Unsaturated Phospholipids Research Articles

Cholesterol Pushes Vitamin E and Polyunsaturated Phospholipids Together

Vitamin E (α-tocopherol) is an essential micronutrient that protects membrane lipids from oxidative damage. Whether this lipid-soluble vitamin preferentially locates in the vicinity of polyunsaturated phospholipids that are most vulnerable to oxidation continues to be debated. We apply MD simulations to address this issue. A key role for cholesterol, ubiquitous in mammalian membranes, is revealed. Binding energies measured for vitamin E in all-atom (AA) umbrella sampling simulations are lower for polyunsaturated phospholipids than less unsaturated phospholipids - they do not indicate preferential interaction with polyunsaturated phospholipids. There is a reduction in the binding energy following the introduction of cholesterol, which we suggest drives vitamin E and polyunsaturated phospholipids together. Support is provided by largescale simulations performed over an extended timescale on vitamin E in mixtures of PDPC (1-palmitoyl-2-docosahexaenoylphosphatidylcholine) with SM (sphingomyelin) and cholesterol that separate into a (raft-like) domain enriched in SM and cholesterol (raft-like) and a (non-raft ) region composed primarily of PDPC (non-raft) but depleted in cholesterol. In coarse-grained (CG) and AA simulations vitamin E is excluded from the raft-like domain and thereby placed in closer proximity to polyunsaturated phospholipids. We surmise that the much greater abundance of cholesterol, which has high affinity for saturated SM, relative to vitamin E is responsible.

Effect of cholecalciferol on unsaturated model membranes

Vitamin D plays an important role in many physiological processes, particularly calcium and phosphorous homeostasis. The biochemistry of vitamin D is also complex, encompassing a range of active molecules that may be either endogenous or dietary in origin. The role of lipids and fats in the production, processing and use of vitamin D is an interesting one, with a relative paucity of model studies into the interactions of vitamin D with lipidic systems such as micelles and vesicles. Here, we have studied the effect of vitamin D3 in simple unsaturated phospholipid systems. We used NMR and FTIR spectroscopy to investigate the effect of increasing vitamin D concentration on the structure and dynamics of the lipid chains and interfacial region. In order to link these model studies with more complex biomimetic environments, we compare results in the presence of buffer and vitamin D binding protein. We have also used DLS to determine that vitamin D3-DOPC vesicles can retain their size distribution for varying amounts of time in different conditions. We find that the acyl chain region of vitamin D3-DOPC membranes are generally disordered, and that the addition of buffer and/or protein alters the properties of the interfacial region.

Multi-omic analysis unveils biological pathways in peripheral immune system associated to minimal hepatic encephalopathy appearance in cirrhotic patients

Patients with liver cirrhosis may develop minimal hepatic encephalopathy (MHE) which affects their quality of life and life span. It has been proposed that a shift in peripheral inflammation triggers the appearance of MHE. However, the mechanisms involved in this immune system shift remain unknown. In this work we studied the broad molecular changes involved in the induction of MHE with the goal of identifying (1) altered genes and pathways in peripheral blood cells associated to the appearance of MHE, (2) serum metabolites and cytokines with modified levels in MHE patients and (3) MHE-regulated immune response processes related to changes in specific serum molecules. We adopted a multi-omic approach to profile the transcriptome, metabolome and a panel of cytokines of blood samples taken from cirrhotic patients with or without MHE. Transcriptomic analysis supports the hypothesis of alternations in the Th1/Th2 and Th17 lymphocytes cell populations as major drivers of MHE. Cluster analysis of serum molecules resulted in six groups of chemically similar compounds, suggesting that functional modules operate during the induction of MHE. Finally, the multi-omic integrative analysis suggested a relationship between cytokines CCL20, CX3CL1, CXCL13, IL-15, IL-22 and IL-6 with alteration in chemotaxis, as well as a link between long-chain unsaturated phospholipids and the increased fatty acid transport and prostaglandin production. We found altered immune pathways that may collectively contribute to the mild cognitive impairment phenotype in MHE. Our approach is able to combine extracellular and intracellular information, opening new insights to the understanding of the disease.

Heterogeneous Ganglioside-Enriched Nanoclusters with Different Densities in Membrane Rafts Detected by a Peptidyl Molecular Probe.

The specific features of the lateral distribution of gangliosides play key roles in cell-cell communications and the onset of various diseases related to the plasma membrane. We herein demonstrated that an artificial peptide identified from a phage-displayed library is available as a molecular probe for specific ganglioside nanoclustering sites in caveolae/membrane rafts on the cell surface. Atomic force microscopy studies indicated that the peptide specifically binds to the highly enriched monosialoganglioside GM1 nanodomains of reconstituted lipid bilayers composed of GM1, sphingomyelin, cholesterol, and unsaturated phospholipids. The ganglioside-containing area recognized by the peptide on the surface of PC12 cells was part of the area recognized by the cholera toxin B subunit, which has high affinity for GM1. Furthermore, the peptide bound to the cell surface after a treatment with methyl-β-cyclodextrin (MβCD), which disrupts membrane rafts by removing cholesterol. The present results indicate that there are heterogeneous ganglioside clusters with different ganglioside densities in caveolae/membrane rafts, and the peptidyl probe selectively recognizes the high-density ganglioside nanodomain that resists the MβCD treatment. This peptidyl probe will be useful for obtaining information on the lipid organization of the cell membrane and will help clarify the mechanisms by which the lateral distribution of gangliosides affects biological functions and the onset of diseases.

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph–mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.

The study of lipotropic action of extract from fruit prunus domestica «Prunofit» byanimal models of alcoholic liver diseases

Searchingof drugs that normalize the function of the digestive glands are important because disruption of digestion underlie the pathogenesis of many diseases such as gastroesophageal reflux disease, hepatitis, gastric ulcer, irritable bowel syndrome, cancer etc. The use of herbal objects containing fibres is a promising direction for solving of the mentioned problem. We were attracted by fruits of Prunus domestica which are rich in fibres (homo- and heteropolsaccharides) and are used in folk medicine as a laxative and hepatoprotective agent.
 The aim of this experimental study was to investigate the lipotropic properties of «Prunofit» extract. The study of lipotropic properties of the «Prunofit» was carried out in the conditions of subacute toxic liver damage caused by the introduction of ethanol.The content of total lipids (TL), total cholesterol (TCh), triacylglycerols (TGs), unsaturated fatty acids (UFA) and total phospholipids (TPL) were determined in liver and blood serum.
 Obtained results of the lipotropic properties study of the «Prunofit» extract at a dose of 200 mg/kg against the background of alcoholic liver damage showed a decrease in the intensity of lipolysis, fatty hepatosis, and manifestations of hyperlipidemia. It has occurred due to a decrease in the content of total lipids, cholesterol, triglycerides and free fatty acids in the homogenate rat liver by 27.9%, 7.2%, 27.5% and 38%, respectively, and rat serum by 28%, 42.2%, 8.15%, 47.1%, respectively, compared with the control pathology. Against the background of model pathology, «Prunofit» extract tended to increase the content of total phospholipids in the liver homogenate by 37.34% and in serum by 30.2% compared with the control pathology. According to its ability to inhibit fatty liver infiltration, the «Prunofit» extract was at the level of the reference drug «Methionine» at a dose of 155 mg/kg.

2D and 3D Control of Lipid Bilayer Membranes for Molecular Sensors

The lipid bilayer structure is the main component of cell membranes, therefore, the physical properties of such structure have been studied for many years. Recently, the applications of lipid bilayers have been studied, in addition to fundamental studies. For instance, a transistor-like device was developed (Ma et al., ACS Omega, 2019).As a part of such studies, we show the 2D and 3D control of lipid bilayer membranes. The first one is the effect of local anesthetics, Dibucaine hydrochloride molecules, on the phase behavior of ternary lipid membranes, which corresponds to the 2D control of lipid bilayer membranes (Yoshida et al., MedChemComm, 2015). The lipid membranes composed of a saturated phospholipid, unsaturated phospholipid, and cholesterol induce lateral phase separation. We revealed that the phase behavior is affected by the insertion of local anesthetic molecules into bilayer structures. Another one is the effect of toxic polar molecules, acetonitrile molecules, on the shape of lipid bilayer vesicles, which corresponds to the 3D control of lipid bilayer membranes (Yoshida et al., Colloids Interfaces, 2018). Insertion of acetonitrile molecules induces the membrane deformation.We are trying to utilize such behaviors for soft and bio-inspired molecular sensors. We show the trials for such applications.

Modifying Effect of Low Salinity on Ni-Induced Alterations of the Lipid Composition in Mussels Mytilus edulis L.

The composition of lipids and fatty acids (FAs) in mussels Mytilus edulis exposed to various concentrations of nickel salt under reduced salinity of seawater (15‰) has been investigated. Alterations of the lipid composition in mussels in response to reduced salinity and the exposure to Ni have been observed. It is noted that changes in the content of phosphatidylserine and cholesterol and in phospholipid unsaturation in the gills and hepatopancreas of mussels under low salinity, most likely, indicate the development of pathological processes and activation of lipid peroxidation. Under normal salinity (25‰), similar changes in the lipid composition of the mussels studied have been observed at the initial stages of the experiment; however, by the end of the experiment, the phospholipid unsaturation is shown to be restored and the content of phosphatidylcholine increases.

Light-activatable liposomes for repetitive on-demand drug release and immunopotentiation in hypoxic tumor therapy

External stimuli-responsive nanomedicine with desirable repetitive on-demand drug release character is postulated to greatly accommodate patients' flexible medication regime. To this object, light-activatable liposomes (Pt/Ce6-LP) integrated with both a Ce6 photodynamic component and a tetravalent platinum prodrug (Pt(IV)) chemotherapeutic component are engineered. This multifunctional system was rationally designed using unsaturated phospholipid to achieve repetitive on-demand drug release under discontinuous light irradiation, thus performing chemo-photodynamic therapy effect and immunopotentiation in hypoxic tumor. Furthermore, glutathione (GSH) consumption during transformation from Pt(IV) prodrug to Pt(II) can avoid depletion of reactive oxygen species (ROS) in photodynamic therapy (PDT). Note this positive feedback loop appears to remodel the redox balance of H2O2 and GSH in tumors, alleviating the hypoxic tumor microenvironment. The alleviated hypoxia is found to be critical to the enhancement of PDT efficacy, reversal of cisplatin resistance in tumors, and polarization of tumor-associated macrophages (TAMs) to the immunocompetent M1-phynotype. Pt/Ce6-LP with light radiation demonstrates significant antitumor effect and persistent post-medication inhibition in patient-derived tumor xenograft model of hepatocellular carcinoma.

Effect of Head Group on Low-Level Ozone Oxidation of Unsaturated Phospholipids on a Water Surface

Abstract The effect of the head group on the oxidation of unsaturated phospholipids under low-level ozone was investigated. The phospholipids with head groups of glycerol and choline show different surface-pressure profiles but similar degradation kinetics of the C=C moieties was observed by heterodyne-detected sum frequency generation spectroscopy.

Modulation of Membrane Fluidity to Control Interfacial Water Structure and Dynamics in Saturated and Unsaturated Phospholipid Vesicles

The structure and dynamics of interfacial water in biological systems regulate the biochemical reactions. But, it is still enigmatic how the behavior of the interfacial water molecule is controlled. Here, we have investigated the effect of membrane fluidity on the structure and dynamics of interfacial water molecules in biologically relevant phopholipid vesicles. This study delineates that modulation of membrane fluidity through interlipid separation and unsaturation not only mitigate membrane rigidity but also disrupt the strong hydrogen bond (H-bond) network around the lipid bilayer interface. As a result, a disorder in H-bonding between water molecules arises several layers beyond the first hydration shell of the polar headgroup, which essentially modifies the interfacial water structure and dynamics. Furthermore, we have also provided evidence of increasing transportation through these modulated membranes, which enhance the membrane mediated isomerization reaction rate.

Oxidation of Unsaturated Phospholipids: A Monolayer Study.

Lipid oxidation does strongly influence the self-organization of plasma membranes; the detailed influence is not yet clear. In this work, phospholipid monolayers at the air/water interface were used as model membranes. Oxidation was induced by the reactive oxygen species formed in a H2O2-enriched solution. The reaction was found to be diffusion-limited; the concentration of the reactive oxygen species was about 50 nM. Isotherms were recorded for different phosphatidylcholines with saturated and unsaturated acyl chains. For unsaturated lipids, the isotherms showed a constant relative molecular area increase after oxidization, independent of the molecular area and dependent on the degree of peroxidation. Similarly, the compressibility modulus was unchanged, but shifted to larger molecular areas. The correlation between peroxidation and changes of the interaction forces between the lipid molecules is discussed.

Spectroscopic, molecular dynamics simulation and biological studies of Flavin MonoNucleotide and Flavin Adenine Dinucleotide in biomimetic systems.

The present study describes a comprehensive investigation of the spectroscopic characteristics, stability and in vitro antioxidant and cytotoxic properties of the Flavin MonoNucleotide (FMN) and Flavin Adenine Dinucleotide (FAD) in Dextran70 (Dx70) and Dx70/phospatidylcholine (PC) biomimetic systems by means of the UV-Vis absorption, fluorescence spectroscopy, chemiluminescence and Neutral Red assay. The affinity of FMN, FAD and the precursor riboflavin (RF) to an unsaturated phospholipid bilayer model as well as the location of the probes within the lipid bilayer were assessed from united-atom molecular dynamics simulations carried out on an unsaturated phospholipid bilayer model system, and the theoretical and experimental characterization of the two probes within biomembranes was complemented with the light microscopy survey of the cell morphology of L929 fibroblast cells cultivated in the presence of various dosage of FAD/FMN. In lipid bilayers, FMN/FAD resulted in a noticeable improvement of the antioxidant activity (the scavenging of reactive oxygen species up to 40%) and a significant effect on cellular viability in the L929 fibroblast cells. The results are important in the oxidative stress process concerning the redox reactions of flavins in humans as well as in further studies on different systems belonging to the category of flavoenzymes/flavoproteins, required for cellular respiration.

Thermodynamic quantification of sodium dodecyl sulfate penetration in cholesterol and phospholipid monolayers

Sodium dodecyl sulfate (SDS) is one of the extensively used surfactants in bioprocesses. Present work analyzes the penetration of SDS in pure and mixed Langmuir monolayers consisting of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol. A series of isotherms are obtained by compressing the monolayers over subphase containing varying concentrations of SDS. The extent of penetration of SDS is quantified in terms of its mole percentage in the monolayer by applying various thermodynamic equations derived from Gibbs adsorption equation. Analysis of pure monolayers shows that SDS penetration is directly correlated with the monolayer fluidity and the lipid packing density. We find that POPC monolayers, which do not pack closely on compression due to a kink in one of the acyl chains, exhibit higher SDS penetration than DPPC monolayers which are more rigid due to two saturated acyl chains that pack very efficiently. The SDS penetration is also analyzed in mixed lipid monolayers in which the lipids are present in molar proportions similar to that in the plasma membranes of mammalian cells. Thermodynamic quantification shows that mixed monolayers of DPPC with POPC (in 2:1 molar ratio) exhibit penetration similar to pure POPC monolayers indicating dominating effect of unsaturated phospholipids on membrane packing. We find lower SDS penetration in mixed cholesterol-POPC monolayers than pure POPC monolayers at all surface pressures and for all SDS subphase concentrations used. This confirms with the well-known condensation effect of cholesterol on POPC monolayers. Cholesterol is known to have a rigidifying effect on DPPC monolayers at low surface pressures and a fluidizing effect at high surface pressures. Consistent with these experimental observations, our thermodynamic quantification predicts lower SDS penetration at low pressures and higher penetration at high pressures when compared to pure DPPC monolayers. Thus, thermodynamic theories are able to accurately predict the experimentally observed trends for penetration in mixed monolayers of cholesterol with POPC and DPPC. Our work demonstrates that thermodynamic quantification is a reliable technique to estimate extent of penetration of various additives in pure and mixed lipid monolayers.

Nicardipine Loaded Solid Phospholipid Extrudates for the Prevention of Cerebral Vasospasms: In Vitro Characterization.

The aim of the study was to develop nicardipine loaded phospholipid extrudates as an alternative for PLA/PLGA-based implants for the prevention of cerebral vasospasms. Extrudates of different mixtures of saturated and unsaturated phosphatidylcholine (PC) were produced and characterized by DSC, microscopy and texture analysis. Single phospholipid components were identified by ELSD-HPLC. Extrudates of 2 mm diameter were obtained by twin screw extrusion temperatures below 50 °C. The ratio of unsaturated and saturated phosphatidylcholine components determines the physicochemical properties of the extrudates as well as the rate of erosion. Nicardipine loaded phospholipids extrudates released the drug over several weeks in vitro. The phospholipid composition of the remaining extrudate changed during the release, the content of unsaturated phospholipids decreased faster compared to the saturated ones. In conclusion, solid phospholipid extrudates are promising materials for the development of new parenteral controlled release systems.

Hepatic Levels of DHA-Containing Phospholipids Instruct SREBP1-Mediated Synthesis and Systemic Delivery of Polyunsaturated Fatty Acids.

SummaryPolyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and arachidonic acid (ARA), play fundamental roles in mammalian physiology. Although PUFA imbalance causes various disorders, mechanisms of the regulation of their systemic levels are poorly understood. Here, we report that hepatic DHA-containing phospholipids (DHA-PLs) determine the systemic levels of PUFAs through the SREBP1-mediated transcriptional program. We demonstrated that liver-specific deletion of Agpat3 leads to a decrease of DHA-PLs and a compensatory increase of ARA-PLs not only in the liver but also in other tissues including the brain. Together with recent findings that plasma lysophosphatidylcholine (lysoPC) is the major source of brain DHA, our results indicate that hepatic AGPAT3 contributes to brain DHA accumulation by supplying DHA-PLs as precursors of DHA-lysoPC. Furthermore, dietary fish oil-mediated suppression of hepatic PUFA biosynthetic program was blunted in liver-specific Agpat3 deletion. Our findings highlight the central role of hepatic DHA-PLs as the molecular rheostat for systemic homeostasis of PUFAs.

Biophysical parameters of the Sec14 phospholipid exchange cycle – Effect of lipid packing in membranes

Sec14, a yeast phosphatidylinositol/phosphatidylcholine transfer protein, functions at the trans-Golgi membranes. It lacks domains involved in protein-protein or protein-lipid interactions and consists solely of the Sec14 domain; hence, the mechanism underlying Sec14 function at proper sites remains unclear. In this study, we focused on the lipid packing of membranes and evaluated its association with in vitro Sec14 lipid transfer activity. Phospholipid transfer assays using pyrene-labelled phosphatidylcholine suggested that increased membrane curvature as well as the incorporation of phosphatidylethanolamine accelerated the lipid transfer. The quantity of membrane-bound Sec14 significantly increased in these membranes, indicating that “packing defects” of the membranes promote the membrane binding and phospholipid transfer of Sec14. Increased levels of phospholipid unsaturation promoted Sec14-mediated PC transfer, but had little effect on the membrane binding of the protein. Our results demonstrate the possibility that the location and function of Sec14 are regulated by the lipid packing states produced by a translocase activity at the trans-Golgi network.

Sphingomyelin Acyl Chains Influence the Formation of Sphingomyelin- and Cholesterol-Enriched Domains

The segregation of lipids into lateral membrane domains has been extensively studied. It is well established that the structural differences between phospholipids play an important role in lateral membrane organization. When a high enough cholesterol concentration is present in the bilayer, liquid-ordered (Lo) domains, which are enriched in cholesterol and saturated phospholipids such as sphingomyelin (SM), may form. We have recently shown that such a formation of domains can be facilitated by the affinity differences of cholesterol for the saturated and unsaturated phospholipids present in the bilayer. In mammalian membranes, the saturated phospholipids are usually SMs with different acyl chains, the abundance of which vary with cell type. In this study, we investigated how the acyl chain structure of SMs affects the formation of SM- and cholesterol-enriched domains. From the analysis of trans-parinaric acid fluorescence emission lifetimes, we could determine that cholesterol facilitated lateral segregation most with the SMs that had 16 carbon-long acyl chains. Using differential scanning calorimetry and Förster resonance energy transfer techniques, we observed that the SM- and cholesterol-enriched domains with 16 carbon-long SMs were most thermally stabilized by cholesterol. The Förster resonance energy transfer technique also suggested that the same SMs also form the largest Lo domains. In agreement with our previously published data, the extent of influence that cholesterol had on the propensity of lateral segregation and the properties of Lo domains correlated with the relative affinity of cholesterol for the phospholipids present in the bilayers. Therefore, the specific SM species present in the membranes, together with unsaturated phospholipids and cholesterol, can be used by the cell to fine-tune the lateral structure of the membranes.

Lipoprotein ability to exchange and remove lipids from model membranes as a function of fatty acid saturation and presence of cholesterol

Lipoproteins play a central role in the development of atherosclerosis. High and low-density lipoproteins (HDL and LDL), known as ‘good’ and ‘bad’ cholesterol, respectively, remove and/or deposit lipids into the artery wall. Hence, insight into lipid exchange processes between lipoproteins and cell membranes is of particular importance in understanding the onset and development of cardiovascular disease. In order to elucidate the impact of phospholipid tail saturation and the presence of cholesterol in cell membranes on these processes, neutron reflection was employed in the present investigation to follow lipid exchange with both HDL and LDL against model membranes. Mirroring clinical risk factors for the development of atherosclerosis, lower exchange was observed in the presence of cholesterol, as well as for an unsaturated phospholipid, compared to faster exchange when using a fully saturated phospholipid. These results highlight the importance of membrane composition on the interaction with lipoproteins, chiefly the saturation level of the lipids and presence of cholesterol, and provide novel insight into factors of importance for build-up and reversibility of atherosclerotic plaque. In addition, the correlation between the results and well-established clinical risk factors suggests that the approach taken can be employed also for understanding a broader set of risk factors including, e.g., effects of triglycerides and oxidative stress, as well as local effects of drugs on atherosclerotic plaque formation.

Interactions of cryoprotective agents with phospholipid membranes - A Langmuir monolayer study

The influence of four common cryoprotectants (dimethyl sulfoxide, glycerol, ethylene glycol and dimethylformamide) on monolayers of four common phospholipids (DPPC, DOPC, POPC and POPE) have been studied using Langmuir isotherms and monolayer insertion experiments. The cryoprotectant concentrations were chosen to be directly relevant to cryoprotection. We show that DMSO causes an expansion of the DPPC area per lipid (in contrast to previous work at higher concentrations). However, it caused compression for POPC, and had little effect for POPE or DOPC. As most previous studies have involved only DPPC, this highlights the importance of studying different lipid types as these may have a significant effect on the interactions. We show that both ethylene glycol and glycerol cause a small expansion of the monolayer at fixed pressure, implying that they insert into the headgroup regions, regardless of lipid species, and consistent with their ability to penetrate membranes. By contrast, dimethylformamide causes monolayer compression for all lipid species, implying it dehydrates the lipid head groups. Membrane insertion experiments at physiological values of lateral pressure highlight that DPPC is the most difficult lipid to penetrate, implying that the penetrating action of cryoprotectants may only occur for unsaturated phospholipids. Thus, extrapolations of results based solely on the DPPC need to be made with care.