Debouzy et al.indd ORIGINAL RESEARCH Correspondence: Jean-Claude Debouzy, Centre de Recherche du Service de Santé des Armées, 24, Avenue des maquis du Grésivaudan, BP 87, 38702 La Tronche Cedex France. Tel: (33) 4 76 63 69 39; Fax: (33) 4 76 63 69 22; Email: jcdebouzy@crssa.net Copyright in this article, its metadata, and any supplementary data is held by its author or authors. It is published under the Creative Commons Attribution By licence. For further information go to: http://creativecommons.org/licenses/by/3.0/. Study of Alkylglycerol Containing Shark Liver Oil: A Physico Chemical Support for Biological Effect? Jean-Claude Debouzy1, David Crouzier1, Bertrand Lefebvre2 and Vincent Dabouis1 1Unité BCM Centre de Recherches du Service Santé des Armées, 24, avenue des maquis du Grésivaudan, BP 87-38 702 La Tronche Cedex, France. 2Unité de Biospectrométrie Centre de Recherches du Service Santé des Armées, 24, avenue des maquis du Grésivaudan, BP 87-38 702 La Tronche Cedex, France. Abstract: Shark liver oil (SLO), is used in natural medicine as immunity stimulant, cardiovascular protector and anti ageing reagent. These properties were related with the high amounts of alkylglycerols (22%) obtained from Greenland shark liver. After a control of the mean SLO composition by NMR and MS, surface and membrane interactions and antioxidant properties were investigated using NMR, ESR and ST measurements and the in vitro consequences on erythrocytes and cells were studied. An estimation of the composition of this extract was performed. Moreover, SLO was found not haemolytic (A concentration induc- ing 50% haemolysis, HC50 could not be reached) and superfi cial tension measurements revealed slight tension active properties. The 31P and 2H-NMR and ESR studies of phospholipid dispersions (dimyristoyl phosphatidyl cholin, DMPC) in the presence of SLO showed a signifi cant increase in membrane fl uidity at low temperature (below phase transition temperature) predomi- nantly observed at the surface level. The anti oxidant activity was also confi rmed, similar as that observed for vitamin E. Keywords: alkylglycerol, membrane fl uidity, ESR, NMR, anti oxidant properties 1. Introduction Greenland sharks, and especially somnosius microcephalus are very robust species well adapted to hard envi- ronmental conditions such as deep and cold surroundings. In empiric traditional Scandinavian medicine, meat and oil from Greenland shark have been extensively used for healing of wounds, physical stress tolerance, and also immune stimulation and antitumor properties. These uses continuously faded out during the 19th century until early 20th when specifi c lipids (up to 50% in shark livers [1]) were identifi ed as alkylglycerols [2] (1-O-alkyl-2,3-diacylglycerols and their metoxy derivatives [3]). This led to the fi rst trials of A.Brohult [4] who evidenced increased production of granulocytes and thrombocytes and proposed the use of alkylglycer- ols to counterbalance the bone marrow depletion after radiotherapy in the therapy of carcinomas of the uterine cervix [5]. Later, alkyglycerols have been found to inhibit the growth and spread of transplanted or chemically induced tumors [6]. Immunological system stimulation was also identifi ed, leading to bacteriostatic properties. It is noteworthy that the same substance, shown to be effective per os, exhibited both immunoreactivity stimulating and antitumor activity. Among the different mechanisms proposed, the ability of alkylglycerol to penetrate the cell membranes would stimulate the body’ s own defense system, mainly the macrophages. Besides, the results found when alkylglycerol was given before radiotherapy would also support the hypoth- esis of a direct interaction with radio induced free radical production. However, no precision of the mechanism involved was clearly proposed at the molecular level. This led us to investigate the biophysical properties of shark liver oil (SLO), the commercial form, by using both NMR and ESR spectroscopies, and assignment biophysics methods both in synthetic systems (phospholipidic membranes) and in red blood cells. Experimental Materials ALKYROL® ALKYROL® oil extract from Greenland shark liver was purchased by NUTRILYS® Company (Divonne les bains, France) and used without further purifi cation. As this extract is a natural mixture, the amounts Drug Target Insights 2008:3 125-135 125 http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ Debouzy et al Drug Target Insights 2008:3 of SLO are better expressed in this paper in mg rather than in mM concentrations (even if the apparent density was estimated at 0.71). This product was characterized by NMR (see Fig. 1 for peak assignment) and MS (ES+) analysis. As major MS peaks were found at m/z = 701; and also 633; 517; 351(m/2z), and from 1H-NMR peak integration and 13C direct spectra and DEPT datas, an very coarse estimation of the mean apparent molecular weight M#650 was proposed SLO. The corresponding molar ratios might be considered as only indicative and close to W/W ratios (molecular mass for Dimyristoylphos- phatidylcholine, DMPC, is 678). Chemicals Dimyristoylphosphatidylcholine (DMPC), egg yolk phosphatidylcholine (EPC), and deuterated solvents were purchased from Sigma (La Verpillère, France) and were used as received. Chain perdeu- terated DMPC-d54 was from Interchim, Montluçon, France. Multibilayers (MLV) DMPC liposomes for 31P experiments were pre- pared in pure deuterated water by successive freezing and thawing cycles [7] until an homogenous milky sample was obtained. [8] The suspensions were degassed under nitrogen gas then introduced into NMR tubes and sealed. The fi nal lipid con- centration was 50 mM (in 500 μL samples), while SLO/DMPC in mixed systems was ranged from 1/50 to 1/25, mg/M. The same procedure was used for multilayers for 2H-NMR experiments, except that 25% DMPC with perdeuterated chains were used (DMPC-d54) to build the liposomes. Methods Haemolytic activity All procedures were in accordance with the stan- dards for animal care established by our institute and were approved by our animal use ethic com- mittee (decree 87-848 19 October 1987). Blood from male Sprague-Dawley rats was col- lected in heparinated tubes and washed twice using isotonic NaCl solution; the hematocrit was then brought to 10%. 1 mL cuves were fi lled with the SLO solutions to test (0 to 32 μL) in 50 μL of DMSO and with 100 μL of the diluted blood in saline. The samples were stocked for 1 hour at 37 °C, then cen- trifuged at 2400 rpm, 4 °C for 10 minutes. Absorp- tion measurements were fi nally performed on a Shimazu MCS-2000 absorption spectrometer at 540 nm, as described elsewhere. [1, 9] The haemolytic activities were expressed in terms of HC50, the concentration giving 50% hae- molysis as referenced to i) the total haemolysis induced by triton X-100 addition or on sonicated samples ii) the absence of any haemolysis (0% haemolysis) evaluated on samples where only isotonic NaCl (0.9% W/W) solution was added. NMR experiments All NMR experiments were recorded on a Brüker AM-400 spectrometer. 1H-NMR spectra in D2O were acquired at 298 K using a presaturation of the water resonance and a spectral width of 10 ppm. The chemical shifts were referenced by setting the water resonance at 4.75 ppm. 1H-NMR control spectra were recorded using classical 1D and 2D (COSY, TOCSY (Sanders, 1989) experiments at 300 K, 2 mg, in perdeuterated di methyl sulfoxide (DMSO-d6). In 1H-NMR T1 and T2 measurements in water preparation (1 mg, D2O, 298 K) used the inversion recovery method [10] with a 10 ppm line width and a 5sec recycling delay to ensure relaxation. Figure 1. Top: proton nomenclature used for acyl chain labeling; middle: 1H-NMR spectrum of SLO; 1 mg in CDCl3, 298 K: bottom: 1 mg in D2O, 298 K; Glycerol proton labeling: G1, G3, methylenic groups, G2, methinic group. 126 Study of alkylglycerol containing shark liver oil Drug Target Insights 2008:3 Partition coefficient (LogP) calculation was realized by using an NMR method derived from the classical Shake-fl ask method [9]: A fi rst 1H-NMR spectrum was recorded acquired as previously in water, while using. 1 mg SLO in 1 mL D2O to ensure that the NMR observation area is fully included in the aqueous solution. Then an equal volume (1 mL) of perdeuterated octanol was added, and the sample vortexed and centrifugated to allow phase separa- tion. The octanol phase was then removed. Another spectrum was the acquired and the intensity signal I (that of CH3 at 0.89 ppm) compared to the intensity of SLO in pure water, (Io). Thus, if R = I/Io, i.e. the remaining fraction of SLO in the water, P = (1-R)/R gives the partition in the sample, and the partition coeffi cient LogP is obtained by LogP = log((1−R)/R) 31P-NMR experiments were performed at 162 MHz. Phosphorus spectra were recorded using a dipolar echo sequence (π/2-t-π-t) [11] with a t value of 12 μsec and a broadband two levels proton decoupling π/2 pulse was 4.8 μs, recycling delay of 5sec. Phosphoric acid (85%) was used as external reference. Undecoupled spectra and partial continuous wave proton low level decoupling (24L) were also used to measure phosphorus- proton coupling constants. 2H-NMR experiments were performed at 61 MHz. MLV were formed as for 31P experiments whereas in deuterium depleted water. Deuterium spectra were recorded by using a quadrupolar echo sequence (π/2-t-π/2-t) with a t value of 20 μsec; π/2 pulse was 8 μs and recycling delay of 15sec. The free induction decay was shifted by fractions of the dwelling time to ensure that its effective time for the Fourier trans- form started at the top of the echo. Surface tension measurements Measurements were done on a Tensiometer CSC-Du Nouy (CSC N°70535) using the ring method of measurement in 20 ml of water. Pure water from MilliQ (18.2 MΩ.cm) was used as reference (75.8 mN/m at 293 K). Electron spin resonance (ESR): spin trapping investigation Anti radical activity was assessed by in vitro spin trapping experiment. Reactive oxygen species were generated immediately before ESR experiment by a Fenton reaction (FeSO4, 0.1 mM and H2O2, 0.1 mM). The formation of short-lived radical spe- cies (.OH) was evidenced by addition of Water Soluble α-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN) (Sigma, France) at 150 mM (in DMSO/ H2O solution 5% V/V) spin trapping agent. Reac- tion was performed in an Eppendorf tube, 100 μL of FeSO4 were mixed with 100 μL of 4-POBN spin trap and with 2 μL of Alkyrol®. The trigger of reaction was performed by adding 100 μL of H2O2. Reference samples were prepared by replacing Alkyrol® by distilled water, Anti radical properties were also compared by replacing Alkyrol® by 2 μL of Vit E. The samples were transferred in 20 μL Pyrex capillary tube, an placed in 3 mm diameter quartz holder. The spectra were acquired using the continuous wave mode with a ESP 380 (Brucker) ESR spec- trometer, operating at a microwaves frequency of 9.71 GHz. The instrumental parameter were: microwave power of 10 mW, modulation frequency at 100 kHz with a modulation amplitude of 0.51 G, receiver gain was 6.30 × 104 and scan range was 70 G with magnetic fi eld centred at 3430 G. Each sample was scanned 3 times at controlled tem- perature 295 K, with the following acquisition parameters: Time constant 20.48 ms, conversion time 20.48 ms and 5 repetitions. Figure 6 B shows typical ESR spectra of the control groups with the 4-POBN spin trap. An estimation of free radical promotion was obtained by measuring the amplitude of the central doublet. ESR spin label study The fl uidity of rat red cell membrane was investi- gated by ESR spin label experiments. Two spin labels (Sigma France) were used: 5 nitroxide stearate (5 NS) and 16 nitroxide stearate (16 NS). This fatty acids self incorporate the membrane and the nitroxide groups provide information of motional freedom of the label in biological mem- brane. So the former probes the superfi cial part of the membrane layer, the latter in its hydrophobic core. [12] The experiments were performed on rat red cells. The erythrocytes were isolated from fresh b l o o d b y c e n t r i f u g a t i o n a t 4 ° C ( 1 0 m i n , 1000 × g), then rinsed using saline, recentrifuged, 127 Debouzy et al Drug Target Insights 2008:3 this procedure being repeated until a clear supernatant was obtained, then brought to 30% packed cell volume. 2 μL of Alkyglycerol solu- tion were added in each 1 mL sample and then labelled with 20 μL of spin label solution (5 ns 10−3M or 16 ns 10−3M). After 30 min incubation at room temperature, sample were transferred by capillarity in 20 μL Pyrex capillary tube. This tube was placed in a 3 mm diameter quartz holder, and insert into the cavity of the ESR spectrometer. The ESR spectra were recorded at different controlled temperature (288, 293, 298, 303, 310 and 315 K) with the following conditions: micro- wave power 10.00 mW, modulation frequency 100 kHz, modulation amplitude 2.05 G, receiver gain 6.105conversion time 40.96 ms, time constant 20.48 ms. Sweep range was 160 G with a central fi eld value of 3435 G. The complete membrane incorporation of the spin labels was ascertained by the absence on the spectra of the extremely resolved ESR lines cor- responding to free rotating markers. 5 NS experimentations: The value of outer and inner hyperfine splitting were measured (2T// and 2T⊥ respectively), on ESR specra (Fig. 5B), and order parameter S was calculated following the equation: [13] S T T C T T C = × − ⊥ +( ) + ⊥ +( ) 1 723 2 . // // With C T T= − × − ⊥( )1 4 0 053. . // The increase of the order parameter value means a decrease of local membrane fl uidity. 16 NS experimentations: The changes in free- dom motion of 16 NS were analyzed with the calculation of τc, the rotational correlation time. τc was calculated following the formula: [14] Tc K W h h= × ( ) −( )−Δ 0 0 1 1/ With K = 6.5 × 10−10 s.G−1 In this formula, ΔW0 is the peak-to-peak line width of the central line; h0 and h−1 are the peak high of the central and high-fi eld lines respectively (Fig. 5C). The decrease of the rotational correlation time means a decrease of local membrane fl uidity. Mass spectroscopy The ES-control spectra were acquired in CH2Cl2 as solvent with 1% formic acid, using a VG.Quatro II spectrometer from Micromass/Waters, and treated with the Masslink 4.00 V software. The capillary tension was 3.88 kV, and the cone tension and ion energy 88 V and 1.8 V, resolution values were set to 15.2, and the multipliers 1 and 2 set to 650 V. Investigations and Results SLO structure evaluation in solution and in water samples Chloroformic solution As expected a true solution of SLO was obtained in chloroform (see Fig. 1 top trace) and the con- trol of SLO main composition could be easily obtained from standard 1D and 2D 1H-13C-31P NMR and ES-MS experiments. Especially, no other hydrophobic components such as phospho- lipids, sterols an squalene were detected and the resonances of glycerol moiety (labeled G1,2,3) and those of the chain (see the nomenclature on the Fig. 1) were clearly identifi ed. As relaxation times were quite homogenous (relaxation times T1 and T2 close to 1sec) [15] an estimation of the average length and insaturation of the chains was obtained by building indexes from 1H-NMR peak integrals as follows: As shown on Figure 1 the resonance labeled (4) at 5.2 ppm is representative of methynic group; however, since this resonance is com- pletely overlapped by glycerol signal (G2), the unambiguous resonance of methylenic groups (3) nei ghbouring methynic (4) was used. The resonances (5) were representative of polyun- saturation, and terminal methyle peaks (7) of number of chains. For each group, the value of the integral was divided by the corresponding number of protons (3, for methyl, 2 for methy- len) to allow a count of the number of groups. Finally, an estimation of the chain length refer- ence, A, was obtained by adding all the weighted resonances 1,2,3,4,5,6 and 7, with subtraction of half the contribution of G1 methylenic group of glycerol (at 4 ppm) to overcome the G2 (CH) contribution at 5.2 ppm. 128 Study of alkylglycerol containing shark liver oil Drug Target Insights 2008:3 Within the different samples controlled, no variation exceeded 10% from the following values: Number of groups per chain; A/(7) = 19 +/− 2 Chain insaturation index: (3)/A = 10% +/−1% Chain polyinsaturation index: (5)/A = 4.9% +/− 0.5%, thus indicating a good homogeneity within the different samples used. MS spectra confi rmed this homogeneity by giv- ing exclusively a dominant line at m/z = 351.9, another of half the intensity at m/z = 517 and four minor components at m/z = 301-417-467-633. Aqueous samples Depending on chain length and insaturation found, partial apparent solubilization in the water was not excluded. [16] Hence, 1H-NMR lines were detected on the NMR aqueous sample containing 1 mg SLO (see Fig. 1, bottom trace). However, the linewidthes measured (from 30 to 50 Hz) suggested the that supramolecular assemblies had been formed, such as micelles or droplets. This led to measure T1 and T2 relaxation times. These parameters are closely related to the correlation time τc and the volume of the system as classically described following the relations: [17] 1/T1 = R1 = A . τc . [(1/(1 + ω 2τc 2) + 4/(1 + 4ω2τc 2)] (1) 1/T2 = R2 = 6A . τc . [4 + 9/(1 + ω 2τc 2) + 6/(1 + 4ω2τc 2)] (2) with ω = 400 MHz; A = γ4.(h/2π)2/r6; γ the gyro- magnétic factor, (h/2π) Planck’s constant and r the inter spins distance. Similar relaxation values were found within the molecule (T2 = 30+/− 10 ms, T1 = 420+/− 20 ms) except for terminal methyl groups (resonance 7, T2 = 50 ms, T1 = 310 ms). This allowed to calculate the range of correla- tion time by using the ratio T1/T2 as follows: R1/R2 = [(1/(1 + ω2τc 2) + 4/(1 + 4ω2τc 2)]/ 6[4 + 9/(1 + ω2τc 2) + 6/(1 + 4ω2τc 2)] (3) Relation in ω2τc 2 simplifi ed in a second degree equation giving ωτc.limits (from 1 to 3). Then, assuming a spherical approximation for the molecular assembly, the Stockes-Einstein relation allows an evaluation of the average apparent volume: τc = ηV/kT, soit V = kT. τc/η (4) where η = 0.9 × 10−3P, (N.s/m2 at 298 K), k = 1.38 × 10−23J/kg, T = 297 and V the volume (m3). Finally, 6 � V � 12 nm3 corresponding to a 50Å diameter. Such assemblies are signifi cantly smaller than small unilameller vesicles of phos- pholipids (typically of 10–20 nm radius, with T1 in the 450–900 ms range and linewidths of 40 to 120 Hz) [18]. From these features, collective properties of SLO could be studied by using these aqueous dis- persion in biological medium that organic solutions precluded. By considering the great importance of interfacial systems in biology, such as cell surfaces, complementary physico chemical tests were then performed. Surface properties The partition coeffi cient (LogP) was calculated as described in the method section. The value LogP = 1.3 well confi rmed that, even if the solubil- ity in organic solvent—octanol- is more than tenfold that in water, the presence of SLO at the interfacial area is highly probable. As a conse- quence, possible tensioactive properties had to be tested. The result is shown on Figure 2: starting from 75.8 mN/m (pure water at 293 K) successive additions of SLO resulted in progressive diminution Figure 2. Superfi cial tension (dG-G°, mN/m), 298 K as a function of SLO concentration (mg/mL) (•), and percentage of haemolysis fol- lowing the concentration of SLO (• ). 129 Debouzy et al Drug Target Insights 2008:3 Figure 3. 31P-NMR of DMPC: Column: A) typical spectrum of (top) DMPC bilayers (50 mM) close to transition temperature (296 K) (bottom) and in the presence of SLO (SLO/DMPC = 1/25 W/W) ; column B) spectra of ghosts prepared from rat erythrocytes (top), and in the presence of 3 mg SLO (bottom). Bottom traces: tem- perature dependence of the chemical shift anisotropy for pure DMPC (•), and SLO/DMPC systems, 1/25 W/W ( ) and 2/25 W/W (∆). The arrow indicates the point of the curve corresponding to the top traces. of surface tension down to ST = 53 mN/m around 5 mg/mL. Higher amounts of SLO induced no fur- ther decrease of ST. Such a limited evolution runs counter any detergent effect or soap-like interactions of SLO, as found for instance for SDS or amphiphi- lic [19] molecules like cyclodextrins. [20] However, these negative tensioactive properties suggest interactions with membranes. This point is the topic of the next section. Membrane structure and dynamics study by 2H and 31P-NMR 31P and 2H-NMR spectroscopies of phospholipid dispersions (MLV) were used to observe the struc- tural and dynamics consequences of the presence of SLO at the polar head (31P) and chain (2H) lev- els of the membrane. The polar head group level As shown Figure 3A (bottom of column), the spec- trum of pure DMPC dispersion (MLV) is typical of an axially symetric powder pattern, with a chemical shift anisotropy of 69 ppm, classical of DMPC bilayers in their liquid crystallin phase around (296 K) phase transition7 The chemical shift difference between the lowfi eld and the highfi eld edges of the 31P-NMR spectrum is called Chemical Shift Anisotropy (CSA, ppm) and is directly related to the fl uidity-reorientation- at the polar head level where the phosphorus nuclei are located. On such spectra a mobile phosphorus group gives a single narrow resonance (several Hz) as detected in true solution or for small structures (micelles), while solide state phosphorus gives extremely broad contributions (more than 100 ppm). Note that membrane fl uidity increases (and CSA decreases) with temperature, with a special jump at the transi- tion temperature between gel phase and liquid crystal structure (around 297 K for DMPC),). Thus the plot of CSA as a function of temperature pro- vides a good overview of membrane dynamics at the polar head level where phosphorus nuclei are located, while the lineshape allows to identify the overall membrane organisation (bilayer, hexago- nal, isotropic phases). Such plots are presented on the bottom traces of the Figure 3: for pure DMPC dispersions and for SLO containing MLV (SLO/ DMPC weigh ratios of 1/25 and 2/25 mg/mg) As expected a CSA decrease (around 18–20 ppm) was observed on pure DMPC systems with the transition-related jump around 297 K. Such was also the case for the spectra recorded under the same conditions on SLO containing systems at various temperatures. Especially, no isotropic contribution typical of detergent effect was observed. However, a signifi cant reduction in CSA value were measured at low temperature (under transition temperature, see Figure 3A, bot- tom trace); this increase in local fl uidity was not detected at higher temperatures while transition temperature was found unaffected by the presence of SLO (297 K). The presence of structural rear- rangements was also supported by this decrease in CSA at low temperature, with a normal transition temperature (297 K) and CSA values close to those of DMPC at higher temperatures. 130 Study of alkylglycerol containing shark liver oil Drug Target Insights 2008:3 The acyl chain level 2H-NMR lineshape Figure 4A (top) shows the spectrum of DMPC-d54 (dimyristoyl phosphatidyl choline with perdeu- terated chains) dispersions. This spectrum is typical of phospholipid bilayers in the liquid crystal phase close to transition temperature (296 K) [21]. Such a spectrum appears as a super- imposition of symetrical doublets, each doublet corresponding to a methylenic CD2 group of the acyl chain. For a given doublet, the splitting (ΔνQ) is directly related to the local order fol- lowing the relation: ΔνQ = [A*(3*cos2θ–1)]/2, where A is 170 kHz (for the CD2 bound in DMPC) and θ the averaged value of the solid angle of reori- entation. This splitting can be used in a fi rst approx- imation as an order parameter. As the acyl chain fl uidity decreases from the terminal methyl group (CD3, as shown on the expanded part of the spectrum m on Fig. 4B) to the methylenic groups close to the polar head of the lipids (the so called “plateau region”, from C-2 to C-8 of the chain), the resulting spectrum consists of i) an inner doublet with a quadrupolar splitting of 3800 Hz attributed to the CD3 methyl group, a is found, ii) doublets with increasing quad- rupolar splittings assigned to successive CD2 groups from C14 to C9; iii) the external edge doublet, attrib- uted to the deuterium of the C2-C8 plateau region where a 29 kHz quadrupolar splitting is measured. Figure 4. 2H-NMR spectrum of A) pure DMPC-d54 dispersions at 296 K (the spectrum is expanded in B to show the splitting of CD3 groups of pure DMPC –top- and in the presence of 1 mg SLO—bottom-), Bottom traces temperature dependence of the half quadrupolar splitting (kHz) for pure DMPC (•), and SLO/DMPC systems, 1/25 W/W ( ) and 2/25 W/W (∆), for plateau resonances (C) and terminal CD3 group resonances (D). 131 Debouzy et al Drug Target Insights 2008:3 The main spectrum recorded under the same conditions (296 K) in the presence of SLO (R = 1/25 and 2/25 W/W) also shows a dramatic reduction in quadrupolar splittings both at the superficial level (the plateau region) and in the deep part of the membrane (right traces and curves Fig. 4). Besides, no other contribution i n d i c a t i v e o f i s o t r o p i c r a p i d m o t i o n w a s found. From this part, one can conclude that SLO induces an overall fl uifi zation of synthetic mem- brane, exclusively present below transition tem- perature, without inducing any detergent effect and membrane structure and dynamics modifi cation over phase transition. The following step was to test the relevance of these observations in biological systems, i.e. red blood cells, by using macroscopic haemolysis tests and ESR biophysical measurements. Biological relevance of biophysical results Haemolytic activity The haemolysis curve is shown on the Figure 2. By comparison with well identifi ed haemytic mol- ecules (for instance natural β-cyclodextrin has a 50% haemolytic concentration of 13 mM,) [22] SLO haemolytic activity is found very low, since the maximum haemolysis obtained was 6.6% (21 mg/mL SLO) and 50% haemolysis could not be obtained. 31P-NMR of erythrocyte ghosts The Figure 3B (top) shows a typical spectrum of red blood cell membranes (100 mg ghosts in D2O for a total sample volume of 500 μL) recorded at 296 K with the same parameters as DMPC dispersions. Due to cellular organization (cyto- skeleton, proteins) the overall membrane struc- ture is signifi cantly more rigid than synthetic systems, according with a CSA (chemical shift anisotropy) of 110 ppm. The addition of 3 mg SLO results in a signifi cant reduction of this value (83 ppm), revealing an increase in collec- tive fl uidity without local membrane damages that should have been evidenced by the presence of isotropic line at 0 ppm. However, this effect required at least 2 mg SLO and was not observed for lower amounts. ESR Spin labeling experiments Spin label experiments were then realised to inves- tigate the red cells membrane fl uidity in different temperature conditions. Two probes were sepa- rately used, 5 NS gives information about super- fi cial membrane fl uidity, while 16 NS concerned the inner membrane region. The results are shown on Figure 5. An increase of the mobility of the two probes contribution could be observed in SLO groups, related to a global enhancement of the membrane fl uidity. Furthermore, at low temperature (288 K and 293 K), a drop in the order parameter was mea- sured in the SLO group compared to control, that disappeared at the physiological and at the above temperature (up to 315 K). The same observation was done for the rotational correlation time of the 16 NS probe with a decrease of τ in SLO group. This feature indicated an overall increase of the membrane fl uidity of the erythrocytes induced by SLO at the lowest temperature. This effect was not noticeable at physiological temperature. ESR spin trapping experiments The Fenton reaction in presence of 4-POBN yields characteristic six-line spectra (Fig. 6B showing 4-POBN results). The spin adduct hyper- fi ne splitting constants were aN = 15.73 G and aH = 2.57 G. According to Finkelstein et al. [23] value: 4-POBN spin trap: aN = 15.60 G and aH = 2.55 G and to Augusto et al. [24] who found aN = 15.50 G and aH = 2.50 G; these hyperfi ne splitting constants correspond to a α-hydroxy- ethyl adduct. This is stable adduct results from a reaction between hydroxyl radicals initially gen- erated and the spin trap. The histogram presented on Figure 6A, shows the free radicals promotion for 3 different concen- trations of SLO (8 mg/mL, 0,8 mg/mL and 0,08 mg/ mL) versus control and Vitamine E (8 mg/mL). Statistical comparisons were achieved using non- parametric tests (Kruskal-Wallis). Vitamine E is a reference antioxidant molecule able to recombine with free radical. In the presence of Vitamine E and Alkylglycerol in the same concentration, a signifi - cant strong decrease in the trace amplitude com- pared to control was observed (−26% SLO and −28% Vit E). For lower concentrations of SLO (0.8 and 0.08 mg/mL) no signifi cant decrease in spin adduct detection could be shown. 132 Study of alkylglycerol containing shark liver oil Drug Target Insights 2008:3 Figure 5. ESR spin labeling experiment. (A) 5 NS and 16 NS results. Left Y axis: temperature dependance of the order parameter (5 NS) for control red cells (black diamond) and Red cell in presence of SLO (grey square). Right Y axis: temperature dependence of the rotational correlation time (16 NS) for control red cells (black triangle) and Red cell in presence of SLO (grey circle). (B) Typical 5 NS spectrum, parameter used for order parameter estimation are inner (2T┴) and outer hyperfi ne (2T //) splitting. (C) Typical 16 NS spectrum, parameter used for rotational correlation time was central peak intensity H0, High fi eld peak intensity and the with of the mid-fi eld line W0. 0 5000 10000 15000 20000 25000 Control Alkyrol 8 mg/mL Alkyrol 0,8 mg/mL Alkyrol 0,08 mg/mL Vit E 8 mg/mL A rb it ra ry U n it s A * * B Figure 6. ESR spin trapping experiment. (A) Mean free radical production after exposure using spin trap N-tert-Butyl-α-(4-pyridyl)nitrone N’oxide (4-POBN). For each group the value was the average of 3 measurements ± SD. (B) Typical ESR of N-tert-Butyl-α-(4-pyridyl)nitrone N’oxide (4-POBN) radical adducts following Fenton reaction. (*) represents P � 0.05. 133 Debouzy et al Drug Target Insights 2008:3 Discussion Beside the well established effects of alkyglycerols and polyunsaturated fatty acids on platelet aggre- gation and infl ammatory reactions, the aim of the present work was to investigate physico chemical properties of SLO, especially membrane interac- tions. This work could be undertaken due to two initial conditions fulfi lled: - the average composition was found homoge- nous between numerous samples tested; this was also in agreement with previous analysis showing a composition exclusively made of alkylglycerols and polyinsatured fatty acids [25] (PUFA); - due to relatively amphiphilic properties, SLO exhibits a signifi cant solubility in the water, by the way of self-organisation in supramolecular assemblies with an average diameter of 50Å. This point allows the use of SLO in aqueous preparation without requiring to other organic cosolvents (DMSO…) or special preparations (encapsulation…). The fundamental part of the study, performed on phospholipidic synthetic membranes allowed to identify the fl uidifi zation of the membrane, as evoqued elsewhere. [26] An intercalation of the oil into the hydrophobic core of the membrane have been observed, affecting the order, packing and overall mobility of the lipid acyl chains. This effect was only observed when the chains are in the gel phase and ordered. This intercalation in the mem- brane, coupled with possible antioxidant effect should constitute the basis for a reasonably model for its action. However, our results clearly show that a dramatic fl uidifi zation is obtained at low temperature, while this effect completely vanishes at temperature (over 296 K). This feature observed both in synthetic systems and in erythrocytes would be of interest in cold environmental conditions if related with the clinical effects expected (a better resistance to intense training…). However, it is worth to note that temperature regulation in sharks is very limited, even at very low temperature. Under this point of view, increased fl uidity at low temperature would contribute to maintain cell and tissue functions in extreme environments where sharks live in. By the way of contrast, homeo- therms such as humans generally maintain their internal temperature around 37 °C by active meta- bolic mechanisms such as increasing blood pres- sure, vasoconstriction and active shivering (from 37 to 34 °C) [27]. Lower temperatures lead to collapses and severe hypothermia to death by ventricular fi brillation. Here the observed effect of SLO at low temperature could appear of limited practical interest. However, in cold environments (e.g. high mountain training, outside work in winter…) cutaneous and subcutaneous tempera- ture are often dramatically lower and SLO proper- ties would play here a physiological role. This is particularly true in some pathologies such as Raynaud’s syndrom or microcirculation abnor- malities (Malan’s syndrome) where both blood cell viscosity and capillary membrane fl uidity are involved [28]. Also, cardiac surgery frequently uses extra corporal circulation systems: during operating time, central temperature is set down to 15 °C to protect the brain from the consequences of long lasting hypoxia (20–30min or more). Possible benefi ts in these circumstances remain to study. SLO antioxidant properties are also of interest: hence, vitamin E is routinely used as adjuvant agent in radiotherapy, used for its antiradical properties in the prevention of radio induced fi brosis [29]. Another advantage is the apparent extremely low toxicity as tested by insignifi cant hemolytic activ- ity and complete absence of detergent effect. Another point of interest is that the amphiphilic properties of SLO are very favorable to overcome biological barrier (cellular membranes, intestinal wall…) by allowing both surface binding and spontaneous cell integration as shown by paramag- netic broadening experiments in cells (not shown). [30] Also, the anti oxidant properties were found similar as those of vitamin E and were related mainly to the general properties of polyinsatured fatty acids. [31] Finally, the presence of non specifi c membrane properties of SLO, associated with its low toxicity is consistent with the great diversity of biological effects evoqued in the past, [32] such as anticancer, antioxidant and anti-infl ammatory properties. A promising way for future research would be to evaluate the specific applications for work or physical effort in cold environments. The extreme environmental conditions (pres- sure and cold) met in all day life of Greenland sharks would also been related with an adaptative Evolutional process leading to optimize biochem- ical composition of this species. From this point of view, the properties of SLO under high pressure conditions (diving) should also be evaluated. 134 Study of alkylglycerol containing shark liver oil Drug Target Insights 2008:3 Aknowledgments Thanks to J.Morin and T.Lerond for stimulating discussions, and Prof. J.Hàn-peuh-Pluu for manu- script relecture. Abbreviations 4-POBN: α-(4-pyridyl-1-oxide)-N-t-butylnitrone; 5 NS: 5 nitroxide stearate; 16 NS: 16 nitroxide stearate; CSA: Chemical Shift Anisotropy; EPC: egg yolk phosphatidylcholine; ESR: Electron Spin Resonance; DMPC: dimyristoyl phosphatidyl cholin; HC50: Hemolytic constant 50%; MLV: Multibilayer Vesicle; ES-MS: Electron Spray-Mass spectroscopy; NMR: Nuclear Magnetic Resonance; PAF: Platelet activating factor; SLO: Shark liver oil; ST: Superfi cial Tension; Vit E: Vitamin E. References [1] Hallgren, B. and Larsson, S. 1962. The glycerol ethers in elasmo- branch fi sh. Lipid Res., 3:31–8. [2] Tsujimoto, M. and Toyama, S. 1922. Studies on Unsaponifi able Frac- tions from Some Fish Liver Oil. Tokyo Inst. Technol., 16:471–510. [3] Hallgren, B. and Stallberg, G. 1967. Methoxy-substituted glycerol ethers isolated from greenland shark liver oil. Acta. Chem. Scand. B., 21:1519–29. [4] Brohult, A., Brohult, J. and Brohult, S. 1972. Effects of alkylglycerols on the frequency of injuries following radiation therapy. Experientia, 57:79–85. [5] Brohult, A., Brohult, J. and Brohult, S. 1978. Regression of tumour growth after administration of alkoxyglycerols. Acta. Obstet. Gyne- col. Scand., 57(1):79–83. [6] Hallgren, B., Niklasson, A., Stallberg, G. and Thorin, H. 1974. On the occurrence of 1-O-(2-methoxyalkyl)glycerols and l-O-phytanylglycerol in marine animals. Acta. Chem. Scand. B., 28(9):1035–40. [7] Dufourc, E.J., Mayer, C., Stohrer, J., Althoff, G. and Kothe, G. 1992. Dynamics of phosphate head group in biomembranes. Biophys. J., 61:42–57. [8] Girault, L., Lemaire, P., Boudou, A., Debouzy, J.C. and Dufourc, E.J. 1996. Interaction of inorganic mercury with phospholipid micelles and model membranes. A 31P-NMR study. Eur. Biophys. J., 24:413–21. [9] Rappel, C., Galanski, M., Yasemi, A., Habala, L. and Keppler, B.K. 2005. Analysis of anticancer platinium(II) complexes by microemul- sion electrokinetic chromatography: separation of diastezreoisomers and estimation of octanol-water partition coeffi cient. Electrophoresis, 26(4–5):878–84. [10] Sanders, J.K.M., Constable, E.C. and Hunter, B.K. Modern NMR spectroscopy, ed. O.U. Press. 1989, Oxford. [11] Mavromoustakos, T., Daliani, I. and Matsoukas, J. 1999. The application of biophysical methods to study drug: membranes interactions, bioactive peptids in drug discovery and design: medical aspects 13–24. [12] Debouzy, J.C., Crouzier, D. and Gadelle, A. 2007. Physico chemical properies and membranes interactions of Per (6-Desoxy-6-Halogenated) cyclodextrins. Annales Pharmacologiques Francaises, 65:331–41. [13] Gaffney, B.J. Pratical considerations for the calculation of order parameters for fatty acid or phospholipid spin labels in membranes., in Spin Labelling. Theory and Applications, R.J. Berliner, Editor. 1976, Academic Press: New York London. 567–71. [14] Gornicki, A. and Gutsze, A. 2000. In vitro effects of ozone on human erythrocyte membranes: an EPR study. Acta. Biochim. Pol., 47(4):963–71. [15] Kaplan, J.I. and Fraenkel, G. NMR of exchanging systems, ed. A. Press. 1980, New York. [16] Dennis, E.A. and Plückthun, A. P31-NMR of phospholipids and micelles, in Phosphorus 31P-NMR: Principles and applications, Gorenstein, Editor. 1984, Academic Press: London 423–80. [17] Canet, D., Boubel, J.C. and Soulas, E. La RMN, concepts, méthodes et applications, ed. Dunod. 2002, Paris. 235. [18] Neumann, J.M., Zachowski, A., Tran-Dinh, S. and Devaux, P.F. 1985. High resolution proton magnetic resonance of sonicated phospholip- ids. Eur. Biophys. J., 11:219–23. [19] Gremy, F. and Leterrier, F. Elementrs de biophysique, Flammarion, Editor. 1981: Paris 295–304. [20] Runhua, L., Jincheng, H., Hanqing, W. and Luinhui, L. 1997. Surface tension measurements and 1H-NMR studies of inclusion complex of beta-cyclodextrin with sodium alkyl sulfonate. J. of inclusion phe- nomena and molecular recognition in chemistry, 28:213–21. [21] Douliez, J.P., Léonard, A. and Dufourc, E.J. 1996. Conformational order of DMPC sn-1 versus sn-2 chains and membrane thickness: an approach to molecular protrusion by solid-state 2H-NMR and neutron diffraction. J. Phys. Chem., 100(18):400–57. [22] Leray, E., Leroy-Lechat, F., Parrot-Lopez, H. and Duchene, D. 1995. Reduction of the haemolytic effect in biologically recognisable beta- cyclodextrin. Supramol. Chem., 5:149–51. [23] Finkelstein, E., Rosen, G.M. and Rauckman, E.J. 1982. Production of hydroxyl radical by decomposition of superoxide spin-trapped adducts. Mol. Pharmacol., 21(2):262–5. [24] Augusto, O., Weingrill, C.L., Schreier, S. and Amemiya, H. 1986. Hydroxyl radical formation as a result of the interaction between primaquine and reduced pyridine nucleotides. Catalysis by hemoglo- bin and microsomes. Arch. Biochem. Biophys., 244:147–55. [25] Centre regional de lutte contre le cancer and Montpellier., c.V.-d.A., Métabolisme des lipides pharmacologiques de l’huile de foie de requin. Compte rendu du Comité d’interface de recherché clinique, 1999. [26] Singh, G. and Chandran, R.K. 1988. Biochemical and biological effects of fi sh and fi shoils. Prog. food nutr. Sci., 12(4):371–419. [27] Meliet, J.L. 2000. Eléments de médecine de la plongée. Bull Med. Sub. Hyp., 10:116. [28] Malan, E. Morphogenesis of pezripherical blood vessel-angiodysplasias, ed. C.E. Fond. 1974. [29] Delanian, S., Porcher, R., Balla-Mekias, S. and Lefaix, J.L. 2003. Randomized, placebo-controlled trial of combined pentoxifi llin and tocopherol for regression of superfi cial radiation induced fi brosis. J. of clinical Oncology, 21(13):2545–50. [30] Debouzy, J.C., Neumann, J.M., Hervé, M., Daveloose, D., Viret, J.J. and Apitz-Castro, R. 1989. Interaction of antiagregant molecule ajoene with membranes. Eur. Biophys. J., 17:211–6. [31] Descher, E.E., Lyttle, J.S., Wong, G., Ruperto, J.F. and Newmark, H.L. 1990. The effect of dietary omega-3 fatty acids (fi sh oil) on azowy- methanol induced focal areas of dysplasia and colon tumor incidence. Cancer, 66(11):2350–6. [32] Pugliese, P.T. and Heinermann, J. Devor disease with shark liver oil, ed. I.C. Inc. 1999, Green Bay WI. 135 << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJDFFile false /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.1000 /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 150 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 150 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName (http://www.color.org) /PDFXTrapped /Unknown /Description << /JPN /FRA /DEU /PTB /DAN /NLD /ESP /SUO /ITA /NOR /SVE /ENU >> >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice