature strain suggest a mechanism for peptide-mediated lipid organization and trafficking within the dynamic lung environment.     

Estimation of amniotic fluid phospholipids by high-performance liquid chromatography

We have developed a high-performance liquid chromatographic (HPLC) method for the analyses of surface-active amniotic fluid phospholipids, lecithin (L), sphingomyelin (S), phosphatidyl glycerol (PG), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), and phosphatidyl serine (PS), which are important in the prediction of fetal lung maturity. The method incorporates an internal standard in the amniotic fluid extract, and utilizes a 10-μl aliquot of a 2:1 chloroform—methanol extract of amniotic fluid injected onto a 5-μm DIOL or CN HPLC column, and a variable-wavelength detector set at 203 nm.Amniotic fluid phospholipid estimations were determined on 40 amniotic fluid samples by the HPLC method and by the routine thin-layer chromatographic (TLC) method. Good agreement was observed between the two methods for the L/S ratio, PG, and PI (r_PG 0.94, r_PI 0.95, r_L/S 0.97).The advantages of the HPLC procedure include: (i) Selective separation for PG, PI, PS, and PE, as well as L and S at the same time. (ii) The internal standard allows individual concentration of phospholipids to be estimated. (iii) The procedure is rapid: 16 min for a single assay compared with 50 min for the standard TLC procedure.     

Disappearance of prostaglandins F2α and 15-methyl F2α from amniotic fluid as measured by radioimmunoassay

A sensitive and relatively specific radioimmunoassay for 15 (S) 15 methyl prostaglandin F_2α was used to determine the levels of the drug in amniotic fluid after it had been injected intra-amniotically for termination of second trimester pregnancy. The disappearance of the free acid (tham salt) and methyl ester of the prostaglandin analogue were similar. The results of this preliminary study suggest that the drug rapidly equilibrates in the fluid and this is followed by a slow removal from the amniotic sac. A comparison with a similar study with PGF_2α, revealed that the analogue had a longer half-life in the amniotic fluid.