Vibrational spectroscopic imaging for the evaluation of matrix and mineral chemistry.

Metabolic bone diseases manifesting fragility fractures (such as osteoporosis) are routinely diagnosed based on bone mineral density (BMD) measurements, and the effect of various therapies also evaluated based on the same outcome. Although useful, it is well recognized that this metric does not fully account for either fracture incidence or the effect of various therapies on fracture incidence, thus, the emergence of bone quality as a contributing factor in the determination of bone strength. Infrared and Raman vibrational spectroscopic techniques are particularly well-suited for the determination of bone quality as they provide quantitative and qualitative information of the mineral and organic matrix bone components, simultaneously. Through the use of microspectroscopic techniques, this information is available in a spatially resolved manner, thus, the outcomes may be easily correlated with outcomes from techniques such as histology, histomorphometry, and nanoindentation, linking metabolic status with material properties.

Imaging the distribution of sodium dodecyl sulfate in skin by confocal Raman and infrared microspectroscopy.

PURPOSE: To image SDS distribution across different skin regions, to compare the permeability difference between porcine and human skin, and to evaluate the interaction between SDS and skin. METHODS: Full thickness porcine and human skin was treated with acyl chain perdeuterated SDS (SDS-d(25)) at room temperature and at 34 °C for 3, 24 and 40 h. SDS distribution in skin was monitored by confocal Raman and IR microspectroscopic imaging. Permeation profiles of SDS-d(25) in skin were derived from the band intensities of the CD(2) stretching vibrations. The interaction between SDS and skin was monitored through the CH(2) and CD(2) stretching frequencies and the Amide I and II spectral region. RESULTS: SDS-d(25) penetrates both porcine and human skin in a time and temperature-dependent manner, with slightly higher permeability through the stratum corneum (SC) in porcine skin. When SDS permeates into the SC, its chains are more ordered compared to SDS micelles. The secondary structure of keratin in the SC is not affected by SDS-d(25). CONCLUSION: The spatial distribution of SDS-d(25) in skin was obtained for the first time. Infrared microscopic imaging provides unique opportunities to measure concentration profiles of exogenous materials in skin and offers insights to interaction between permeants and skin.

Infrared spectroscopic studies of sodium dodecyl sulphate permeation and interaction with stratum corneum lipids in skin.

The barrier function of skin is primarily provided by the lamellar lipid matrix of the stratum corneum (SC), which has been shown in previous infrared (IR) and related studies to consist predominantly of ordered lipids packed in orthorhombic and hexagonal domains. In the current work, we investigate the effects of the anionic surfactant, sodium dodecyl sulphate (SDS), on SC lipid packing and phase behaviour, using FT-IR spectroscopy. The use of acyl chain perdeuterated SDS allows unequivocal spectroscopic detection of both endogenous lipid and exogenous material in intact tissue. IR spectra were acquired as a function of temperature from isolated human SC exposed to SDS for various incubation periods at 34°C. SDS is found to enter the SC and is observed to be in a more ordered state in the SC than in solution, indicating that the SDS interacts with the ordered SC lipids. The results reveal that SDS reduces the amount of orthorhombic phase in the SC and increases the amount of hexagonally packed lipid at physiologically relevant temperatures. In addition, a small decrease in the lipid T(m) (acyl chain melting temperature) is observed. Furthermore, these SDS-induced changes were found to be strongly dependent on the time of exposure.

Real-time detection of neonatal seizures improves with on demand EEG interpretation.

OBJECTIVE: Under- and overtreatment of seizures may result in adverse outcomes; thus, early, reliable seizure identification is crucial. Continuous conventional ElectroEncephaloGram (cEEG) is the gold standard, but amplitude integrated EEG (aEEG) is most widely-used in the Neonatal Intensive Care Unit (NICU). We aimed to determine whether a novel pathway combining cEEG and aEEG for seizure detection would improve real-time seizure identification. METHODS: A single-center, prospective quality improvement project cohort. Patients at-risk of seizures were monitored by cEEG and aEEG concurrently, with the option for the neonatologist to contact a neurologist for real-time cEEG interpretation. The primary outcome was correct identification of seizures by the new combined pathway compared to aEEG alone. RESULTS: Seizure detection using aEEG had a sensitivity of 46.2%; specificity of 93.3%; PPV of 66.7%; and NPV of 85.7%. Utilizing the new on-demand, real-time cEEG interpretation by a neurologist, correct seizure identification increased by 27% (95%CI: 10-56%). Over-diagnosis of seizures was avoided in 33.3% (95% CI: 12.1-64.6%) and misuse of anti-seizure medication was prevented. CONCLUSIONS: Combining aEEG with on-demand cEEG interpretation improved accurate seizure detection in a high-risk NICU population, with the potential to avoid over-treatment. SIGNIFICANCE: We describe a novel combined EEG monitoring pathway to improve seizure detection, and prevent unnecessary treatment.

Maturational changes in dentin mineral properties.

In this study the changes in properties of the maturing mantle and circumpulpal dentin were quantitatively analyzed. Sections from six fetal bovine undecalcified incisors were used. Regions of mantle and circumpulpal dentin of sequential maturation stages were identified on spectroscopic images acquired by Fourier Transform Infrared Imaging. Spectroscopic parameters corresponding to mineral properties at these stages were analyzed and reported as a function of distance from the cervix of the incisor, the latter representing tissue age. Mineral parameters were correlated with distance from the cervix. Values of these parameters in mantle and circumpulpal dentin were compared. A multi-phasic pattern of changes was found for all the parameters examined, with most of the alterations occurring in the initial maturation period. The patterns of temporal variation in mantle and circumpulpal dentin mineral properties show distinct developmental stages and were not identical for the two dentin compartments. The study showed that mineral maturation in dentin is not a linear process and that mantle dentin is developmentally distinct from circumpulpal dentin, presenting at certain stages different physicochemical events during the maturation of the tissue.

Thermal denaturation and photochemistry of bacteriorhodopsin from Halobacterium cutirubrum as monitored by resonance Raman spectroscopy.

Resonance Raman studies of the thermal denaturation of bacteriorhodopsin from Halobacterium cutirubrum show that the N-retinylidenelysine moiety present in the chromophore is N-protonated. This corroborates an earlier suggestion of Lewis et al. ((1974) Proc. Natl. Acad. Sci. U.S., 71, 4462-4466). The widely differing excitation profiles of two -C=C- stretching modes are explained in terms of the light-initiated reaction cycle in the molecule. Glutaraldehyde fixation of bacteriorhodopsin has no effect on the intensity ratio of the two modes, suggesting that no large motion of the protein is necessary for the photoreaction cycle to occur.

Use of deuterated phospholipids in Raman spectroscopic studies of membrane structure. I. Multilayers of dimyristoyl phosphatidylcholine (and its -d54 derivative) with distearoyl phosphatidylcholine.

The temperature dependence of the Raman spectrum has been studied for binary phospholipid mixtures of dimyristoyl phosphatidylcholine (and its chain deuterated -d54 derivative) with distearoyl phosphatidylcholine. Two distinct melting regions are observed for the 1 : 1 mole ratio mixture. The use of deuterated phospholipid permits the identification of the lower (approximately 22 degrees C) transition with primarily the melting of the shorter chain component, and the higher (approximately 47 degrees C) transition primarily with the melting of the longer chains. The C-H stretching vibrations of the distearoyl component respond to the melting of the dimyristoyl component, an apparent consequence of alterations in the lateral interactions of the distearoyl chains. These changes in the C-H spectral region suggest that phase separation does not occur in the gel state for this system. The results are in reasonable accord with recent calorimetric studies (Mabrey, S. and Sturtevant, J.M. (1976) Proc. Natl. Acad. Sci. U.S. 73, 3862-3866). The feasibility of using deuterated phospholipids to monitor the conformation of each component in a binary phospholipid mixture is demonstrated.

Fourier transform infrared spectroscopic identification of gel phase domains in reconstituted phospholipid vesicles containing Ca2+-ATPase.

Ca2+-ATPase from rabbit sarcoplasmic reticulum has been isolated, purified, and reconstituted into lipid environments containing as primary components 1,2-dielaidoylphosphatidylcholine (DEPC) and acyl-chain perdeuterated 1,2-dimyristoylphosphatidylcholine (DMPC-d54). Differential scanning calorimetry (DSC) has been used to elucidate the phase behavior of this lipid pair while Fourier transform infrared spectroscopy (FT-IR) has been used to monitor the state of each lipid component in the presence of protein. The lipid mixture shows gel state miscibility over at least most of the composition range, a result in good accord with Van Dijck et al. (Biochim. Biophys. Acta 470, 58-69 (1977)), for the binary mixture with proteated DMPC. Acyl chain perdeuteration thus does not greatly alter the miscibility properties of the lipid pair. Reconstitution of Ca2+-ATPase with this lipid pair proceeds with moderate efficiency. Up to 80% of the endogenous lipid can be replaced depending on the lipid composition. Unusual composition-dependent protein-induced effects on lipid melting properties are noticed. At low levels of DMPC-d54, both the DEPC and DMPC-d54 components have their melting processes broadened and shifted to lower temperatures, compared with binary lipid mixtures of the same composition. This suggests that protein perturbs both lipids in similar fashion. At high levels of DMPC-d54, the DEPC component exhibits a highly cooperative melting process at temperatures close to that for pure DEPC. This strongly indicates that domains of DEPC are present (at least at low temperatures) in the bilayer, and that Ca2+-ATPase is excluded from these domains. The protein thus exhibits preferential interaction with the DMPC-d54 component. This work demonstrates the utility of FT-IR for identification of the molecular origin of particular domains in reasonably complex lipid mixtures. The relevance of this work to native membrane systems where lipid domains have been observed by several groups is discussed.

Deuterated phospholipids as Raman spectroscopic probes of membrane structure. Phase diagrams for the dipalmitoyl phosphatidylcholine(and its d62 derivative)-dipalmitoyl phosphatidylethanolamine system.

Raman spectroscopic techniques have been used to construct phase diagrams for the binary phospholipid systems, DPPC-d62/DPPE and DPPC/DPPE (DPPC, dipalmitoyl phosphatidylcholine; DPPE, dipalmitoyl phosphatidylethanolamine). For the former, the half-width of the C-2H stretching modes of the deuterated component near 2100 cm-1 serves as an indicator of phospholipids fluidity. The phase behavior is described semi-quantitatively using regular solution theory with the following non-ideality parameters: rho(1)0 = 0.75 kcal/mol and rho(s)0 = 1.05 kcal/mol. The use of deuterated phospholipids as one component of a binary mixture permits direct evaluation of the conformation of a particular component in the mixture throughout the phase separation region. The approach is demonstrated with the help of a simple model correlating the half-width of the symmetric C-2H stretching mode with the fraction of DPPC-d62 hydrocarbon chains in the liquid crystalline state. The effect of chain perdeuteration on the phase behavior of DPPC with DPPE is evaluated by comparison of the phase diagram of the DPPC-d62/DPPE system with that of DPPC-DPPE. The latter has been constructed previously from both probe and calorimetric techniques, and is created from the Raman spectroscopic data using the I(1130/1100) ratio to characterize the transgauche population ratio in non-deuterated hydrocarbon chains. A reasonable fit to the phase behavior is obtained using: rho(1)0 = 0.85 kcal/mol and rho(s)0 = 0.90 kcal/mol. The similarities of the non-ideality parameters in the two phase diagrams indicate that the effect of perdeuteration on the phase behavior of DPPC is not extensive. The use of deuterated phospholipids as essentially unperturbed components of a model membrane system is justified.

A comparison of differential scanning calorimetric and Fourier transform infrared spectroscopic determination of mixing behavior in binary phospholipid systems.

Fourier transform infrared (FT-IR) spectroscopy and differential scanning calorimetry (DSC) have been used to elucidate the phase behavior of two binary lipid mixtures, acyl chain perdeuterated 1,2-dipalmitoylphosphatidylethanolamine (DPPE-d62)/1,2-dielaidoylphosphatidylcholine (DEPC) and acyl chain perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC-d62)/1,2-dimyristoylphosphatidylethanolamine (DMPE). The former shows gel state immiscibility over most of the composition range. The FT-IR data indicate that one of the solid phases is essentially pure DEPC, while the other solid phase contains both lipids. The DPPC-d62/DMPE pair are miscible over the entire composition range. The use of deuterated lipids as one component in the mixture permits the melting characteristics of each component to be separately determined in the FT-IR experiment. The FT-IR data are used to assign the endotherms observed in the DSC to particular molecular components. For the DPPE-d62/DEPC system, two endotherms are observed at compositions between 10 and 67 mol% DPPE-d62. The lower transition is assigned to the DEPC component, while the higher event contains contributions to the enthalpy from both lipids in the mixture. The midpoint of the DEPC melting occurs substantially below that for DPPE-d62. For the miscible pair, each of the lipids melt over approximately the same temperature range. The complementary and consistent nature of the information available from FT-IR and from DSC is demonstrated from the current work.

Conformational disorder in unsaturated phospholipids by FTIR spectroscopy.

Conformational disorder in liquid alkenes and in the L alpha and Hparallel phases of some unsaturated phospholipids has been monitored by FTIR spectroscopy. The CH2 wagging region (1330-1390 cm-1) in saturated chains contains vibrations of particular 2- and 3-bond conformational states as follows: 1341 cm-1, end-gauche (eg); 1352 cm-1, double gauche (gg); 1368 cm-1, the sum of kink and gtg states. In unsaturated chains, this spectral region revealed an additional band at 1362 cm-1 and (occasionally) a feature near 1348 cm-1. The 1362 cm-1 band is tentatively assigned to the wagging of CH2 groups adjacent to the C = C bond. Substantial populations of both gg and (kink+gtg) states are evident in the L alpha phases of unsaturated phosphatidylcholines (PC's). Unsaturated phosphatidylethanolamines (PE's) are more ordered than their PC counterparts, and possess fewer gg and eg states. Chain disorder in the Hparallel phase of PE's approaches that in L alpha phases of unsaturated PC's. Changes in conformer distributions during the L alpha-->Hparallel transition in 1,2-dioleoylphosphatidylethanolamine (DOPE), 1-palmitoyl,2-oleoylphosphatidylethanolamine (POPE), 1,2-dielaidoylphosphatidylethanolamine (DEPE), and N-methyl-DOPE(N-MeDOPE) were semi-quantitatively estimated. For DOPE and DEPE, slight cooperative increases in both gg and (kink+gtg) states occur, for POPE only the gg population increases and for N-MeDOPE only the kink+gtg populations increase. These disorder increases are consistent with the small calorimetric delta H for this transition. Difficulties in quantitative determination of conformational disorder in unsaturated chains are discussed.

Fluctuations in IR spectral parameters detected in mixed acyl chain membranes of Acholeplasma laidlawii B.

Acholeplasma laidlawii B cells were grown at 37 degrees C on three binary C16:0-d(31)/C18:1 fatty acid mixtures at initial mol ratios of 3:2, 1:1, and 2:3. These mol ratios produced final C16:0-d(31)/C18:1 lipid acyl chain mol ratios of 1.66 +/- 0.23 (n=6), 1.3 +/- 0.20 (n=6) and 0.58 +/- 0.09 (n=10), respectively, in the membrane of the microorganism. Membrane conformational order for the deuterated and proteated acyl chains in intact cells was monitored by FT-IR spectroscopy through the thermotropic response of the acyl chain CD2 and CH2 stretching frequencies. Intact cells and isolated membranes revealed broad phase transitions centered well below the growth temperature. This result differs from previous studies (Moore, D.J. and Mendelsohn, R. (1994) Biochemistry 33, 4080-4085) of cells grown on a single saturated fatty acid source, where Tm was close to the growth temperature. Fluctuations in IR spectral parameters from the liquid crystalline phases were detected in ten separate samples of cells grown on a 2:3 mixture (final mol ratio 0.58:1) of C16:0-d(31)/C18:1, and in no other cell preparation. These were manifest by reduced precision in the measurement of CH2 and CD2 stretching frequencies and are attributed to fluctuations in the membrane conformational order. In addition to conformational order fluctuations in intact cells, similar behavior was noted for the simple binary phosphatidylcholine (PC) mixture, DOPC/1-C16:0-d(31),2-C18:1 PC (2:1 molar ratio). In this instance, the fluctuations were also detected through the temporal and thermotropic response of the relative intensity of the 1341 cm(-1) band assigned to end-gauche conformers about the penultimate C-C bond in the oleoyl chains. The relationship of these observations to the Raman spectroscopic detection of packing fluctuations in highly unsaturated PC's (Litman, B.J., Lewis, N., and Levin, I.W. (1991) Biochemistry 30, 313-319) is considered.

Do clinical levels of general anaesthetics affect lipid bilayers? Evidence from Raman scattering.

We have used Raman spectroscopy to investigate the effects of the general anaesthetics halothane and chloroform on lipid bilayer order. Clinical concentrations of these anaesthetics had no significant effect on the hydrocarbon chain conformation in multilamellar vesicles of dimyristoylphosphatidylcholine/cholesterol. This result was obtained with a technique sufficiently precise to monitor changes in the acyl chain trans-gauche population ratio associated with a 1-2 K alteration in temperature. Very high levels of anaesthetics caused a marked disordering of the hydrocarbon chains. The danger of inferring an effect at clinical concentrations from data obtained at much higher levels is illustrated by a statistical analysis of our dose-response curves.

Fourier transform infrared and differential scanning calorimetric studies of a surface-active material from rabbit lung.

A surface-active material with a chemical composition consistent with lung surfactant and with the ability to lower surface tension on a Wilhelmy balance to about 6 mN/m, has been isolated from rabbit pulmonary lavage. The thermotropic properties have been characterized with the techniques of Fourier transform infrared spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC). FT-IR melting curves were constructed from the temperature-dependence of the lipid CH2 symmetric stretching vibrational frequencies near 2850 cm-1. A broad gel-liquid crystal phase transition with an onset temperature of about 22 degrees C, and a completion temperature of about 38 degrees C was observed, with slight sample-to-sample variations in temperatures. A similar completion temperature was noted in DSC endotherms. Ca2+ (5-10 mM) increased the onset temperature of the lipid-melting event, and induced an ordering of surfactant and of its lipid extract at all temperatures studied. The effect on the lipids was suggestive of a Ca2+-induced phase separation caused by ion binding to phosphatidylglycerol and other acidic components. Evidence for a direct interaction between Ca2+ and the phosphate groups was suggested through small Ca2+-induced shifts in the 1090 cm-1 symmetric PO2 stretching frequency. Removal of most of the protein component from a 10:1 (lipid/protein, w/w) sample caused an ordering of the resultant lipid fractions. In contrast, removal of most of the protein component from a 20:1 sample resulted in no change in lipid order or thermotropic behavior. These observations are discussed in light of the roles played both by Ca2+ and protein in the spreading of surfactant. The power of FT-IR to acquire useful structural information from complex biological tissues is demonstrated.

The effect of sonication on the hydrocarbon chain conformation in model membrane systems: a Raman spectroscopic study.

Raman spectra are presented for egg lecithin above and below the gelliquid crystal phase transition, and several regions of the Raman spectrum are shown to be sensitive to conformational changes in the hydrocarbon chains. These regions are used to investigate the effect of sonication on the structure of egg lecithin and dipalmitoyl lecithin vesicles. Sonication of both egg lecithin above Tm, and dipalmitoyl lecithin above and below Tm produces no change in the relative population of trans and gauche isomers in any of the systems studied. Sonication does however appear to effect interchain interactions, a possible consequence of imperfect packing towards the center of the bilayers in vesicle systems.

Deuterated fatty acids as Raman spectroscopic probes of membrane structure.

Raman spectra are reported for the C-D stretching region of stearic acid-d35 bound in egg lecithin multilayers. The temperature dependence of the spectra shows that the linewidth of the C-D stretching bands is a sensitive and non-perturbative probe of membrane hydrocarbon chain conformation. The utility of this approach for studying lipid conformation in membranes containing a significant fraction of non-lipid component is discussed.

Effects of cholesterol on the interaction of Ca2(+)-ATPase with 1-palmitoyl-2-oleoylphosphatidylethanolamine. An FTIR study.

Ca2(+)-ATPase from rabbit skeletal muscle has been isolated, purified, and reconstituted into vesicles containing binary mixtures of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE)/cholesterol. Fourier transform infrared spectroscopy (FTIR) was used to investigate the effect of protein on the thermotropic behavior of POPE in these reconstituted ternary complexes. The CH2 symmetric stretching modes of the phospholipid acyl chains near 2850 cm-1 served as an index of the melting process. The thermotropic transition of the POPE component in a 103:12:1 (POPE/cholesterol/Ca2(+)-ATPase) complex was shifted to lower temperatures compared with a protein-free binary lipid mixture of the same relative proportions. When combined with differential scanning calorimetric (DSC) data for the binary (POPE/cholesterol) lipid systems, this observation suggests that Ca2(+)-ATPase preferentially sequesters 15-35 molecules of POPE from the lipid mixture and therefore excludes cholesterol from its immediate environment. Higher levels of cholesterol in ternary complexes progressively eliminate the cooperative POPE melting event.

Labeling of tyrosines in proteins with [15N]tetranitromethane, a new NMR reporter for nitrotyrosines.

Lysozyme and ribonuclease were used as model proteins to explore the feasibility of detecting protein-bound nitrotyrosines by 15N-NMR spectroscopy. The reporter group was introduced via synthesized [15N]tetranitromethane. Several experiments for detection of the 15N resonance in the model [3-15N]nitrotyrosine demonstrated a substantial pH-dependence of the chemical shift. When lysozyme was nitrated, either two or three 15N resonances were detected, depending on the extent of nitration. The pH-dependence of the detected resonances clearly described an apparent microscopic pK in accord with reported values, while addition of Gd(III) gave selective line broadening, indicating that the 15N reporter group could also monitor relative distances from paramagnetic sources. Nitration of ribonuclease showed five 15N resonances, of which three persisted in the purified monomer. The pH-dependence of these resonances also described apparent microscopic pK values. The [3-15N]nitrotyrosine model was reduced to the [3-15N]aminotyrosine and its 15N resonance was easily monitored by several methods, including selective population inversion. When the protein-bound nitrotyrosines were similarly reduced, much sample decomposition resulted, a possible result of photooxidation, and/or reduction of disulfide bond(s), thereby making interpretation difficult.

Palmitoylation of lung surfactant protein SP-C alters surface thermodynamics, but not protein secondary structure or orientation in 1,2-dipalmitoylphosphatidylcholine langmuir films.

Pulmonary surfactant-specific protein, SP-C, isolated from porcine lung lavage, has been deacylated to investigate the role of the two thioester linked palmitoyl chains located near the N-terminus. Surface thermodynamic properties, secondary structure, and orientation of native and deacylated SP-C in 1, 2-dipalmitoylphosphatidylcholine (DPPC) monolayers has been characterized by combined surface pressure-molecular area (pi-A) isotherms and infrared reflection-absorption spectroscopy (IRRAS) measurements. The isotherms indicate that deacylation of SP-C produces more fluid monolayers at pressures less than 30 mN m-1. The helical secondary structure and tilt angle (70-80 degrees relative to the surface normal) of SP-C remained essentially unchanged upon deacylation in DPPC monolayers at a surface pressure approximately 30 mN m-1. The results are consistent with a model that acylation of SP-C may influence the rapid protein-aided spreading of a surface-associated surfactant reservoir, but not the structure of DPPC or SP-C in the monolayer at higher surface pressures.

Some relationships between membrane phospholipid domains, conformational order, and cell shape in intact human erythrocytes.

A novel method developed in this laboratory [D.J. Moore et al., Biochemistry 35 (1996) 229-235; D.J. Moore et al., Biochemistry 36 (1997) 660-664] to study the conformational order and the propensity for domain formation of specific phospholipids in intact human erythrocytes is extended to two additional species. Acyl chain perdeuterated 1,2-dilauroylphosphatidylethanolamine (diC12PE-d46) was incorporated preferentially (in separate experiments) into the inner leaflet of stomatocytic erythrocytes and into the outer leaflet of echinocytic erythrocytes, while acyl chain perdeuterated 1,2-dipentadecanoylphosphatidylcholine (diC15PC-d58) was incorporated into the outer leaflet of echinocytic erythrocytes. The conformational order and phase behavior of the incorporated molecules were monitored through FT-IR studies of the temperature dependence of the CD2 stretching vibrations. For both diC12PE-d46 and diC15PC-d58, the gel-->liquid crystal phase transition persisted when these lipids were located in the outer leaflet of echinocytic cells, a result indicative of the persistence of phospholipid domains. In each case, the transition widths were broadened compared to the pure lipids, suggestive of either small domains or the presence of additional molecular components within the domains. The conformational order of diC12PE-d46 differed markedly depending on its location and the morphology of the cells. When located predominantly in the inner membrane of stomatocytes, the phase transition of this species was abolished and the conformational order compared with pure lipid vesicles at the same temperature was much lower. The current results along with our previous studies provide a sufficient experimental basis to deduce some general principles of phospholipid conformational order and organization in both normal and shape-altered erythrocytes.